Final Exam Study Guide -All Lectures :D

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Need help with cell cycle?? Crash course -easy map at the end!

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Key Ideas: Pink
Numbers: Green
Topic: Blue
Vocab Words: Orange

● (title of each slide)

Topic 26: Microtubules in Mitosis

● Roles of MTs in Interphase and Mitosis
○ In Mitosis
■ 1. Cease Interphase Roles: Organelle transport and positioning
■ 2. New Role: Attach to, Position, and Move Chromosomes
○ What happens during transition?
■ A lot active time and in s phase we get duplication
■ mitosis= separate chromosomes/genetic material
■ S phase we duplicate centrosome and get two spindle poles and become
active and form spindles and two centrosomes move apart from center
toward the edges and chromosomes condense envelope breaks down and
spindles reach to chromosomes and line up in the center and we get
separation of the two sister chromatids- each new cell gets copy
■ Appreciate that chromosomes condense-
● Picture
○ When you see Interphase- decondensed- can't trace path of dna
○ Appreciate- chromosomes condense
○ Mitosis- high condensed
○ Spreads- see chromosome- only in mitosis
○ Interphase- decondensed - can't see individual dna
● Interphase/prophase and mitosis
○ We have transcription from the 2 nm-30 nm- decondensed and partially missing
histones as we describe but it can be packaged in higher ordered structure and it
gets more
○ Key: we know little/nothing of the higher ordered packaging past the 30nm
● Picture
○ Proteins have been extracted
○ You see string molecules which is the dna
○ Individual threads are dna molecules and there is massive amount in a single
nucleus
● DNA compaction: angel hair pasta in lincoln theater
○ Scale up the amount of dna and size of cell to a million fold so we can
comprehend
○ 2 x 3 x 10^9 bp x0.34 nm/bp = 2 meters dna/nucleus
■ Scale by 1,000,000
■ Nucleus: 10 um 10 meters
■ Dna diameter: 2nm 2 millimeter
■ Dna length: 2 meters 2000 kilometers (1200 miles)
○ Uiuc to orlando, florida (Disney to be specific in case)= 1066
○ Amount of dna in cell- like size of room- you would have 1200 miles that you
would have to segregate into daughter cells
○ Take bowl of spaghetti and separate so it doesn't tangle and broken
■ How do you do that?
■ What cell faces
● Events in Mitosis
○ Chromatids are isolated so dna doesn't get tangles
○ Allows efficient segregation of sister chromatids
○ Rebuild nucleus and go onto the next cell cycle
● Regulation of Dynamic Instability in Mitosis
○ Balance of MAPs v. Catastrophe Factors -> Dynamics
○ Increased Gamma- Turc at centrosomes
○ Regulate proteins that are involved in mt assembly and disassembly
● Three classes of mitotic microtubules
○ Kinetochore
■ Centromere where two sis chromatids are held together
■ Proteins and dna
■ One set of mt that reach out and attach to kinetochore and attachment
helps it to reel in sis to appropriate spindle pole
○ Astral
■ Extending from the spindle pole out towards the edges of the cell
■ Looks like a star- radiating out
○ Polar
■ They extend toward the center of the cell but do not connect to
kinetochore
○ All of these are involved in moving chromosomes
● Chromosome segregation general features
 ○ Protein dna complex at centromere- have multiple mts attach to one sis and the
other mt to attach to the other sis
● Structure of kinetochore
○ When we look at the kinetochore we can see there are two parts the outer layer
and the inner layer
● EM of MTs attached to the kinetochore
○ Kinetochore itself looks like an oreo in a side view
■ GO over picture
● Spindle assembly and function
○ 2 (opposite) types of chromosome movements
■ 1. Prior to metaphase- move chromosomes to middle
● A. capture chromosomes
● B. move chromosomes to middle
■ 2. Anaphase
● Move chromosomes to poles
○ Key: to understanding mitosis realize that there are two types of chromosomal
movements-
■ 1.Chromosomes line up on metaphase plate- first movement get
chromosomes to line up in the center of plate- towards the center of the
cell
■ 2.Second is opposite- once we go to divide sis chromatids have to move
out toward the edge of the cell
○ Metaphase is split into two parts
■ Before anaphase - chromosomes move to center
■ Anaphase- ii chromatids separate from each other
■ After- sis move out toward spindle
■ Prior to metaphase- mts from spindle pole have to reach out and capture
sis chromatids and then move to center, go to metaphase to anaphase
● Proper attachment to 2 spindles
○ Proper attachment -> tension -> stabilize MT at kinetochore
■ Stabilize = capture
○ Improper attachment -> no tension -> de-stabilize MT
■ De-stabilize = release
○ One feature that has to happen - how do we properly attachments to spindle poles
■ Not minor issues
■ How mts reach to one sis and the mts from other spindle reach towards the
other sis?
■ Two sis chromatids are identical - kinetochores are identical - how do you
tell one from the other?
● They have equal probability
■ Cell does trial and error process
 ○ If it doesn’t get it right it's starts over and tries again
■ Key: only when mts are properly attached that mts from one spindle pole
attaches to one sister and the other - do we get tension between the sis
chromatids? Once mts attach to kinetochores they start reeling- properly
do this then we have tension the two sis chromatids are being pulled away
from each other- any other arrangement does not cause tension -
■ Incorrect attachments- break them and try again
● Sensing tension
○ Sense tension by chromosome passenger complex (CPC)- lies between both sis
chromatids and it has aurora kinase that can phosphorylate proteins of the
kinetochore- when it is phosphorylated we can have attachment of mts but if the
proteins are phosphorylated the mts will let go and try again
○ Proper arrangement so we can pull - the kinetochores will be pulled away and
physically separated and not close to the aurora b kinase- proteins will be
dephosphorylated and connections to mts will be stabilized
○ Illustrates outer layer and inner layer of kinetochore
○ Inner layer- proteins bound to dna - cenp-a is modified version of histone
■ Another member of histone h3
■ Specialized kind of nucleosome that is part of centromere
■ Cenp-a binds to other proteins that are part of inner layer
○ Outer layer
■ Proteins that are made up of inner and outer layer are connected by
proteins that have coiled coil domains - or long thin cables
■ Another set of proteins
● Opposing Forces Act on Chromosomes During Congression
○ Mts are interacting with chromosomes in multiple ways not just kinetochore, but
whole length of arms
○ Experiment
■ Take laser and break some sis chromatids in half
■ Prior to anaphase- half that contains centromere and kinetochore will
move toward spindle - part that lacks moves to the center of cell
■ Lots of motors proteins that are associated with the arms- move down mts
to the plus end
■ Minus are anchored in spindle and plus are radiating out toward the center
of the cell
■ Prior to metaphase we have two opposing motions
● Pulling on kinetochore toward spindle pole
● Moving arms toward center of cell
■ Prior to anaphase
● Motor proteins on the arms that moving chromosomes toward plus
end win!
 ● They are stronger than the pulling on kinetochore
■ Anaphase
● We turn off those motors that are moving toward the center
● And sister chromatids are pulled to the spindle poles
■ We are pulling on kinetochores and pushing on chromosome arms
● Forces that move to the center is stronger
■ THis is called congrationcongressional = moving things to the metaphase
plate
■ Has to be Kinesins because- moving chromosomes toward plus ends
which is the center of cell- kinesins win PRIOR to metaphase
● Note how similar this process looks to “centering” of mts in interphase
○ Idea that centrosomes have centering activity- find there way to center of cell
○ Structure of spindle pole looks like centering- here is one nucleus and here is the
other and we have spindle poles in center of that structure
○ Like they form wall in the middle -sis chromatids
○ Fighting with each other- allows them to center in what will become the nucleus
● “Spontaneous” Spindle Assembly in the Absence of Centrosomes
○ If you take mitotic chromosomes and mt in extract that lacks spindle poles you
will spontaneously form spindles
○ You will end up with chromosomes lined up in virtual metaphase plate
○ Plus end is with chromosomes and minus ends towards a spot as if there were a
spindle pole there
○ This happens because of the action of motor proteins acting on chromosomes and
mts
○ One set of kinesins that are bound to 2 mts at a time and moving to plus end
■ The mts will slide past each other and motor stays in place
■ Two minus ends are pulled away
■ And plus are brought close
○ Kinesins bind to chromosomes on one end and move mts to the plus end and that
will move chromosomes to the plus end - other kinesins we will have plus end in
middle
○ Other set of proteins that bind to one mt and slide while the walk on another mt
■ Move down minus end and pull both mts together at minus end
■ Kinesins that
○ Two forces that generating spindles
■ Nucleation
■ Antiparallel cross-linking by kinesin- 5
■ Outward push by kinesin -4,10
■ Focusing of poles by dynein and kinesin -14
● How do we get more mts in the vicinity of the chromosomes than in the remainder of the
cell? How do Mts “find” the chromosome
 ○ If you take a look at mts there are higher density towards the center of the cell
than elsewhere
○ Number of polar, and kinetochore- high density in the middle
○ How is it that the mts reach out and grab towards the chromosomes?
● How is the nucleo-cytoplasmic trafficking regulated?
○ Ran= Ras related nuclear protein
○ Small GTPase of 24 kDa
○ There is no nucleus so there is no nuclear export/inport
○ Ran
■ We have gef and gap
■ Where was gef located for ran? - it was bound to chromatin
■ Gef bounds to chromatin even in mitosis
■ Gap was distributed in cytoplasm - in mitosis it is everywhere in the cell
equally distributed
● Role of Ran in Spindle Assembly
○ Ran-GTP-> signals MAPS locally ->
■ Stabilizes MTs and promotes assembly of mts
○ Ran gef bound to chromosomes -> high concentration of rangtp around
chromosomes
■ Stabilize and promote assembly near chromosomes
■ Part of capture mechanism in vivo
○ Ran GAP evenly distributed in cytoplasm, therefore only high concentration of
RANGTP is around the chromosomes
○ What happens is that ef will convert ran to the active form
■ Gtp is active form
■ Only place it can be active is when it runs to ef -
■ Ef bound to chromatin- you have active ran surrounding chromosomes
■ As you have diffusing ran - it runs into gap and turns to gdp form
■ You have high concentration of actin ran around chromosomes and all
other ran is inactive the one that isn’t near chromosomes
■ Ran does then, it stabilizes mt
■ As a result, any mt that are in vicinity of chromosomes get stabilized, the
ones that aren’t near- grow and shrink
■ Any microtubule that ends up going in the right direction gets stabilized
capture chromosomes at kinetochore or we can stabilize polar mts that
surround the chromosomes
■ The ones that go wrong way - can try again - dynamic instability \
■ Any mt that ends up in that zone can keep going- basically mechanism to
stabilize mt that are going in the right direction
■ Metaphase plate
● Is an effect of where polar mts overly each other
 ● That zone is the zone where mts are stabilized by concentration
gradient of ran
● Goes out of zone- it is no longer stabilized and break down
● Mp is defined by concentration gradient of ran that allows mts
from one spindle to overlap the mts from other spindle
● Ran gtp facilitates search and capture - forms stable overlap of mts
that define the metaphase plate
● How do the mts that are attached to the kinetochores “reel in” the chromosomes
○ How do we change direction of motion where we go out towards
■ They overlap so they can form the metaphase plate- when they overlap
you allow zone so the chromosomes can line up
● More MT Dynamics
○ 1. In prophase, prometaphase and metaphase MTs treadmill
○ 2. In anaphase, MTs depolymerize from both ends
■ -> key point: energy of depolymerization contributes to movement of
chromosomes in anaphase A
○ Mts themselves- as it turns out mts are primarily growing as the move toward the
center of the cell, in fact they are treadmilling - kinetochore mts are treadmilling
○ Once we hit anaphase- mts are depolymerized from both ends- part of process of
reeling chromosomes in towards spindle poles
● SPeckle microscopy
○ Treadmilling
○ Kinetochore mts treadmill
● Treadmilling prior to anaphase
○ Have chromosomes in brown in metaphase plate
○ We have spindle at top and bottom
○ White box - picture of that slice and look at it over time
○ Add a small amount of fluorescent tubulin and see what happens
○ We have dot of tubulin near the kinetochore and you can see dot moving out over
time
○ Moves from kinetochores to spindle pole
○ Adding new monomers and kinetochore
○ Growing at plus end at kinetochore and shrinking at minus end at spindle pole
■ Puzzling why?
■ Minus end is anchored in spindle pole- if minus end is anchored- wouldn't
it be capped
■ If plus end is at kinetochore wouldn't it be capped?
■ But it's not….- but then how is it anchored?
● Tubulin treadmilling
○ How do both treadmilling and anchoring occur simultaneously?
 ■ If we are treadmilling we continuously adding to plus and removing at
minus
■ They both have to be free but capped…
● Anaphase A: Motors and De-polymerization
○ Energy of Depolymerization moves the chromosomes
○ Building the plus end prior to metaphase and anaphase- and after anaphase we are
depolymerizing from plus end- energy moves sisters during anaphase
● Picture
○ Give solutions to problem
○ At kinetochore we have two sets of proteins that are bound to the dna
■ That is the inner layer
■ Proteins that are the outer layer of the kinetochore
● Both held by long thin proteins
■ No one is hanging on to the end of mt- that end is between the inner and
outer layer - and inner is grabbing to the sides of the mt near the end- hold
on to the mt near the end- but it can be free to grow or shrink
■ Kinetochore attached to mt - free to grow or shrink
○ Key: the left half of the slide is before anaphase- and the right is after anaphase
■ Left- set of movements that is moving towards center
■ Right- moving chromosomes to spindle poles
■ You are not holding to very ends of mt
● On the left half - events prior to anaphase and right is after
anaphase
● Key: proteins comprise inner layer of kinetochore
○ Space in between long thin proteins that connect proteins of
inner and outer layer
○ What phase are sis chromatids are pulled apart- beginning
of anaphase or prior- during- pulled apart
● Answer: Ends of mts reside between inner and outer layer- ends
are not capped so we can have exchange of monomers
● Proteins of outer are bound to sides of mt near the end- hold onto
the mt without capping the end
■ Part of the story of how sis chromatids move- function of dynein
● Connected by long proteins that connect to printer of inner
kinetochore
● Dynein moves down mt- the chromosomes is pulled behind
● Dynein- ski boat
● Inner and chromatid- water skiers
● Vine connecting boat to skier
● Proteins are off prior to anaphase and on to the right
 ● These proteins like kinesin 13- mt disassembly are on in anaphase
so we have net assembly prior to anaphase and disassembly after
● Switch in anaphase turns off mechanisms that move chromosomes
to middle and turn off when we switch to anaphase and turn on the
ones that will move chromosomes toward the end of cell
● More mt dynamics
○ Key: There are actually two types of movements
■ 1. Dynein motor proteins that pull sis toward spindle pole using atp
hydrolysis to move dyneins
■ 2. Depolymerization
○ In yeast if you delete all motor proteins that are involved i sis chromatids, they
still move just fine- energy is SUFFICIENT
● How to harness the energy of de-polymerization
○ Ring proteins identified genetically in yeast
■ Yellow protein - dam 1
■ Green- kinetochore mt
■ Dam one
● Forms polymer so each that looks like vertebrae is dam one protein
- each bind to make a ring that surrounds mt
● Dam1 Rings on Yeast MTs
○ What happens when mts depolymerize
● Sliding of rings during disassembly of mts
○ Rings slide together
○ Count all rings at the top, then what happens at 2 min to 5 min it shrinks, the rings
before it shrinks you end up with the same number after
○ Rings do not fall off when mt shrinks - but the rings slides away from the ends
that are depolymerizing towards the center
● Dam1 Ring “surfs” down depolymerizing MT
○ Energy of de-polymerization drives chromosome movement in anaphase a
○ Proteins in inner layer of kinetochore that are attached to sis chromatids
○ Long thin proteins connecto to proteins in outer layer
○ Proteins in inner layer there is the dam1 ring
○ End of mt is between inner and outer layer- depolymerize, protofilaments spill out
like making waves which push on ring and moves
○ Pulls the sister chromatid behind it
○ You want to segregate chromosomes efficiently
○ 10 to the 5th cell division you have a mishap - if something happens with dam1
ring, then you have dynein
○ Even at rate- there is disease from mishap
● Mt end is in between the inner and outer layers of the kinetochore
 ○ The arrangement provides the answer to how the kinetochore can hold onto the
“end” and also allow treadmilling
● Picture
○ Dam one rings aren't shown but the would be between depolymerizing and the
dyneins -- ring in between plus end and dynein
● Picture - anaphase B
○ Anaphase a
■ Early part where sis chromatids down depolymerizing mt to spindle pol
■ Moving sister towards to spindle poles
○ Anaphase b
■ Movement of spindle poles away from each other
■ Results of depolymerizing astral mts- shrinking put spindle toward edges
and pushing on polar so we push spindle to opposite sides or apart from
each other
● Motors and Movements in anaphase b
○ Kinesins that are bound to polar mts where they overlap
○ Kinesin-14 only one one
○ Kinesin 5- both- push on both
○ Those things bind to one mt and move along another - motor proteins move
towards plus end
○ Push mt out toward the edges which pushes the centrosomes towards the edge
○ Dyneins at edge of cell attach to plasma membrane and those move towards the
minus end because dyneins fixed in place pull mt passed them minus ends in
centrosome move toward the dyneins, kinesins pushing the outwards and dyneins
is pulling centrosome outwards
○ Depolymerization of astral as depolymerization of kinetochore mts
● Picture
○ Depolymerizing at plus and minus end
○ Plus depolymerization that drives movement of ring protein and pulls sis
chromatid behind them
○ Kinetochore- plus and minus- depolymerizing so mts are shrinking
○ Tips of astral- depolymerizing so they are shrinking and dyneins pulling mts
toward the edge
○ Middle- polar- kinesins that pushing mts apart from each other and push spindle
poles away from each other
○ This will all be turned on when we go from metaphase to anaphase transition

Topic 27: Integrating Cells into Tissues Part 1 Cadherins and Cell Adhesion
● Types of tissue
○ Epithelial
○ Connective
○ Muscle
○ Nervous- or neural
● Epithelia
○ Typically layered of cells have tight contact between individual
○ Sheets of cells that can be one cell thick or stratified- multiple cells stacked
○ Find these at transition of inside and outside
○ Skin is example; so is lining of gut- inside and outside - lining of your lungs -
lining of secretory organs
○ Cover or coat things
○ Lining of gut is single layer of cells - absorbing nutrients
○ Skin is stratified- mechanical barrier between you and outside world
○ You can have it where it's not all tight- where there is not a lot of mechanical
stress
○ Keratinized vs soft
■ Hard because it faces a lot of mechanical stress
■ Soft - not a lot of stress
○ Lots of functions
■ Absorption, exchange, and protection
■ Absorption exchange secretion protection- waterproofing
○ Allows for exchange of materials but selective- keeps out the bad stuff
○ You are water proof- jump in pool you don't swell up - barrier from letting water
coming and and leaving
○ Keeps things where they're supposed to be
● Connective
○ Connective tissue is largely non-cellular
○ Made up of extracellular matrix - ecm
○ Dense connective tissue
■ Very few cells
■ Majority of tissue is ecm- proteins that are excreted outside of cell
■ Epithelia- densely packed cells next to neighbors
○ Examples
■ Cartilage,bones, ligaments, tendons,
■ Blood- mostly serum fluid with few cells floating around in it-ecm around
blood cells
■ Fat tissue- connective
○ Connect things together- attach muscle to bone or bone to bone or muscle to
muscle
■ Anchor things
 ■ Support
■ Provide skeleton that supports body
■ Cushioning- like cartilage
● Major functions of fat tissue
● Muscle
○ Skeletal muscle
■ Voluntarily contract
○ Cardiac muscle
■ Very similar to skeletal- not voluntarily
○ Smooth
■ Do not control with nervous system
■ Dictated by involuntary type of signals
● Neural tissue
○ Have neurons that send signals-
○ Bunch of other cells like glial cells that are specialized
● Multicellular
○ mOtion of cells working together- key transition point for it to occur
● Events over evolution
○ Focus t bottom
○ 3.5 billion - life
○ Most life initial life was single celled life
○ One can ak when you had multi organisms
○ Fundamental transition - single to multi organisms
○ Key: order of 500-600 million years ago - not very long- last 8t or 9th that life and
planet has existed
● Some major transitions in evolution
○ 3.5 billion years ago
■ Unicellular life emerges
■ Photosynthetic bacteria begin to release oxygen
○ 2 - longer ago than 2.7 bya
■ Eukaryotes emerge
○ 555 million years
■ Multicellular organisms common in the sea
■ Single cell organisms -> multicellular organisms predates this time
○ 500 million
■ Vertebrate fish
○ 450 million
■ Arthropods move onto land
■ Descendants become spiders,mites, scorpions and millipedes
○ 420 mya
■ Land plants evolved/ emerge
 ○ 360 mya
■ 4-limbed vertebrates move onto land as seed plants and large forests
emerge
○ “Philosophical or lifestyle differences between unicellular organisms and
multicellular organisms
○ Transition of unicellular to multicellular is relatively recent key:
■ Key: that transition represents philosophical change in how cells do
business
● C. Elegans: development follows an invariant lineage
○ During development, approx 12 percent of cells undergo apoptosis
○ 1090 cells formed
■ 131 undergo apoptosis to get out of way
○ Enormous difference how single organisms act because you don't want to lose
cells- but multicellular it can benefit the organism
○ Apoptosis- one unique feature in multicellular organisms
○ Key: appreciate huge transition that has to accompany from unicellular organism
to multi
● Early experiments to understand cell adhesion
○ Basic features is that cells stick together
○ Sort from each other to form tissue
■ How do they do that
○ H.v. wilson- sponge cell adhesion
■ Focused on sponges
■ Take sponges- pass through sieve or mechanically dissociated cells and
have individual cells where they are not attached
■ Let them sit in solution with each other- they will bump with one another
and stick together and reform sponge- cells come together and adhere to
one another
● Use two different species
○ One in red and yellow, and disrupt adhesion of both sponges and mix both
populations together, they sort out from each other
○ Blue and yellow don't mix, yellow will go with yellow and blue will go with blue
○ There is specificity to cell-cell adhesion
○ Whatever is driving adhesion - something unique about yellow that lets it bind to
yellow and blue bind to blue
● Embryogenesis
○ Red- ectoderm
■ Green- mesoderm
■ Blue- neural
■ Yellow- endoderm
 ○ Three layers and region that makes neural tissue, cut out chunks and see if red
sticks to red, and vice versa
● Reaggregation of amphibian embryonic cells
○ Cut in chunks and mix them and you see ectoderm stick to each other and not
other different types of cells
○ Consistent with the idea- general property- different tissues within organism have
different specificity so cells from one and from another hold together but not each
other- a will attach to a but a will not attach to b
○ Hint: when we mix cells together and adhere to each other , they sort out
■ Here ectodermal cells are on the outside and neural cells end up in the
inside
■ Remarkable- that's how they are actually organized!
■ Possible that cell adhesion drives sorting of tissue during development
○ Adhesive properties of cell- ability of cells to sort out during development
■ If that's the case-many different types of tissue to sort out in same pattern
● Sorting in amphibian embryonic cell aggregates
○ If you go through variety of different cells that's how things arrange - neural cells
in center- epidermal on the outside
○ Consistent with the idea that adhesive properties of cells is CONSISTENT with
adhesion playing a role in arrangement of the tissues
● Cell sorting in embryonic organs
○ Taking cells from embryonic organs
○ Specificity and cells that are outside are on the outside
○ Steps
■ Dissociate embryonic organs
■ Combine
■ Cells reaggregate
■ Sort according to organ type
■ Specific patterns of aggregation
○ Differential adhesion hypothesis Steinberg:
■ Cells sort due to differences in adhesive properties (could be quantitative
or qualitative)
● How can one identify the hypothetical adhesion molecules?
○ Doesn't tell us what molecules are involved
○ This experiment tells us mcb- goal to identify proteins on surface of cells that
interact with other cells to hold them together
○ Question if that is simple explanation how do we identify those proteins?
○ Steps
■ 1. First need an assay in vitro aggregation
 ● Purify proteins - take cells that you can dissociate and put in
buffer- petri dish- and if cells bump and stick they form cell
aggregates
● Ability to form aggregates when you shake cells and allow for
bumping
■ 2. People discovered that easy way of dissociating cells if removing
calcium
● Presence of calcium - cells stick and vice versa
○ Strategy to identify proteins
■ If you can find antibodies that bind to adhesion molecule and blocked their
function, antibodies should prevent aggregation of cells
■ Look for specific antibodies that block aggregation because they are
attached to the adhesion molecules
■ Throw in antibodies and look for the ones that do not aggregate
■ Massive amount of work - took whole cells and injected those cells into
mice
● Mice made antibodies against al lot of proteins on surface of cell
and made 100s of monoclonal antibodies- recognized proteins on
surface of cell- screen through 100s of antibodies to find small
number that blocked reaggregation
■ Upshot- identified antibodies that could block cell adhesion
● Adhesion blocking antibodies were used to identify the ca2+ dependent adhesion
molecules
○ How can antibodies be used to identify the gene encoding a protein of interest?
○ Screen cdna expression
○ The cdna encodes a putative cell -adhesion molecules (cam); how do you
convince yourself that it really is a CAM?
○ Question: we have antibody that binds to adhesion protein but how do we know
what that protein is and what the sequence of the gene is?
■ Screen an expression library
● Genomic library
○ A library is when you take set of clones and clone them into plasmids or viruses
and that set of clones represent all the parts of the genome
○ We have genomic dna and cut it up into thousands of tiny pieces using restriction
enzyme
■ Clone little pieces into a plasmid so we have large set of plasmids that
takes up different piece of human genome
○ Any given plasmid can fit only a certain amount of dna - 20k base pair of dna
○ Cutting up 3 billion into 10-20k base pair and then cloning them
■ We can then transform those pieces into e coli bacteria
 ■ Endup with thousands of bacteria each that has different part of human
genome
● Plating for single cells- 6 min lecture 3
○ Take bacteria that we transformed and then plating for single cells
○ All cells in a colony are identical
○ Each colony contains a different clone
○ Take dilute solution and plate on petri so cells are separated
■ Whole series of individual cells
■ Let them grow into colonies that have billion of cells
■ If we start with single cells than all cells in this colony will be identical to
each other
■ They all contain one peice of human genome in plasmid- each has same
piece of dna
■ Each colony has different part of gnome
● Genomic library
○ Refer to different things- chopped up genome but more commonly refers to
plasmids that we ligated things into or colonies on a plate
○ Or you can take all colonies and put in a mixture together
○ Or purify dna of mixture and have lots of copies of plasmids- refer to a library
○ Libraries are almost always made in e coli
○ Libraries are collections of ( a mixture of ) clones
○ Libraries can refer to bacterial colonies or to dna purified from pooled bacterial
colonies
○ Chopped up human genome in a lot of pieces but which one has clone that you are
interested for? Gene of adhesion protein
■ Refer to screening a library
● Looking at many individuals for rare clone that contains gene
interested
■ To find the clone you are interested in within the library you need a way to
search through all the clones in the library for the rare clone that is the one
you are interested in. you need a way to screen the library
● Cdna library
○ Not using genomic library- simplest one to understand and it's nice because we
chop up whole human genome- we have genes and promoters and enhancers
■ It contains complete set of dna from the genome
○ Cdna
■ Copy - it starts with mrna as starting material than genomic dna
■ Enzyme from retrovirus reverse transcriptase- it converts single stranded
rna to double stranded dna
■ Makes dna copy that has same sequence- don't need to worry about how it
works
 ■ Start out with messenger rna and purify it from other rnas and copy it into
double stranded dna and clone those pieces into our plasmid
■ Great thing- they contain entering coding region on a single piece of dna
■ Coating on fragments of dna that are contained in our library
○ Genomic
■ Human genes are large - given human gene can be 100k base pairs in size
so average is too big to be on an individual clone
○ Cnda- introns already spliced out and end up with pieces of men's that are in the
size that can be cloned and have all of the coding region or exons
● Outlines cloning cdna
○ Add linkers- short sequences that have restriction enzymes and ligate to end and
clone it into the vector
○ Start with mrna and make it double stranded dna and clone those pieces
● Expression library
○ Each bacterial colony with the library will produce the human protein
corresponding to the particular cDNA clone in that colony
○ Library in which cdna clone can be expressed, transcribed and translated in e coli
○ Start out with plasmid that contains bacterial promoter and shine dalgarno
sequence ( translation start site for bacteria)
■ Make an mrna and ribosome can jump on and translate and convert cdna
into a protein
■ Each one of our clones contains cdna for different protein
■ Expression library- make different human protein
■ Libraries almost always made in e coli
○ Human library
■ Start with human dna or messenger - white
■ Whatever library - the dna or rna is the starting source
● Screening a library
○ To find the clone you are interested in within the library you need a way to search
through all the clones in the library for the rare clone that is the one you are
interested in. You need a way to screen the library
○ We have expression library, we have thousands of clones of bacteria on a plate
and each clone has different dna from different messenger rna and each makes
different protein - how do we screen through all of these colonies??-
○ Screen Expression library using antibody the one that disrupted adhesion by
binding to protein
● Experiment- colony lift-screening and expression
○ Start out with dozens of petri dishes that contain bacterial colonies that have
different colony from cdna library
○ Take piece of filter paper
 ■ Usually nylon and we cut a piece and lay it down on top of the colonies-
some of the cells from each colony transfer onto the nylon membrane
■ Some of the cells stay on the plate-so we have our plate saved so we can
go back anytime
■ Cells from each colony transfer on membrane
■ Treat cells on membrane with various reagents that break cells open- and
those proteins will stick onto the membrane - where the cell was
■ Incubate the membrane with our antibody and secondary antibody and
only bind to colonies in which protein that is recognized is produced
■ Make antibodies radioactive and get black spots that have our protein of
interest
■ Go back to master plate and know that that colony is the one we are
interested in and we know sequence of dna we are interested in
● How would you identify a protein (a band on a gel) in the genomic era?
○ Use antibody to precipitate the protein of interest or to identify which band on a
gel is your protein
○ Cut out band on gel or take precipitated protein and subject it to mass spec
○ Identify protein based on protein composition and mass compared to a database of
information on each and every protein in the genome
○ Take antibody and precipitate protein of interest
■ Take protein and fire it into mass spec
● Key: it is very sensitive measure of molecular mass
■ Take protein and chop it up into pieces using protease
■ Put each piece in mass speck and determine molecular mass
■ Known sequence of genome, have computers search for the open reading
frames
■ Computer predicts mass of each protein and each fragment of each protein
■ Take mass of all proteins and compare it to data base that has every mass
of every protein and that will identify the protein we shot into the mass
spec
■ Requires 2 things:
● purified proteins into mass spec and sequence of the genome
● Adhesion blocking antibodies were used to identify the ca2+ dependent adhesion
molecules
○ How can antibodies be used to identify the gene encoding a protein of interest
○ Screen cdna expression
○ The cdna encodes a putative cell-adhesion molecules (cAM) how do you
convince that it actually is?
● Expressing cadherins in a fibroblast confers cell - cell adhesive properties
○ Fibroblasts and certain cancer cells show very little cell-cell adhesion even in the
presence of ca2+
 ○ Transfecting these cells with a cdna encoding for e-cadherin confers the property
of calcium dependent cell-cell adhesion
○ Disruptive cell adhesion
○ Number of particles in assay- if these molecules adhere from 12 to one particle
adhering -
● Discovery of cadherin
○ Protein = cadherin
○ What you do to show that you really have cell adhesion
■ Start with fibroblast- they are normally surrounded by ecm - don't adhere -
like islands in ecm
■ So they have low adhesive properties on their own
■ Actually cloned a protein of cell-cell adhesion take cadherin and express it
in fibroblasts
■ When he did this - fibroblasts stuck to each other- proved that clone
identified was capable of producing protein that allowed cells to stick
together
■ Adhesion was calcium dependent - and protein that mediated calcium
dependent adhesion
● An rnai experiment demonstrating that cadherins are necessary for strong cell-cell
adhesion
○ Experiment
■ Generated rnai that targets this cadherin and injected into frog eggs
■ Asked what happens if we eliminate cadherin protein by eliminating
messenger rna
■ Blastocyst- they are like balls and opening the top of it
■ On the left- we have rnai - the cell spill out
■ On the right- cells stick together
■ This tells us that cadherin can mediate adhesion - but cells do not adhere if
cadherin is gone in regular cells
● Different tissue types segregate from one another during development. Separation of the
neural tube from the overlying ectoderm is a classical case
○ As we develop different tissues- different set of cells adhere to each other-
○ We end up with different patterns of adhesion in different cells
○ Early embryology experiments showed that different cell type selectively adhere
to one another
○ e - cadherin confers cell-cell adhesive properties to a non-adherent cell
○ Might animals express multiple cadherins to help drive tissue sorting?
○ People thought at that time didn't realize there were so many gene families - are
there proteins that mediate different tissues and are they related?
● Cadherins comprise a family of adhesion molecules with differential tissue distribution
○ The ectoderm expresses e-cadherin
 ○ The neural tube expresses n-cadherin
○ Lots of antibodies and look for the ones that disrupt and have expression library
○ Discovered family of protein that were expressed in different tissues
○ Given different names based on location
● Expressing two different cadherins is sufficient to drive cell sorting
○ Express p-cadherin in one population and label them green
○ Express e-cadherin in another and label them red
○ Mix and add calcium
○ Populations sort out over time
○ Later time points- e sorts away from P.
○ In one fibroblast he expresses e cadherin
■ Those cells are green
■ And make e cadherin in red cells
■ They do sort out from each other
○ Cadherins can mediate cell-cell adhesion and required in living organisms,
properties are sufficient for cell sorting
● The cadherin superfamily
○ Cadherin superfamily is characterized by a cadherin motif called an EC domain
○ Based upon this, humans express at least 80 different cadherin superfamily
proteins
○ Are they all adhesion molecules? Why this complexity?
○ Key: what they have in common is the ec domain - outside of cell the
extracellular domain - domain on the outside that interacts with neighboring cell
■ These cadherins are transmembrane proteins that span plasma membrane -
outside part interacts with other cell and they have cytoplasmic domain
○ Divide superfamily into subfamilies- subfamily is the classic cadherins
■ There are other family members- protocadherins we know little
■ Some of them have anti-adhesion properties
■ First cadherins discovered were the “classic cadherin” and these have a
conserved domain structure
■ Classic cadherins mediate cell-cell adhesion
■ Research on the other types of cadherin family members is just getting
started. Thus far, the few protocadherins that have been studied in detail
are anti-adhesive
● Differential cadherin expression demarcates regions of the developing brain
○ Ask which family members are produced in which cells in mouse brain
○ Key: different parts of mouse brain expresses different types of cadherins
○ Takeichi and others think that the diversity of cadherin family members might
help explain complex segregation of different cell types. Different cadherins, for
example, are expressed in discrete regions of the brain.
○ Key: not enough cadherins to explain the wiring of brain at neuron-neuron level
 ○ Not enough cadherin family members to explain all the wiring
○ Different family members specify different regions of the brain
○ Two levels of specificity - region of brain becoming - in that region more
complex structure that does neuron to neuron wiring
● Model of cadherin structure
○ Extracellular domain is made up of 5 domain repeats
■ These regions are calcium dependent and binds between the balls/domains
■ When calcium binds- it goes into a straight conformation and without it it
becomes floppy
■ If you want to dissociate cells you take out calcium - not case in living
organism - there is always calcium outside of cells
■ Calcium is always around and functioning and causing cells to adhere-
only experimentally does this work
○ On the left is a model of how cadherin may function
■ Each of these molecules is a cadherin molecule - they cluster in patches on
surface of cell and that's due to interactions with cadherins in an individual
cell- binding to another cell
■ Cadherin binds to another cadherin on another cell and to the neighboring
cadherin
■ Like a patch of velcro 0 - little tiny hooks where each individual velcro
molecule hooks to the other material - and they in a patch have millions of
little hooks that - two pieces are held tightly - millions of cadherins in a
cluster on surface that interact with millions of cadherins in the
neighboring cell
○ Model structure part two
■ In other textbook, the repeat domains overlap the repeat domains of the
other- instead of the tips interacting- these are from differences in the lab-
not clear how this functions
● How is cadherin binding specificity determined?
○ DOmain swapping experiments
○ Exchange different portions of the extracellular repeats, assay binding
○ N-terminal 113 aa essential for specificities -1st cadherin repeated
○ Domain swap experiment
■ Took e cadherin that had all 5 repeats and p is the repeats farthest- if you
swap the four domains closest to membrane and change it with p instead
of e, then that chimeric protein behaves like a p cadherin - stick to p not e
■ Specificity for these interactions are in the domain that is farthest from the
membrane
■ If we have p - and 5th one being e- it sticks to E
■ So the last one or 5th domain is the one that determines p or e or the
specificity
Topic 28: Integrating cells into tissues Part 2 cadherins and other cams

● Different cadherins are expressed in different cell types

○ E cadherin: epithelial
○ N-cadherin: neuronal, heart, skeletal muscle
○ P-cadherin: placental, breast epithelium
○ VE-cadherin: endothelial cells
○ How does cadherin-mediated adhesion work?
○ Express e-cadherin in L cells; label w/ green fluorescence
○ Express N-cadherin in L cells: label w/red fluorescence
○ Qualitative difference in adhesion molecules can mediate cell-cell interactions
● Three ways in which surface molecules can mediate adhesion
○ 1.Function as homodimers- can interact with identical copy of itself on
neighboring itself
○ 2.Heterophilic- binds to distinct
○ 3.Or they can interact with an intermediate
○ Aggregation experiments defined two types of adhesions:
■ 1. Ca2+ dependent
■ 2. Ca2+ independent
● Cell sorting can be driven by differential adhesive strength
○ Lowr level of n-cadherin in red
○ Higher level of e-cadherin in green ( e or n cant remember sorry)
○ One type of cell that does not adhere to itself express single cadherin and same in
other - red and green - but green makes more cadherin
○ Mix them -they sort out as if they were making different cadherins
○ Cell sorting may be due to both different types of cadherins and different levels of
cadherins
○ Other levels of regulation such as ptm?
○ Higher level end up in the middle- have tighter adhesion
○ We don't have to have different cadherins to mediate these effects
● These models based analysis of the extracellular domain alone
○ Does the cytoplasmic domain contribute to adhesion?
● Does cadherins cytoplasmic domain contribute to adhesion?
○ Cytoplasmic domain is conserved
○ Experimental strategy
■ Seems odd but see results in 2 slides
○ Start with epithelial cells in culture
■ Overexpresses cytoplasmic domain of n-cadherin in those cells
○ Took a clone that expressed only cytoplasmic domain of cadherin and
overexpressed it - cell type that could adhere and produced high levels of cd
 ○ And that disrupted adhesion
● Overexpression of a cadherin cytoplasmic tail disrupts cell-cell adhesion
○ Hypothesis- cd binds to something and that something is required for adhesion -
so that when we express a lot of cd it binds to whatever thing is and soaks it all up
so that its not available to bind to the wild type cadherin -
○ Dominant negative effect
○ How might overexpression of cytoplasmic domain disrupt adhesion
■ Hypothesis: cd sequesters or binds a component that is necessary for cell
adhesion in such that the component is not available to the wild type
cadherin
○ Prediction:
■ 1. Cd binds something required for adhesion
■ 2. The cytoplasmic domain itself is required for or contributes to adhesion
● Is cadherins conserved intracellular domain required for adhesion?
○ Deletion analysis- take a clone of cadherin and here is region of clone that
encodes cd and making series of clones that lack part of gene and produce a
protein that lacking some amino acids
■ Cut off a little at end and then more - and make truncated protein - we cut
section in the middle -
■ When you delete two different parts- the cadherins can mediate adhesion-
and those two parts are needed
○ Delete different portions of the intracellular domain
○ Intracellular domain is required for adhesion
● What does cadherins intracellular domain interact with?
○ Immunoprecipitation ( and coimmunoprecipitation)
■ 1. Add antibodies that recognize protein of interest
■ 2. Add beads containing antibody binding protein
■ 3.Put it into cell extract
■ 4.Isolate beads; analyze to see if other proteins are bound
○ Generate antibody that binds to cd of a cadherin
■ Incubated with cell extract and then we add beads that attach protein that
bind to antibodies
■ Precipitate antibody and whatever it's bound to
■ Precipitate cadherin that is connected to antibody
■ And precipitate any other protein that is bound to the cadherin
■ Ask what those extra proteins are
○ This identified three proteins
■ Use anti-cadherin antibodies to immunoprecipitate cadherin:
● Pull down three other proteins that bind cadherin:
○ Alpha; beta; gamma-catenin
○ Catena- chain in latin
 ■ These proteins link the IC domain of cadherin to the actin cytoskeleton
■ Idea is that these proteins for chain between cadherin and something else
in the cell
● Proteins that link cadherin intracellular domain to the actin cytoskeleton
○ Bind to actin cytoskeleton
○ If you want to attach both cells, make a strong connection, is connect cytoskeleton
of one cell to the other cell
○ You have actin cytoskeleton connected to canten that is connect to cadherin which
is connected to cadherin of the other cell to catenin to actin cytoskeleton
○ Other proteins involved we don't need to know
● Cadherins are sometimes normally clustered with actin filaments inside cells
○ Punctate staining= clustering
■ There isn't uniform aining- there are spots where there is intense and not
○ Co-localization with actin
○ They coincide with one another- alpha, beta, and gamma suggest that all three act
together
● Role of cadherins conserved intracellular domain?
○ Same deletion leads to not binding to actin cytoskeleton - catenins to actin
cytoskeleton
● Major families of CAMS
○ FOur families
■ 1. Cadherins
● Mediate cell-cell adhesion
■ 2. Ig-superfamily CAMS
● Ig repeat domains found in antibodies
● Mediate interactions of proteins
■ 3. Integrins
● Mediate adhesion between cell and extracellular matrix
■ 4. Selectins
● Bind to sugars that are expressed on surface of other cell
● Immunoglobulin (IG) superfamily: ca2+ independent adhesion
○ Neural cell adhesion molecule
■ Like cadherin originally identified by antibody inhibition of aggregation:
mediates homophilic interaction
■ At least 20 forms generated by alternative splicing
■ Carries large amounts of negatively charged sugar, poly-sialic acid or PSA
■ NCAM+ PSA aggregates less well than NCAM-PSA amount of PSA
decreases during development
● What does this mean
○ Intercellular adhesion molecules (ICAMS): leukocyte movement into tissues
 ○ Early in development, these are modified by sugar - reduces the stickiness- idea
that early in development you don't want to be sticky until you make proper
connections and remove acid- and become more stable
● Ig Superfamily
○ In general, play important roles in proper targeting of neurons
○ Hundreds of family members - 750 in human genome
○ Why so many - complexity of brain - help in wiring
○
■ 100 x 10^9 neurons - 1000 connections
■ 100 x 10^12 connections
● Cadherins in developing nervous system
○ Cell movement and adhesion are related- inversely proportional
○ To move you have to stop adhering
○ We regulate level of adhesion when cells need to move
○ Developing organism- formation of neural tube
■ Invagniation and form neural tube
■ Becomes different parts of body- peripheral nervous system- stop adhering
and move to different parts of the body
■ In order to move you have to stop adhering to your neighbors
● Cadherins and Cancer
○ Metastasis
■ Loss of e cadherin correlates with metastasis
■ Re-expressing e cadherin in a highly metastatic epithelial cancer cell
suppresses metastasis
■ 1. Mix a normal heart fragment with normal epithelial cells. Epithelial
cells encapsulate the heart fragment
■ 2. Mix a normal heart fragment with metastatic epithelial cells. Epithelial
cells invade the heart fragment
■ 3. Force expression of e- cadherin in the metastatic epithelial cells.
Epithelial cells once again encapsulate the heart fragment
■ Cancer migrates- but it has to stop adhering to its neighbors
○ Experiment
■ Take a chunk of heart tissue H and take some epithelial cells -N
■ Incubate epithelial with heart, epithelial form dark ring around heart
tissue- standard
■ In center - we incubate cancer - those cells with heart, they invade the
heart tissue - if you take soame cancer cells and you over express cadherin
- they adhere to each other and form ring around heart
■ Suggesting for invasion of metastasis you have to have a low level of cell-
cell adhesion
Topic 29 Cell Junctions

● Types of Cell Junctions

○ First defined by EM
○ 1. Anchoring junctions: attach cells to each other or to extracellular matrix
■ Adherens junction: cell/cell via actin
■ Desmosomes: cell/cell via IFS
■ Hemidesmosomes: Cell/ECM via IFS
■ Focal adhesion: cell/ecm via actin
○ 2. Occluding junctions
■ Function as barriers
■ Tight junctions (zonula occludens) in vertebrates separate junctions in
vertebrates
○ 3. Communicating junctions
■ Gap junctions
■ Synapses (neurons)
■ Plasmodesmata (plants)
● Types of anchoring junctions
○ Actin- actin
○ If- of one cell to if of other cell
○ Local adhesion - proteins to ecm
○ Hemidesmosome - if through proteins to ecm
● Original description of junctional complexes in epithelia
○ By em, characterized tripartite junction complex in numerous different types of
epithelial cells:
■ Mucosal epithelia of stomach, gall bladder, oviduct, uterus
■ Glandular epithelia of liver, pancreas, stomach, thyroid
■ Epithelia of pancreatic, hepatic, and salivary ducts, kidney
○ Palade- define secretory pathway- pulse chase experiment - rough er than golgi
○ Did em- he noticed cell junction and described them
● Adherens junction
○ Extracellular adhesion molecules: cadherins
○ Linked to actin cytoskeleton via adaptor proteins
○ Found in epithelial cells
○ Same connections- actin cytoskeleton connected to cadherins that connect to
cadherins to neighboring cell and then catenins
○ Different about this type of adhesion, in the epithelial cells we form an adhesion
belt!
■ A ring of actin filaments that go around the cell
■ On each side of cells - will be connected over full length of that belt
 ■ All points - attached to neighboring- they also have adhesion belt so they
are all tight close together
● Proteins that link cadherin intracellular domain to the actin cytoskeleton
● Desmosomes
○ Very similar
○ But instead of belt they form SPOT WELTS
○ Have proteins that connect to each other- and they connect through set of proteins
to the Filaments
○ Not forming belt, but they are forming these zones where cells are stuck together
○ What is the adhesion molecule?
○ There were antibodies that made in patients with autoimmune disorders and had
blisters- you see blisters on skin when we disrupt function of IF
○ These antibodies made are targeting proteins of surface of cell that disrupt
connection to neighboring cells- and those cells are connected to the IF
○ Clue came from autoimmune disorders:
■ Pemphigus vulgaris, blistering of skin and mucous membranes
■ Auto-antibodies disrupt adhesion between epithelial cells
● Treat cells with antibodies: lose adhesion
■ What is the antigen? How would you figure it out?
■ Identified new cadherin family member: desmoglein 3
■ Pemphigus foliaceous: another autoimmune disorder leading to blistering
of superficial epidermis
■ Autoantibodies directed against cadherin family member:t welts
○ Identified protein- take antibodies and screen expression library
● Desmosome structure
○ We have cadherins that are interacting with cadherins of neighboring cell and
their cd is acting with iF than actin cytoskeleton
○ Proteins that act as adaptor between cadherins and IF-
■ Plakoglobin
■ Desmoplakin
■ Plakophilins
● Desmosome components
○ We have adaptors that attach cd of cadherins to if cytoskeleton
○ Extracellular adhesion molecules:
■ Desmogleins and desmocollins the desmosomal cadherins
○ Intracellular domain binding proteins:
■ Plakoglobin: similar to beta-catenin; plakophilin: similar to gamma-
catenin
○ Linkage between IFs and intracellular domain binding proteins: desmoplakin
● Structures at Cell/Cell or Cell/ECM Junctions
○ Tight junctions are at the top site of cells - epithelial cells- lining gut
 ○ They are single cell layer thick
○ We have this basal lamina- ecm matrix connected on one side and the other side
facing gut we have microvilli
○ Tight junction
■ Connection between cells below microvilli but above adhesion belt
■ Adhesion belt is supporting the tight junction
○ Desmosome acts like spot welds connecting cells together
● Polarity of epithelial cells
○ Key: cells have to be divided into two parts
○ Facing into gut has to function different than the surface of cells pointing into
body
○ One role is to take up nutrients from gut- we have to have receptors and transport
proteins that will take up nutrients
○ Key feature of cells: they have to be divided into two parts
○ Apical structure
■ Part on top
■ Lamen of gut
○ Basal lateral
■ Bottom
○ These two parts have to be different so glucose or nutrients from gut enter through
apical surface and they will uptake nutrients from gut
○ Basolateral- does opposite- releases nutrients which will end up in the blood
○ Divide cell into two parts- one side that will take up and the other side will release
○ Tight junctions divide cell into two distinct halves
● Em views of tight junctions
○ We have repeated structure shown on right- membranes come close together in
repeated fashion - tight junctions
● Freeze fracture EM view of tight junctions
○ Freeze and smash get cracks
○ You strip off the lipids in one half of the bilayer and see chain link fence of
proteins within tight junction
○ Tj strands (between two epithelial cells)
○ Freeze fracture em: exposes proteins within the lipid bilayer
○ Proteins attached to each other and dividing membrane into two parts- lipids on
top and they are distinct from the bottom because they can't cross protein protein
barrier
● TIght junctions
○ Protein in green like chain link fence dividing lipid bilayer into two parts
● Transcellular or paracellular transport
○ Paracellular
■ Molecules can move through proteins that make up the tight junction
 ■ Selective- proteins that make up tight junction decide
■ Molecules move extracellularly through parts of TJS
● Ions, water, small molecules
○ Transcellular
■ Proteins enter cell on one side and exit through other
■ Uptake on one side and release on the other
■ CONTROL FLOW OF SOLUTES
● Role of tight junctions in plasma membrane
○ Know tight junctions it divides it into two halves is this experiment
○ Liposomes (phospholipid bilayer vesicles, made in vitro
○ Fuse 2 different types of liposomes to apical surface of MDCK cells:
■ 1.fluorescein labeled outer leaflet
■ 2. Fluorescent labeled inner leaflet
○ Label lipids so they are fluorescent
■ Make vesicales and label them in the outer layer of bilayer or label them in
the inner
■ Fuse vesicles to the cells
■ And fuse them on the apical surface and asked where they end up
■ If you do this, when vesicle fuses to cell, OUTER lipids become outer
layer of plasma membrane- fuse the lipids fuse into plasma membrane but
cannot cross tight junctions
■ If you fuse this vesical with inner bilayer- it becomes inner of bilayer and
those can get around entire cell
■ KEY: lipids of inner bilayer can move between apical and basolateral side
■ Outer are restricted to the apical or basal side
■ Transmembrane proteins span both bilayers get stuck on one side or the
other-
■ Remove calcium- they can diffuse to other sideockout mouse20
○ Barrier function of tight functions
■ Add dye to apical side it can get between microvilli but can't cross tight
■ From basal, it can go up but not across tight junction
■ Restricting movement of molecules from one cell to another
■
○ Key feature of tight junction
■ Divide plasma membrane into two parts- apical and basal and they can be
functionally different
● Identification of tight junction components
■ They start out with tissue that has tight functions
■ Fractions of membrane with tigh functions
■ Generate monoclonal antibodies
■ Biochemical approach
 ● 1. Start with tissue that has abundant tight junctions
● 2. Isolate membrane fractions enriched in tight junctions by density
gradient centrifugation
● 3. Generate monoclonal antibodies that bind to tight junctions
● Tight Junction Components
○ How do you convince yourself that you're antibody recognizes a tight junction
protein?
○ Immunogold EM is this protein inside or outside of the cell
○ First TJ protein
■ ZO-1 (zonula occludens-1); intracellular.
○ How would you find transmembrane components?
○ Immunoprecipitation identified ZO-2, ZO-3; neither is TM PROTEIN
○ Start all over and identify new TM protein: OCCLUDIN
○ Zona occludens proteins - here is immunogold em of zo-1
■ What you see are spots that are associated with tight junctions but not over
membrane itself
■ Did identify proteins associated with tight junctions in cytoplasm but not
over membrane itself- core components to be transmembrane- first round
did not identify transmembrane proteins -
■ Z01-3 act like catenins- attach to proteins that make up tight junctions to
actin cytoskeleton- they are the adaptors
● Occludin was detected over the points of membrane contact in tjs
○ How to determine whether occludin is required for tight junction function in vivo?
○ If you get rid of them do you lose tight junctions
● Knockout mouse: the goal
○ Delete the gene that codes for occludin and see if we lose tight junctions
○ COnstruct mutant version of occludin gene on a plasmid
■ Take restriction enzyme and cut out big chunk of coding region - use
deletion mutant to replace gene in the chromosome
■ Replace wild type with mutant type- because we cut out entire coding
region it will be null allele- not functional
■ Delete one of the two copies so organism is still alive and generate and
mate with other mice
○ Basically Replace one of the wild type copies of YFG with a null allele
■ Cross heterozygous mice to create a homozygous mouse with two null
alleles of YFG
○ DOne initially
■ Cutout big section of coding region and replace with marker gene-
monitor whether or not we knocked the gene out
○ Steps
■ Take mutant allele in test tube and transform it into mice
 ■ When that dna goes in it can integrate into chromosome- random sites OR
homologous recombination can take place which will take gene mutated
and inserted into chromosome instead of wild type gene
■ Most of clones are random insertion invents-
■ Strategy for detecting the rare event
■ Transfected dna can integrate at random sites (standard transgenic
organism). This is a relatively common event
■ Or the transfected dna can replace the endogenous copy of the gene via
homologous recombination. This is a relatively rare event.
● Homologous recombination
○ Have wild type gene in chromosome- pink is the coding region of wild type and
the yellow and green dna is the sequence that flanks the coding sequence you are
interested
○ In test tube hav gene that has yellow and green but replace target gene with
marker gene
○ If you get random insertion it will hop in chromosome somewhere
○ HR-then you'll get recombination between sequences that flank the gene and the
sequences on your clone
○ As a result your marker will get inserted of the chromosome- wild type will pop
out and degrade
● Knockout mouse
○ Overall strategy
■ Start out with embryonic stem cells grow in tissue
■ Start engineer gene in tissue culture
■ Look through cells for mutant that replaced wild type
■ In vitro and take embryonic stem cells that we engineered and put them
into a developing embryo
● Cells will get incorporated into developing mouse
■ Put embryos back in female mouse and give birth
■ They will have our gene
■ Key: cells you inject into developing blastocyst can be any part of the
mouse
■ Mice known as chimeric- different mice will have different parts of the
body with the occludin gene
■ Look for mice whose reproductive organs have the altered gene
■ Mate mice so next generation you'll end up with one wild type and one
mutant - heterozygous
■ Take two heterozygous and make a homozygous mouse
● Knockout mouse part 2
 ○ How to select for the cells in which the occludin gene is replaced with a mutant
allele ( a null allele) in the face of the fact that most of the transformed dna will
integrate at random sites?
○ NeoR gene makes mammalian cells resistance to the drug G418
○ tkSV makes mammalian cells sensitive to the drug ganciclovir
■ Normally human cells are resistant but it will make it sensitive
○ If you express neor you are resistant to g418
● COnstruction of knockout
○ Here is your favorite gene
■ Normal wild type sequence of coding region and the flanks the gene
■ Cut out big chuck of coding region of gene and replace with neoR
■ We are also going to add tk hsv gene - however tk gene is NOT
INSERTED INTO SEQUENCE OF HUMAN GENOME
■ Inserted into plasmid sequence- not homologous to any region of human
genome
○ Take construct that contains neor gene instead of gene and has the tk gene on it
■ If the dna that we put into cells inserted randomly - that entire piece of dna
with both neo and tk gene will insert into chromosome- the ends of this
that are recombinogenic
■ If we have HR- only neor gene will get inserted- because region of
homology flanks neor but not tk- neor inserted instead of wild type and tk
gets cut off
■ Search for gene replaced with mutant- resistance to g148 and ganciclovir-
have neor but not tk
○ HR
■ Gene targeted insertion-
■ Mutation in gene x cellars are resistant to g418 and ganciclovir because
neor got in but not tk!
○ Non HR
■ Both get in
■ Random insertion
■ No mutation in gene x cells are resistant to g418 but sensitive to
ganciclovir
○ Embryonic stem cells
■ Look for neor gene inserted- cells in green and lack tk gene- by yellow
-take those cells and put them in developing blastocyst and end up with
mutant mice
● Different types of transgenic organisms
○ Different kinds of mutations we can make-
○ 1.We can add gene - dominant allele- inserts randomly doesn't matter where
○ 2.Gene knockout
 ○ 3. Gene replacement-
■ Where you make point mutation in gene and replace the wild type allele
with point mutant - and see what effects happen
● Occludin was detected over the points of membrane contact in tjs
○ Have antibodies that bind to transmembrane proteins- is part of tight junctions
○ What if you get rid of occludin?
○ Made mouse and got rid of occludin- nothing happened and the tight junctions
were perfectly fine-
○ Occludin Part of tight junctions but not essential for function of tight junction
● Claudins
○ Did everything again and identified claudins and knocked them out and tight
junctions function was gone
○ CLAUDINS= ESSENTIAL FOR FUNCTION OF TIGHT JUNCTIONS
○ 20 family members identified in humans
○ Cell-type specific expression
○ Many are co-expressed in the same tissue
○ Ectopic expression can result in TJ formation in cells that lack TJs
○ Different combinations of claudin expression lead to changes in paracellular
permeabilities
○ Two ways to measure paracellular permeability
■ 1. Add small fluorescent tracer to apical or basal surface: measure ability
to pass between layers
■ 2. Measure transepithelial electrical resistance (greater resistance= lower
permeability)
○ Required and sufficient for tight junctions
○ Regulate passage of material between cells
● TIght junctions and medicine
○ Hereditary hypomagnesemia
■ Most mg2+ is reabsorbed from the urine through the paracellular pathway
in the kidney- recycling
■ Mutations in claudin -16 responsible for disease
■ Claudin -16 expressed in specific region of kidney
■ Claudin-16 might form tight junction pores that function as mg2+
channels
■ Mutated- you cant uptake mg and have mg deficiency
○ Tight junctions targets of bacterial pathogens
■ Toxins from vibrio cholera and other pathogenic bacteria alter the
permeability of tight junctions; either bind to tj proteins directly or
regulate their permeability by signaling pathways inside the cell
 ■ Cholera-die from it because you have severe diarrhea and dehydrated-
cells lining gut - waterproof- tight junction responsible- you leak water if
tight junctions aren’t working
● Current view of tight junction structure
○ Occludins part of tight junction not essential
○ Other proteins - JAM -
○ All these proteins make wall that makes up tj
○ Adaptor protein
■ Zo 1,2 ,3 that attach the jams, occludin, and claudin to actin cytoskeleton
to give them strength
● Gap junctions
○ Electrophysiology experiments showed that some cells were coupled electrically -
pathways that required maximal speed, like escape responses
■ Direct transmission of action potential, no elay
○ Also showed that fluorescent molecules, radioactive tracers, essential nutrients
could pass from cytoplasm of one cell to cytoplasm of neighboring cell
○ Find them in tissues and cells that are electrically coupled
○ In your heart you have small set of cells that send signal and remaining are
connected through gap junctions so they can share that signal -and cells contract
at the same time
○ One way you can show that gap junctions exist by injecting dye in that type of
tissue
■ Cel number 3- dye gets into the rest of the neighboring cells by passing
through the gap junctions
● Gap junctions part two
○ Em view of heart muscle cells
○ We have enriched fraction where al individual dots are gap junctions- they look
like donuts with hole in the middle
○ Electron microscopy showed that electrically coupled cells had specialized
junctions; adjacent membranes separated by a gap of 2 nm
○ Coordinate the activities of groups of cells heart- gjs permit rapid cell-to cell
transfer of action potentials, ensuring coordinated contraction of cardiomyocytes
● Methods of study gap junctions
○ To determine size of channel:
■ Microinjected fluorescent tracers of different sizes into connected cells: is
tracer confined to one cell?
○ See dye get into neighboring cells- use different sizes to see opening smaller than
1 kilodalton can move between cells
○ We have metabolites- like amino acids, atp - can get shared especially ions
● Some gap junctions are regulated
○ Neuron and injected dye
 ○ The bright part is cell body of neuron and we have axons spread out- and see cell
bodies of other neurons
○ Inject dye- and gets to other neurons by the gap junctions
○ Some cells are regulated so they can close gap junctions- like in an eye- gap
junctions have been closed off and tracer dye can't get in into neighboring
○ Detect Color and detail in bright light
● Gap junction components
○ Identified in same manner as tj proteins
■ Connexins (vertebrates)
■ 20 different genes in mammals annexins - invertebrates
■ 6 connexin subunits produced 1 connexon- half channel on each cell
■ Connexons may be produced from same monomer or from different
monomers
■ Channels can by homotypic or heterotypic
○ Enrich for fractions and make antibodies- clane and make expression library
○ Plants and invertebrates have different version
○ They form a ring- 6 connexins get together and form cylindrical channel that
spans the membrane
■ Other set of 6 forming channel in neighboring cell and get together to
form a single channel and transport goodies
○ Different tissues you can make different types of gap junctions
● Gap junctions
○ Many of these are regulated by calcium!!!
○ They will CLOSE in presence of CALCIUM
○ Why does that make sense?
○ Where is calcium found? Sarcoplasmic reticulum- but we have higher
concentration outside than inside of cells- why would calcium be important to
close these off?
○ IDEA IS : it is great to be coupled to neighbor and share stuff
■ But...gap junctions can be bad- if cell ruptures and leaking atp, amino
acids…
■ If it is connected to neighbors it will suck stuff from neighbors- but
calcium can get inside the cell so it's isolated from neighbors
● Connexins and medicine
○ Heart doesnt beat right if you screw up connexins in heart
○ Neurosensory deafness- cx26, cx31
○ cataract/heart malformation - cx43, cx46, cx50
○ Charcot- marie tooth disease - progressive neural degeneration cx32

Topic 30: Cell Cycle Embryonic Cycle and Biochemistry

● The eukaryotic cell cycle
○ G1,,s, g2, m
○ Sphase
■ Duplicate dna
○ M phase
■ Segregate sister chromatids
○ Key: whole cell cycle is design to protect genetic material - what job is- you want
to replicate every dna base pair once!
○ S phase and m phase- “action”
○ G1 and g2 are gap phases that are in between
● Flow cytometry to analyze dna content
○ Can't say what stage is in - mitosis- dna is condensed or spindles, but interphase
you can't tell if in g1 or g2
○ Flow cytometer can measure individual cells what stage they are in
○ Steps
■ You add a dye to cells and that dye binds to dna and fluoresces once it
binds
■ Amount of fluorescence proportional to amount of dna
■ G1- diploid- and is the 2n content of dna
■ G2- or m phase- replicated dna and they have 4n content- twice as
fluorescent
■ S phase- have amount in between 2n and 4n - some has been replicated
some hasnt
■ Measures amount of dna or fluorescence in cells and counts how many
cells are in g1, g2/m, and how many have in between - s phase
○ If you have asynchronous pop- all different stages of cell cycle- area under peak is
proportional to length of time that cells spend in that cell cycle
○ Ex. replication takes up half of cell cycle - cells will have dna content between 2n
and 4n at any given time
● Cell fusion experiment
○ Fuse cells together monitor effects
○ Led to idea that proteins that control cell cycle
○ We do cell fusion
■ Take two cells and treat them with stuff that allows them to fuse
■ If you take cell in s phase and another in g1 and fuse them, the g1 nucleus
will immediately undergo replication - implication that s phase cells have
some factor that can drive cells from g1 into s phase
■ If you take cell that is in s phase and fuse it to cell that is in g2, the g2
nucleus DOES NOT go under replication
■ Implication - replication is blocked in g2 nucleus
○ S phase cells have factor that can induce dna replication in g1 cells
 ○ G2 cells do not respond to this factor. Block to re-replication: replicate once and
only once
○ somehow it's blocked in the g2 nucleus- cant get extra round of replication until
after you do mitosis
● Fuse Mitotic Cells with Interphase cells
○ In all cases, the nuclear envelope breaks down and chromosomes condense
○ If you take cell in mitosis with any other cell in cell cycle, in all cases will enter
mitosis right away- envelope breaks down and chromosomes condense
○ Some protein or something in mitotic cells that can drive other cells into mitosis
● Advantages of studying embryonic cell cycle
○ Mater regulatory proteins that drive transitions from g1 into s or any stage into M
○ In embryos, cycles are synchronized
■ Early cycles are rapid
■ Xenopus: 1st
○ A lot of early work was done in xenopus and oocytes - same frog we talked about
early on with tissue
○ Nice thing about cells is that they have early stages of development- same stage of
cell cycle- progress synchronously
○ Cycles are rapid- first one is 75 minutes and the next 11 are 30 minutes each
○ Large cells and they are useful to do biochemistry
○ Provide good source of material for biochem
■ Xenopus egg> 1 mm diameter;
■ 100,000x cytoplasm of human somatic cell
● Embryonic cell cycles
○ Batch of eggs that we fertilized in vitro and they go through cycle synchrony
● Xenopus Laevis
○ Generating one eggs take frogs nine months
○ Frog will stuff into that egg that egg needs to develop into tadpole stage
○ Egg gets fertilized externally and develops externally
● Maturation of xenopus oocytes
○ Arrest- 8 months, grow synthesize materials needed for rapid embryonic
development
○ Progesterone, secreted by follicle cells surrounding egg, triggers maturation
○ Final stage is triggered
○ Key: cell gets to be huge because it gets arrested in g2 and grows until it's time to
become mature egg
○ Progesterone triggers the oocyte- immature egg- in g2 to enter meiosis
○ For our purposes - meiosis and mitosis are similar
○ Cell undergoes meiosis one and arrests in mitosis 2 and egg gets fertilized and
complete meiosis 2 once egg is fertilized and go under sync divisions
○ Progesterone-meiosis one- meiosis 2- fertilized- divisions to created blastula
 ○
● Maturation promoting factor - MPF- Yoshio Masui
○ What people did is an experiment to see what's going on
○ Steps
■ See eggs arrested in mII
■ Take needle and take cytoplasm of one cell and put it in another
■ Take cytoplasm from egg arrested in m2 and inject cytoplasm in oocyte
that is in g2- that triggers the cell the oocyte to go into m phase
■ Transfer of cytoplasm from m2 arrested eggs drives interphase oocytes
into meiosis, as if they had been treated with progesterone
○ Suggests that some something in m phase cell when we put in g2 it drives it to
mitosis - some factor that drives transition
○ One possibility is progesterone because to get cells in m2 they had to get exposed
to progesterone
○ We can say that it’s not progesterone because
■ You take cytoplasm from the cell in m and put in g2 cell, this cell goes
into m phase
■ You can use small amount of cytoplasm that it is unlikely that
progesterone is carried over
■ We can take cytoplasm that has been triggered and put it into another
oocyte- and it also goes into m phase
■ Any progesterone in initial it would have been diluted out know there isn't
any progesterone around
○ Implications:
■ 1. Factor other than progesterone is involved= mpf
■ 2. Mpf may be or regulate an enzyme
■ 3.may be autocatalytic-triggered another cell to go into m phase but you
can take tiny amount of cytoplasm and put into another oocyte- small
amount taken from cell can generate a lot in cell you inject it into
○ Take small amount of cytoplasm from cell in m phase and put in g2 cell and goes
straight away into m phase- small amount of cytoplasm creates a large amount
○ May be an enzyme because small amount can trigger in second cell
● Injections to assay MPF activity
○ Xenopus oocytes, eggs,
○ Take cells that are going through cycle and take cytoplasm and put in g2 oocyte
and see when it makes mpf- that triggers oocyte to go into g2
○ If you take extract from cell in g2 and put in oocyte- does NOT go to m
○ If you take extract from cell in meiosis One that does have mpf activity - inject it
into oocyte in g2 it will go into m phase
○ Back in meiosis 2- mpf are high - g2 goes to m phase
○ Cell cycle- peaks every time we are in m phase and goes away at other stages
 ○ Mpf activity highly conserved:
■ Cytoplasm from mitotically arrested mammalian cells also contains mpf
activity
■ Take extract from humans in m phase and put in frog eggs and goes from
g2 to m
● Things to Remember- we’ll return
○ 1. MPF- drives g2 into m
○ Talk another set of experiments- through 3 different experiments and they all
merge
● Identification of cyclin
○ Experiment to study translation in sea urchin embryos
○ Amphibian eggs have large eggs- you put everything in egg- same basic idea as
frogs
○ You go through early rounds of cell division basically mom puts everything in
egg that it needs for early development- when does transcription and translation
start?
○ Doing experiments on translation in early embryos
○ Steps
■ Took radioactive methionine and see when proteins are being translated
■ Saw that some proteins got translated and some proteins that start
translating 46 min after fertilization and abundance increases
■ One protein cyclin- increased in abundance and then it disappeared- 76
minutes it was gone, then increased again and then decreased
■ When he looked at time points he saw level of cyclin increased and
decreased during cell cycle
■ Hence the name cyclin
● Xenopus egg extract that cycles in vitro
○ Two possibilities: Cyclin regulates cell cycle- need it and then don't or it doesn't
regulate but protein abundance changes in stages- you are required or cell cycle
dependent event
○ Is this protein a regulator?
○ Experiment steps:
■ In vitro assay for cell cycle
■ Normally cell extract is broken in buffer and proteins get diluted in buffer
and make it the same conditions as in the cell but it's hard to do that so
they took frog eggs- oocytes and put them in oil instead- and if you spin
them fast they pop- they break open and cytoplasm leaks out- but in oil
you get just get concentrated cytoplasm
■ Same conditions it was in the egg
■ You take that extract and add sperm - compacted dna- and you will get
cell cycle events and form membrane around the sperm dna
 ■ That will go into mitosis and condense membrane breaks down -and get
spindles form - go through multiple rounds of cell cycle- form nucleus
around dna concerne break down and form spindles and reform nucleus- s
and m phases
■ Dna will get replicated- no g1 or g2 phases
● Role of cyclin B?
○ Use egg extracts: deplete and replenish cyclin B
○ Experiment:
■ 1. Treat extracts with low doses of rnase to destroy mrnas
■ 2. Inactivate rnase
■ 3. Add back sea urchin cyclin B mrna transcribed in vitro
○ Ask what happens- cyclin required for progression through cell cycle?take
extracts that go through cell cycle and treat with rnase- degrades rna- small levels
of rnase efficient to degrade messenger rna and did not disturb other rna
■ Inactivate the rnase- if he did this to get rid of mrnas- extract stop cycling-
some messenger rna is needed for cycling
■ And mrna has to be translated to make protein
■ Rnase he used you can turn it off- calcium dependent remove calcium-
■ Assk if cyclin required for cell cycle 0 added back the cyclin mrna to
extract
■ Only mrna in the extract is cyclin mrna and extract started cycling again
○ Results: clin needs to be degraded
○ When he looked at mpf activity same as it was high he had high levels of cyclin
and vice versa if it was low
○ Cyclin is mpf or somehow related to mpf function
● ROle of cyclin B?
○ Rnase-treated extract + cyclin b mrna
● Cyclin degradation is required for exit from mitosis
○ Cut off different parts of cyclin gene- truncated protein
○ Remove n terminal amino acid, cyclin was no longer degrADED- it went up and
down in cell cycle- and took mutant that couldn't be degraded and stable, the
extract got stuck in m phase
○ Upshyot you have to produce cyclin from g2 to m and get rid of flying in order to
get out of m phase
○ Both aspect cycling- increasing and degraded is needed
○ Add rna encoding mutant form of cyclin B:
■ 90 n-terminal amino acid
■ Induces mitosis but cannot be degraded
● What we have learned so far
○ Mitotic and meiotic cells possess an activity that can drive interphase cells into
mitosis
 ○ This activity mpf is highly conserved among eukaryotes
○ Cyclin protein levels alternate as cells progress through cell cycle and correlate
with mpf activity
○ New cyclin protein synthesis is sufficient to drive extracts into mitosis
○ Cyclin protein degradation is required for exit from mitosis
● Things to remember
○ 1. Mpf
○ 2. Cyclin

TOpic 31: Cell cycle: genetic analysis of yeast cell cycle

● Genetic analysis of phage t4 assembly

○ Key: identify genes that comprise pathways- almost any biological process can be
described as a pathway
○ Used it on phage genetics
○ Steps
■ Looked for mutant phage that was defective in infecting cells
■ Each is a different gene- number
■ Look in em defective in phage assembly - showed that building tail part
ws separate than building the head
■ Different set of genes
■ Order which genes act on which step to see how much of tail you can
assembly or head
■ Coupling genetic analysis looking for genes defective in making- they
derived pathway
■ We have genes that act early or later in pathway or end - we have
branched pathway- head and tail come together that build final phage
■ When people studied cell cycle in yeast ask if these events - what are the
genes involved in each event
● Life Cycle of the budding yeast s. Cerevisiae
○ Key: it can divide mitotically but it can form haploids or diploids- but what's
different is that haploid can divide mitotically - not like sperm or egg- products of
meiosis that is haploid and divide
○ Two types of haploid
■ A cells and alpha cells
■ Haploid a can mate with alphas to make diploid
■ A cant a or alpha with alpha
■ Diploids divide to give rise to more cells
○ Single celled that can divide mitotically - haploid or diploid
○ Generate
● Genetic analysis of the cell cycle in budding yeast
 ○ Yest divides rapidly - every 90 minutes goes through cell cycle
○ It can divide as haploids or diploids- important because isolate recessive mutants
in haploid cells
○ Loss of function mutation - if you are looking at diploid you would have to
knockout both copies- but not in haploid because there is only one so it's much
easier to isolate
○ In addition, all genomic techniques developed using yeast- small genome,easy to
manipulate, and cell cycle is very fast
○ First eukaryote to be sequenced
● Budding yeast
○ Grows by budding
○ Mother cell forms bud and there will be a bud that will grow and before it gets to
the size of mom it separates
○ Nice about yeast- you can tell what stage of cell cycle it's in by looking at them-
flow cytometry to tell for human cells- but not for yeast
○ Forms bud at beginning of s phase- unbudded cells are in g1
○ Small bud- beginning s phase - medium- end of s phase- final bud are ready to
divide-
○ Size of bud- stage of cell cycle
○ And see nuclear divisions with right stains- analyze and see what stage it's in
● Plating single yeast cells
○ Grow yeast and liquid culture or in petri dishes
○ And have different type of media for yeast to grow
○ Steps
■ Take wild type cells growing in liquid culture and plate for single cells
■ Spread on petri dish - where individual cells are spread out from each
other
■ Cells divide and form colonies- takes 2 days- 10 ^7 cells
■ Important feature for plating- all cells within colony are identical because
they all started out life as single cell- identical to original cell that starts
colony
● Replica plating
○ We take petri dish with yeast colony - take lid off petri dish and flip it and press
colony onto sterile velvet cloth
○ Low tech is good- it works-
○ Sterile velvet press plate on top and some cells from each colony will transfer
○ Take plate off- still have original
○ Take new plate and smash it on cloth and some cells transfer onto new plate- and
you will end up with replica of original plate- same colonies in same position
○ Not lot of cells in each so you let it grow and you have a new plate with fully
grown colonies
● Hunting for mutants
○ Look for mutant cells
○ Wild type yeast cells- haploids do something to mutagenize them -
○ Plate for single cells
○ Different cells will be mutated in different genes but because we are plating for
individual cells when we form colonies all cells will be identical- in on colony we
will have mutation in one gene, g1, g2 and g3-
■ So we hunt for mutants that have phenotype we are looking for
■ Screen through 100k to 200k-300k colonies
■ Genetic screen
● Look for lots of individuals for small that have genotype you're
interested in
● Ones that are defective in cell cycle
■ All cells in colony have same gene mutated
■ But different colonies can be mutated in different genes
● Problem:
○ Cells defective in genes required for cell cycle progression should not be able to
divide; these types of mutations will be lethal. How can one screen for and
maintain lethal mutants in a haploid organism?
○ Answer: screen for conditional mutations
● Conditional mutations
○ Temperature sensitive
○ Think about wt gene that makes wt protein
○ Yeast is happy growing at 30 but can grow at 23 or 37
○ We have yeast cell
■ Mutation that affects stability of protein doesn't fold up well
■ It will fall apart at a lower level temperature
■ All wt is stable at 37
■ Get mutation in gene that makes less stable protein so it folds up normally
at a low temperature 23 but denatures at 37
■ We have mutation in gene that behaves like wt at 23 but null allele at 37
■ Refer to temperature sensitive mutations
○ Have wild type and then mutant type or nonpermissive condition- temperature
sensitive- point mutations that affect- fold at low temp but unfolds at high
○ Yeast grows at 30- proteins unfold at37- mutant at elevated temp - allows us to
keep mutants alive because they grow just fine at low temp- and allows to ask
what is phenotype that is associated with mutant at elevated temp
● Screen for mutants with
○ First part look for genes that are essential for life
■ Takes yeast cells and mutagenized and plated for single cells on rich
media that grew into colonies at 23 degrees
 ■ Replicated onto new plate and grew at 37 degrees
■ Mutations - in genes that were essential for life - the ones at 37 - the cells
died - get rid of protein to grow at 37 cells died
■ Identified mutations in genes that are essential for life
● Two stages of cell division cycle- CDC screen
○ Get mutations in genes that are interesting and some aren't
○ We are identifying essential genes that are needed for life but our goal is to find
which ones are needed for cell cycle
○ 1. Primary screen-> screen for ts mutations in essential genes but lot's of genes are
essential, how do we identify the genes that are required for specific events in the
cell cycle?
○ 2. Secondary screen-> search among the ts mutant collection for genes that cause
arrest in specific stages of the cell cycle
○ Rna or dna- can transcribe and die- some don't have anything to do with cell cycle
and look at secondary screen
○ Look among mutations that identified essential genes and look for ones that were
defective in the cell cycle
● Terminal Phenotypes: arrest at a single point in the cell cycle
○ Look at how cell dyes- terminal phenotype
○ Imagine if you have gene that is required for g1 to s but not any other time - then
cells that are in g1 when you elevate to 37 should continue to grow
○ Cells in s phase will keep going than g2 and then m phase and stop growing in
next g1 phase
○ Looked for mutant cells where all die in same stage of cycle- that that protein
identified in mutation was required for that stage of cell cycle -
○ Idea: assume gene that is required for stage in cell cycle
■ Imagine the possibility the cell cycle that protein is needed- not needed in
s phase or g2 or m
■ Such a gene would have following properties
● If we started it growing at 23 than shifted to 37 and inactivated,
cells in g1 would continue through g1- protein not needed in g1,
but once you get to boundary they stop dividing
● Same with s phase - they don't need protein for others- continue
cell cycle until they get to g1-s transition
● If only time this protein is for g1-s transition then they should all
arrest at that time- or terminal phenotype
● Strains where all cells died with same terminal phenotype
● Not the case for mutation in rna polymerase
○ A synchronous - raise temp - and cell dies after 20 the ones
that were in late g2 will die in m phase
 ○ Die in all phases of cycle- mutation and genes that are
essential but not part of cell cycle die at different times
○ Genes needed for cell cycle will die at the same time!
● How does one identify cell cycle mutants? Secondary Screen: look at terminal phenotype
○ Wild type =Cells proceed unaffected through cell cycle
○ Cdc mutant = Cells arrest at one point in cell cycle
○ Mutant in non cell division = Cell cycle is arrested at multiple random times in
the cycle
○ cdc= cell division cycle mutants;
■ Functions required at specific points in the cell cycle
● Budding yeast cdc mutants ( >50 )
○ Cdc28- arrest late in g1 as unbudded
○ Cdc7- conditional arrest any non permissive - right at beginning of s phase
○ Cdc9- arrest at end replication late in s phase
○ Cdc15- arrest as large budded selves in cytokinesis
● Cdc15 arrest point
○ Growing at permissive temperature with all different type of buds
○ Arrest- all cells arrest as large budded cells
● Cell division cycle -cdc mutants
○ Defective in different steps of the cell cycle:
■ Spindle pole body duplication
■ Initiation of dna replication
■ Nuclear division
■ Cytokinesis
■ Cell separation
○ Ok so you’ve got 50+ cc mutants… how do you figure out which one is the most
interesting one to study?
● Budding Yeast CDC Mutants (>50)
○ Cdc9 is not an interesting gene if you want to study cell cycle
■ Codes for dna ligase
■ If you can’t ligate okazaki fragments you can't complete s phase- and die
at the end of s phase
■ Has nothing to do with controlling cell cycle
■ Mere step needed
■ Which one is important to study?
○ Which ones are the regulators of cell cycle?
■ What is regulation??- biological decision making- to do something or to
not do something
● Hartwell defined “start”
○ Start is a critical decision point in g1
○ Steps
 ■ If you took yeast cells and did not feed them they didn't grow
■ If you withdraw food they stopped dividing and sit there until food
appears
■ If you give them food they start dividing
■ Are there enough nutrients to divide or not to divide- no g1- if there are
we go to s phase and replicate and go through cell cycle
■ 2nd. Decision - mating- they each put out pheromone so they arrest in g1 -
idea is that both cells want to be haploid so fuse when g1- mating
pheromones arrested cycle at late g1- seems to b decision point in late g1
where you mate or don't and divide or not if nutrients are available
■ Reasoned if i want to identify gene and protein that regulates start, i want
the protein that is near start
● Pathway analysis
○ Cdc28
■ Same time close to start
■ Suggesting this protein is required for transition to start
○ Wild type goes through start
■ G1 to s phase
■ In phase three things happen
● 1. Dna replication
● 2. Buddingdc28
● 3. Spb duplication
○ Cdc7
■ Arrests without replicating dna
■ Budding
■ Spb duplication
○ Cdc24
■ Dna replication
■ No budding
■ Spb duplication
○ Cdc31
■ Dna replication
■ Budding
■ No spb duplication
○ COnsistent with 28
■ None of the s phase events
○ In cdc28 mutant, none of the downstream events occur
○ Hartwell concluded that this was the key gene required for entry into the cell
cycle
● Some cdc mutant phenotype
○ Cdc28 arrest point was closest to or at start
● Cdc28 is a key regulator of the cell cycle
○ Cdc28 is required for start, start is in g1 prior to the g1->s transition
● Things to remember
○ 1. Mpf (humans, frogs, sea urchins) g2->M
○ 2. Cyclin (frogs, sea urchins) g2->2
○ 3. cdc28 ( s. Cerevisiae yeast) g1 - yes...really mid g1 to late g1
● Genetic analysis of the cell cycle in fission yeast (s. Pombe)
○ Reproduces by fission
○ Used to produce an african bear called “pombe”
○ Grows longer and cuts in middle to make two equal size
○ Pombe means bear in swahili
○ Like budding yeast, grows rapidly; well suited for classical and molecular genetic
analysis
○ Cells divide symmetrically, cell size is very uniform at same position of cell cycle
○ Grow in length, not diameter; easy to measure growth
● How closely are yeasts related?
○ Evolutionary distance= time from last common ancestor
○ Time from lca= amount of time two species have been diverging from each other
● Evolution of primates
○ We last shared ancestor with chimps, then most distant primates
● Evolution in perspective
○ Human and chimps - 5.4 mya
○ Human and fruit flies - 990 mya
○ S cerevisiae and s pombe- 1210 mya
○ We are closer related to mosquitos then the two yeast
○ Cell cycle- and look at evolutionary time- it makes perfect sense to look at
different yeast
○ Just because they are very similar doesn't mean they are closely related
○ Human vs fungi or yeast is 1520 mya
● S pombe cell cycle
○ Like budding, it can divide as haploid or diploid
○ Like budding, you can tell what stage of cell you are by morphology
○ Born - small, most growth occurs in g2
○ Small- g1/s phase- growth g2- m phase- chromosome condensation
○ Visually get good idea of what cell cycle you are in
● Genetic nomenclature
○ Do same thing
○
S. cerevisiae S. pombe
 wild-type CDC28 (uppercase#) cdc2+ (lowercase#+)

Recessive (LOF) cdc28 (lowercase#) cdc2- (lower#-)

Dominant (GOF) CDC28-1 (-1 is the cdc2^D

dominant allele)

Protein Cdc28 Cdc2

● Cdc mutants in s pombe
○ He found strains that were appeared to be defective in cell cycle
○ Cdc25 mutant are bigger than normal
○ Wee one mutant
■ Temp sensitive alleles
■ They were smaller than normal cells on average
■ Looked for other conditional mutants and found that it gave rise to cells
that were a lot larger
■ You go back and do screen to look for similar phenotypes
■ Gradient centrifugation - start with cells mutagenized them and restric
temperature- large- then centrifuge and throw mutants the ones that
weren't large
■ Works on density- he got a bunch of tiny mutants
■ He identified wee one
○ Nurse attempted to enrich for large cells (fail to divide at restrictive temperature)
by sucrose gradient centrifugation
○ At bottom of gradient, found small cells instead
○ These mutant cells (wee1) divided at half normal size
● Wee1 mutants properties
○ Divide at normal size at permissive temperature, but at half wild type sized at
restrictive temperature
○ Wild type cells spend much of their time in g2 growing to critical size before
entering mitosis
○ Wee mutants lose this g2/m growth control and divided at smaller size
○ Nurse looked for more alleles of wee1 by screening for more mutants yielding
small cells; also found mutations in other genes that produced wee phenotype
○ Prematurely leaving g2 -going to m phase earlier than supposed to
● Cdc mutants in s pombe
○ How did nurse figure out which mutants were the most important ones to study
○ Looked for genes in which dominant and recessive alleles (gof and lof alleles)
gave opposite phenotypes
○ Two key genes: cdc2 and wee1
○ Dominant and recessive alleles give different phenotypes
 ○ Switch pathway- decision making-
● Cdc2 phenotypes
○ Cdcd 2+ - wild type
■ Normal size
○ Cdc2-
■ Loss of function mutation
■ Phenotype at nonpermissive temperature - larger than usual
○ cdc2D
■ Gain of function
■ Smaller than supposed to be
○ Implication: cdc2 is a key regulator of the cell cycle- don't have enough you go
into m phase later and too much you go to m phase sooner
● Cdc2 is a positive regulator of the cell cycle
○ Cdc2 drives the g2 to M transition
○ Divide earlier if you have too much
○ Not enough or get rid- you divide too late or not at all
● Things to remember
○ 1.mpf
○ Cyclin
○ Cdc28
○ Cdc2
● Wee 1 dosage effects
○ Panels c and d are god, therefore similar to dominant alleles
○ Loss of function - divide earlier than they should
○ Over expressing wild type of wee 1- put gene on plasmid so we had three copies
of gene than just one
■ Dominant mutation -e xtra week 1 - cells are larger than normal
○ If you have 5x
■ Cells Are even bigger
○ Loss of function you have smaller
○ Acting like regulator of transition between g2 and m
● Wee 1 is a negative regulator of cell cycle
○ Inhibiting
○ If you have extra wee1 you delay transition and grow
○ Get rid of wee1 you divide earlier- remove repressor of transition
○ Wee 1 is negative regulator
● Genetic symbols
○ Key point: the arrow or inhibition symbol indicate the role of the protein in its
active form
○ Wee one inhibits g2 to m
○ Cdc2 promotes transition from g2 to m
● Things to remember
○ Mpf
○ Cyclin
○ Cdc28
○ Cdc2 promotes g2- m
○ Wee1 inhibits g2- m
○ What are cdc28 and cdc2?
■ Clone the wild type genes
● What is cdc2?
○ Screen genomic library by complementation
○ In yeast you clone by complementation
■ Start out with mutant strain in cdc2 in pombe - that strain will not crow in
non permissive temperature- because lacks cdc2 gene
■ Make genomic library from a wild type strain
■ One of those library members will code for wild type cdc2 gene
■ Where other thousand clones won't -they'll have other genes
■ Transform that library into yeast strain and plate for single cells
■ Each cell takes up different part of library and form colony that has one
gen or the other
■ We have a colony that as plasmid that has blue gene, red gne, orange
plasmid, so then we have to screen through transformants to find gene of
interest
■ Replicate that onto plates at non permissive temperature
■ All strains will die except the ones that have cdc2 gene because all strains
lack cdc2 in chromosomes have mutant allele in chromosome - isolate
plasmid from strains and sequence it
■ All will die except the ones that took up cdc2 gene
● Another version
○ Looking at cdc28
○ Random clone those cells will die at non permissive- only the cdc28 will have
function at higher temperature isolate plasmid and clone it
● What do cdc2+ and cdc28 encode
○ Cdc2 from pombe has sequence similarity to s. Cerevisiae cdc28
○ Encodes a protein kinase
○ Next, used functional complementation in s pombe to ask if other organisms have
similar genes
○ Screen s cerevisiae genomic library; cdc28 from cerevisiae restore viability to
pombe cdc2 mutant
○ so , key cell cycle cycle regulators are conserved in these very different yeasts
(separated by 1 billion years of evolution)
 ○ Try bold experiment: is there a human gene that can rescue yeast cdc2 cell cycle
defect? -
○ Yes! They found a human gene with 65% sequence identity to cdc2; this human
gene can function in yeast cell cycle-proving conservation and key regulators of
cell cycle
● COnservation of key cell cycle regulators
○ GUys that had biochemically identified factor mpf that drove cell cycle and
purified it
○ During Purification they discovered fractions that have mpf could phosphorylate
histone 1
○ Based on that analysis- purifying mpf- tiny bit of liter of frog eggs- they got
protein that was hetero dimer that had two subunits -34 and 46 kDa
○ Protein kinase thats a dimer
○ Raise antibodies against the cdc2/cdc28 proteins and those proteins bound to band
in frogs the 34
○ Erodimer made up of cyclin and cdk
○ Mpf is heterodimer- one part is kinase- that was encoded by cdc2 and cdc28
○ Biochemical purification of mpf from xenopus- using 1 liter of frog eggs yielded
a few ug mpf
■ Mpf has h1 kinase activity
■ Consists of two proteins
● 34 kDA and 46 kDA
■ What are they?
● Antibodies raised against conserved sequence in cc2/cdc28
recognize 34 kDa band
● Antibodies against yeast cyclin B recognize 46 kDa band
○ So mpf is a heterodimer consisting of a cyclin B and a cyclin dependent kinase or
CDK1
● THings to remem
○ Mpf is a two subunit heterodimer
■ One part is cyclin
■ The other is kinase that has cdc28 and cdc2
○ Mpf is cdk plus cyclin
■ Cyclin dependent kinase
○ Context dependent
■ Cdk can refer to heterodimer- the complete functional protein
■ Cdk can refer to only the kinase subunit
■ Be aware!
● CDKs (cyclin- dependent kinases)
○ We now know there are multiple cdks
○ In yeast
 ■ The cdk subunit the kinase subunit in red is the kinase that regulates
various steps in cell cycle
■ That subunit phosphorylate proteins needed for a stage
■ Form different cyclins at different parts- they are regulatory subunit
● Dictate specificity of the kinase
■ Cyclin in g1- when it binds to kinase subunit- kinase phosphorylates
proteins in g1
■ Then get rid of blue cyclin and produce other in s phase- orange- that
cyclin will bind to kinase subunit in s phase and give it different
specificity and phosphorylates different set of proteins that are needed for
s phase
■ Get rid of s phase cyclin and produce different one green- bind to kinase
confer specificity and phosphorylates proteins that function in m phase
■ Produce different cdks depending on what cyclin they have - determine
specificity ant targets of kinase
○ Three different cdks
■ G1- only functions in g1-
■ S-
■ M-
● Classic mpf drove cell cycle from g2 into m and that was this
version of cdk
○ All euk cells have three classes of cdks: g1/s-cdk, s-cdk and m-cdk, some also
have g1-cdk
○ Cells usually have more than one member of each class
○ There are more than one cycle of each type
■ There are a couple of them
■ So many targets of proteins
■ Need multiple versions to hit all types
■ More than one s phase or m phase

Topic 32: Cell Cycle Regulation, Checkpoints and Cancer

● Components
○ CDK kinase subunit - Cdc28
○ G1/S cyclins - Cln3, Cln1, Cln2- don't need to know - multiple g1 cyclins
○ S Phase cyclins - Clb5, Clb6
○ M Phase cyclins- Clb1, Clb2, Clb3, Clb4
○ Sic- inhibits s phase cdks
○ SCF - Ub ligase- > degradation of Sic1
○ APC/C-Cdh1- ub ligase -> degradation of s and m cdks
○ apc/c-cdc20 = ub ligase -> onset of anaphase
 ○ wee1= kinase that inhibits m cdks
○ Cdc25 = phosphatase that activates m cdks
○ Whi5 and Rb- inhibits txn of g1/s and s cyclins
○ Cdc14- phosphatase required for exit from mitosis
● Overview of “drives” of cell cycle
○ They will trigger events that move cycle forward
○ 1.G1 Cdks-> Production of S-Cdks
○ 2. S cdks- dna replication and other s phase events
○ 3. M cdks- events of mitosis up to anaphase
○ 4. APC= anaphase and later events in mitosis
● Logic
○ Irreversible switches- once we turn driver off it can't get activated until next cell
cycle
○ Each type of cdk and the apc is switched on and off at different times in the cycle
● Four Key Players: G1/S cdks, Cdks, m cdks, and APC
○ Scdks stay active until mid mitosis
○ Mcdks become active at bending of mitosis and end in the middle of mitosis
○ Apc then comes on and targets s and m cdks for degradation
■ Ligate those cdks to shut them off
■ The logic is that we talk about mitosis as phase of cell cycle-
■ Mitosis as two phases- first half we do things and second we reverse those
events- in order to carry out early m cdks phosphorylate proteins
■ At anaphase we have to turn those events off and reverse them and that's
what apc does-
■ Apc inactivates mitotic cyclins so we can reverse what we did in first half
of mitosis
■ Remember when we looked at mutant cyclin that couldn't be degraded and
couldn't come out- have to degrade m cyclin
● Logic of regulation of cell cycle
○ Idea is that each of components activates activities in that phase
○ each cdk and the apc activates events required for its stage of the cycle]
○ Each cdk and the apc triggers events that lead to the activation of the next cdk or
apc-c in the sequence
○ Each cdk triggers events that lead to its own inactivation
○ G1 turns off apc and turn on s- s turns off g1 and turn on m- and so on and so
forth
○ The mitotic cdks don't directly turn off s but lead to apc to turn on and apc turns
off s and m cyclins
● Additional key points
○ Here is overview
 ○ Another key feature: replicate and segregate genetic material- main phases is s
phase and m phase- when goes to s phase or m phase wants to do it all at once
○ So one way to think about is going to war like invading canada- you want to build
a army so you can defeat canada and build tanks and then when you are ready you
invade
○ Same thing with s and m phase- you want to do it all at once
○ So cell produces large amount of s cdks ahead of time- make them in g1 thats
your army but they are in an inactive state
○ When you have some then you turn them on and invade
○ Same thing goes with m cdks- we make them inactive form in s phase and turn
them one when it's ready
● Nutrients -> translation and stabilization of initial g1/s cyclin initial g1/s cdk-> txn of
additional g1/s cyclin genes -> full activation of g1/s cdks
○ The way mammals and yeast know when to enter cycle is different- small minor
differences- big pic is same
○ Minor has to do with lifestyle
○ Yeast
■ Single cell organism- and it is dependent on nutrients- no food it doesn't
divide and vice versa
■ Nutrients is division deciding point
■ Nutrients g1 cdks
■ One of the cyclin - 3 is unstable protein in absence of proteins- and gets
stabilized - active does transcription of other g1 cyclin
○ Human cells
■ Cells have nutrients all the time
■ Another decision - growth factor- signaling
■ Growth factors that signal initial g1 cyclins to be produced and
transcription turns on of other g1 cyclins
○ Same but different
○ The mechanism we focus on is later g1 cyclin genes are repressed in much of cell
cycle
■ Activator protein to turn them on
■ In humans its e2f and yeast is sbfcaue cyclins are made all the time
■ Whi5 and humans is Rb
● These are inhibitory proteins that inhibit those proteins
■ What happens as signaling and activating of early cyclin
● Initial g1 cdk is made phosphorylate whi5 and rb and removes
inhibitor- and turn on transcription of late g1 cyclin
○ Retinoblastoma is mutated in many cancers
■ LOF of rb leads to driving cell cycle
 ■ Remove inhibitor of e2f and turn on g1 cyclins all the time- cells
continually enter cell cycle
○ Cdc 14 phosphatase resets whi5 at the end of mitosis
○ Gene encoding rb is mutated LOF in many cancers
● Roles of G1 Cdks
○ Nutrients/growth factors ---->initial g1/s cyclin---> later g1/s cyclins
○ g1/s cyclins mid-G1 “low levels→ late g1 high levels of g1/s cyclins--->
activation of s cdks
○ g1/s cyclins mid-G1 “low” levels → production of s cyclins ---> s cdk initially
inhibited →phorsphorylating it
● Start
○ g1/s cdks shut off the machinery that degrades s and m cyclins
○ S phase cyclins produced in g1 but held in inactive form
○ As level of g1/s cdk peaks -> start= activation of s cdks
○ Steps
■ 1. S cdk phosphorylate g1/s cyclin -> degradation of g1/s cyclin (m cdk
also targets g1/s cyclin for degradation)
■ 2. S cdk -> replication
■ 3. S cdk-> txn of M cyclins
○ Sic 1 binds to cdk- inhibits function
■ Bound by inhibitor sic1
○ High levels of g1 cyclin they will phosphorylate sic1
■ Phosphorylation recognized by scf ub ligase and ub sic one for destruction
■ Trigger s phase events
○ That's what trigger start- destruction of sic1
○ S cdks are activated they phosphorylate g1 cdks and they go away or inactivated
○ And we cannot cumulate g1 until get back to next g1
○ S cdks and m cdks phosphorylated g1/s cyclin targeting it for degradation
■ g1/s cyclins can accumulate again until s and m cdks are gone
■ g1/s cdks inactivate apc which degrades s and m cyclins
○ M cyclins are produced in s phase
○ S- cdks activate transcription of genes that code for the m cyclins
■ Mitotic cdks initially held in inactive form via phosphorylation of the m-
cdk by wee1
■ Dephosphorylated is ACTIVE form of wee1
● Figure violates standard rules of genetic diagrams
○ Arrows are nonsense lol
○ We don't yet understand all aspects of m-cdk activation but one way to think
about it is:
■ g1/s cyclin and turn on apc/c-cdc20
● APC= E3 Ubiquitin ligase
 ○ In a way like cdk and cyclin- its heterodimeric and subunits that does degradation
and the other regulates target specificity like cyclin
○ M-cdks activate apc/c-cdc20
○ apc/c-cdc20 function is required for sister chromatid separation
■ apc/c-cdc20 targets s cyclins for degradation
■ apc/c-cdc20 targets a large fraction of the m cyclins for degradation
○ apc/c-cdh1 targets the remainder of the m cyclins for degradation
○ Two forms of apc like cdk
■ Apc in cdc20 form and apc-cdh1
■ Change specificity of ligase
○ Two main features is get rids of m and s cdks and trigger sis chromatid separation
and what happens in second half of mitosis
● Activation of apc/c-cdh1
○ Cdc14 is a phosphatase
○ Activation of cdc14 is required for exit from mitosis
○ Cdc14 activates apc/c-cdh1 which targets remaining m cyclins for romve
phsophate s or have a phsophatasedegradation..which results in activation of sic1
via dephosphorylation
○ This ‘resets’ sic1 to its g1 state
○ Cdc14 dephosphorylates whi5 “resetting” whi5 to its early g1 form
○ Cdc14 leads to repression of txn of m cyclins
○ Does
■ Involved in activation of apc
■ It promotes events of second half of mitosis by binding phosphorylations
that mcd's did- mcd's drive first half phosphorylating- have to reverse it by
cdc14 dephosphorylation everything to reverse process
■ Among targets of cdc14 is sic 1 and whi5
■ Those guys are involved in: sic- inhibits cdks- and we have to
phosphorylate to get off- phosphatase removes p so we can have sic one
and go back into g1 and do the same thing
■ Whi5 and rb- inhibiting transcription of cyclin - phosphorylated them
which removed them so genes can be activated- cdc14 takes ps off so
whi5 and rb can inhibit the function of g cyclin transcription
● Picture
○ Apc activity persist into early g1- cannot accumulate s or mcd's until you have g1
cdks
○ g1/s cdks shut off apc-c allowing accumulation of s cyclins
● Picture
○ Turning off of the apc by the g1 cyclins is by phosphorylation - the cdh1 subunit -
and causes it dissociate from the ligase and becomes inactive
○ Once apc is gone we can accumulate a cylin
 ○ Destruction of m cyclins paves the way for accumulation of g1/s cyclins
○ g1/s cdks inactivate apc/c-ch1; thereby allowing accumulation of s-phase cdks
● Picture
○ We have irreversible switches where you can't turn on until you get to next cycle
○ Like breaking down like sic1- by ubiquitination and destruction of sic1
■ Destroy protein and you can't build up until next cell cycle
○ Apc targets m and s cyclin -once destroyed can have them until next cell cycle
○ - cell cycle can only progress in one direction
● Cell cycle checkpoints = inhibitors of progression
○ g1/trigger transition to s and apc triggers anaphase-
○ Checkpoints inhibit cell cycle and stop it when bad things happen or when earlier
events have finished
○ If dna is not fully replicated there are events where we block m cyclins until
replication is complete
○ Apc is inhibited prevented until all chromosomes are attached to spindle
○ Signals that are sent the inhibit cycle until critical events occur
○ Negative regulators or inhibitors that inhibit drivers!
● Picture
○ Experiment that showed this sort of thing can happen and proteins that block cell
cycle
■ Budding yeast
■ Mutation - temp sensitive in cdc13
■ Non permissive- cdc13 not functioning - cells do not complete dna
replication
■ What happens is those cells arrest in a certain stage in cell cycle
■ Can't complete replication - all arrest in g2
■ If you have double mutant - cdc13 and rad9 null allele- cells continue to
divide and die- go into meiosis before they finish- and lead to damage that
implies that rad9 protein is part of mechanism that dna is not replicated
and inhibits cell cycle until dna is completely replicated
● Lee Hartwell
○ Did original cdc screen
○ Discovered checkpoints
○ Logic that led him to look for checkpoints
■ Rad9 monitors dna damage and halts cell cycle progression until damage
is repaired
■ When you exposed human tissue to radiation - they prolonged g2 phase
■ Does that happen in yeast? Some mechanism that prevents progression
■ Took yeast cells that damaged their dna and that prolonged g2- cells are
sensing dna damage and blocking progression through cell cycle until they
can repair it because you don't want to replicate damaged dna
 ■ Cells in g1 will arrest there until dna is fixed
○ If it's damaged you have to stop replication and prolong that stage until it's fixed
○ Get rid of rad9 the cells won't prolong g2 they won't wait to fix and you get
increased mutation rates and death
■ Rad9 is short for radiation - hypersensitive to radiation suggests that rad9
monitors for dna damage and halt cell cycle progression
● Experimental evidence for checkpoints
○ If we have incomplete replication and undergo mitosis before we are done we die
○ S phase and g2 takes 35 minutes but if we had hydroxyurea the time goes up to 90
minutes- this inhibits enzyme that makes nucleotides
○ Cells wait to go into m until they are done replicating dna
● Screen for genes that regulate the s phase checkpoint
○ S and g2 takes 35 minutes
○ With HU it takes 90 minutes
○ If we remove the proteins that sense dna that it's not replicated- then we might go
after mitosis after 35 minutes and even with HU but you die
○ Genetic screen: look for cells that can grow on media lacked HU but non on
media containing HU
● Dna damage checkpoint
○ Set of proteins that recognize damaged dna and sent signal to stop cell cycle until
we can fix the dna
○ Proteins like atm and atr and ck2 and chk1 are protein kinases
■ Like signal transduction pathway - but internal - sensing damaged dna or
not completely replicated dna
■ A lot of events that can trigger this pathway
● 1. Double strand breaks
● 2. Stalled replication forks
● 3. Dna mismatches
● 4. Nucleotide damage
■ Send signal that inhibits cdks and block transition to next cell cycle
● 1. Repair
● 2. Blocks cdc25
● 3. Tries p53 which leads to apoptosis or p21
■ P53 mutated gene in cancer that controls apoptosis
○ Breakthrough prize
■ Up to 5 win per year
● Checkpoints and cancer
○ Loss of checkpoints -> loss of brakes on cycle= excess proliferation, loss of cell
death, increased genomic instability
○ Damage
■ 1. Cell cycle stops
 ■ 2. High level of damage- apoptosis
■ 3. Turn on transcription of genes that repair
■ 4. Dna repair
○ Main hallmarks of cancer cells is that there genome is screwed up - broken and
rearrangements- when you lose checkpoints you can repair - and whine up with
genomic rearrangements and have higher mutation rate- lead to cancer
○ This is increased genomic instability -have lots of damage
○ If you lose checkpoints you can't undergo apoptosis- and cancer can undergo
apoptosis and accumulate massive amount of genomic rearrangements
● Checkpoints Insure faithful transmission of dna/genetic material
○ Some act when there is a problem
■ Dna damage
■ Stalled replication forks
○ Some act to prevent cell cycle transitions until previous step is complete
■ Ongoing replication
■ Spindle assembly checkpoint
■ Spindle position checkpoint
● Spindle position checkpoint in yeast
○ Activation of cdc14 and the MEN does not occur until the spindles are properly
localized
○ Key: until we position spindles properly in case of budding- one in parent and one
in bud, we don't activate cdc14- it gets sequestered in nucleolus and activation
requires that spindles are properly positioned
● Kinetochroes
○ Sends signal that inhibits the apc until properly attached (under tension)
○ A single unattached spindle sends enough of a signal to inhibit the apc
■ Proper tension stop sending signal to apc and off you go
○ Kinetochores not properly attached send signal that inhibits cdc20 and therefore
the apc. Cdc20 bound by the inhibitor mad2
○ Kinetochores properly attached there is no signal ->cdc20 and apc become active.
Cdc20 released from mad2
○ Spindle send signal ultimate tarte is inhibition of apc until tension - apc gets
activated and under anaphase
● Checkpoints = Brakes on the cell cycle
○ Five things
■ 1. Dna damage
■ 2. Unreplicated dna
■ Dna damage
■ Chromosome unattached to spindle
● Some hallmarks for cancer cells
○ 1. Unregulated proliferation
 ○ 2. Genomic instability - increased mutation rate
○ 3. Metastasis - often sessile- motile
■ To get those things to happen have to mutate half dozen genes-mutations
that can be dominant or recessive give rise to cancers
■ Mutations that give rise to cancer include mutation in checkpoint and have
increased instability
○ Many types of cancer
■ 1. Different cancers -> different genes mutated
■ 2. Approx 6-7 mutations -> cancer
■ 3. Mixture of both dominant and recessive mutations
● Overview of Human Cell Cycle Regulation
○ Few differences than yest
■ Single kinase- cdc2 and cdc28
■ We have multiple and variety of cyclins
■ Works more or less the same but there is more machinery
○ We have checkpoint genes in red and those mutated are in cancer- P53, p21, and
variety of genes pathway that can contribute
● RB is mutated in a lot of cancer
○ Gene mutated in 30 percent of cancer
○ Inhibits transcription of cyclins and in order to go into cell cycle you need it if
you mutate it so you don't have rb than genes are on all the time and you always
enter cell cycle
● Connecting signaling to cell cycle progression
○ One of the genes that is regulated by ras is Myc- mutated in cancer downstream is
cdk and activate and remove RB that produces cdks and we enter cell cycle
○ ras= gof
○ myc= gof
○ rb= lof
○ e2f=gof
○ Oncogenes and tumor suppressors
○ Mutations in genes that contribute to cancer- are classified as oncogenes or tumor
suppressor
○ oncogenes= genes in which gain of function contribute -
■ Like ras, myc, and e2f
● If ras is always on then you get a gain of function
○ Tumor suppressors
■ Genes whose loss of function contribute
■ RB is an inhibitor of cyclin - remove it and get transcribed and rb is a
tumor suppressor
○ These refer to wild type version of gene
○ Gives us pathway if gene is oncogenic or tumor suppressor
MAP OF CELL CYCLE - CRASH COURSE

1. STEP 1: G1
a. Whi5 (yeast) and RB(humans) inhibit the transcription of making late g1 cyclins
i. How to get rid of it? Use initial G1 - comes from nutrients
b. G1 also shuts off APC to get s cyclins
i. How? It phosphorylates it
c. S cyclin is made but it is inactive by the inhibitor sic 1
i. How to get rid of sic 1? Use SBF (yeast) (E2F= humans)
ii. Need High levels of G1 in order to get sbf
2. STEP 2: S
a. Phosphorylate G1/S cyclin in order to get rid of those cyclins
b. Scdks do what?
i. Replication
ii. Transcription of M cyclins
c. The mcdks are inactive here by Wee--1
i. How to activate them? You will eventually have more mcdks than wee-1
can handle, and then mcdks activates the other mcdks
ii. Need cdc25 to activate the mcdk
3. STEP 3: M
a. Phosphorylates g1/s cyclin
b. How does activation start?
i. No one really knows but it is believed that turning off g1/s cyclin and
turning on apc does it
4. STEP 4: APCx2
a. There are two types of apc
b. apc/c cdc20
i. Degrades m and s cyclin
ii. Starts anaphase
c. apc/c cdh1
i. Turned on by cdc14
ii. Degrades rest of m cyclin
iii. Triggers sister chromatid separation
5. STEP 5:
a. CDC 14- DEPHOSPHORYLATES
i. Restarts whi 5 or rb
ii. What is needed to leave mitosis
iii. It is a phosphatase
iv. Restarts sic when apc is on
v. Repression of m cyclins