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4/17 - lec 22:

1.
Using the components discussed in class, propose a signal
transduction pathway that would result in the release of
cortical granules during fertilization. From this pathway,
identify three potential causes of CG-related infertility.
Cortical granules: releasing components that form
fertilization envelope
Signal Transduction Pathway Leading up to CG release:
1. Ligand binds to GPLR receptor on egg cell and activates
it
2. L-R complex triggers conformational change, which is
transduced to G-protein
3. G-protein exchange GDP for GTP and becomes activated
a. It also separates into G-alpha and G-beta,gamma
4. G-alpha (bound to GTP) travels along membrane until it
encounters Phospholipase C
5. Once Phospholipase C is activated, it catalyzes the
hydrolysis of PIP2 (phospholipid in the egg cell
membrane) into DAG and IP3
a. DAG will remain in the membrane, while IP3 is
soluble in the cytoplasm
6. IP3 will diffuse into the cytoplasm and will bind to
calcium channels on membrane of Smooth ER, opening
them and releasing calcium into the cytoplasm by
facilitated diffusion
7. Calcium levels in the cytoplasm will go above the basal
level (10-4 mM)
8. Calcium will then bind to Calmodulin, activating it
9. Calmodulin will then bind to Calmodulin-induced
Kinase ,
10.
Calmodulin-induced kinase will the phosphorylate
(using ATP) myosin
a. Myosin : cytoskeletal motor proteins
11.
Activated myosins will carry cortical granules being
formed up to the plasma membrane for release.
a. Cortical granules made in the endomembrane
system (being secreted outside the cell)
12.
Myosins move CG up to the PM, where their internal
contents will be released and form the fertilization
envelope outside the fertilized egg
Causes of Infertility:
Lack or abnormal CG formation
Misfolded G-protein

2.

Initial receptor is defective or absent


G-protein cannot exchange nucleotides
Calcium levels do not get high enough to activate
calmodulin

Describe an enzyme-coupled receptor. Detail in detail the


structure and function of receptor tyrosine kinases (RTKs).
Enzyme-Coupled Receptors:
Transmembrane proteins
o Subunits consist of singlepass polypeptides
Cytoplasmic domains
o Either have intrinsic enzymatic activity
o Or associated with an enzyme

Receptor Tyrosine Kinases: class of ECRs


Phosphorylate hydroxyl group on tyrosine residues
Involved in autophosphorylation
o Will phosphorylate serine residues on themselves
o Changes properties of molecules (adding polar,
negative group)
3.
Describe growth factor characteristics and the roles of
played by growth factors in cells.
Growth Factors:
Common ligands for RTKs
Small molecules capable of stimulating cell growth,
division, or differentiation
Some can act Broadly
o Affect many classes of cells
Some can have Narrow targets
o Affect one/few cell types (specific)
o EX: Erythropoietin
Proper cell behavior requires a specific combination
of growth factors
o Epidermal Growth Factor: broad range,
stimulates proliferation of many cell types and
acts as an inductive signal during embryonic
development
4.

Describe a monomeric G protein (e.g. Ras).


Ras
Monomeric G-Protein

5.

Signal transduction component and important regulator


of cell growth
Identified in rat sarcomas
Similar in function to heterotrimeric G-protein
Associated with cytoplasmic face of PM (monotopic
membrane protein)
Alternatively binds to GDP and GTP
Inherent GTPase activity
o can convert GTP back to GDP by hydrolyzing
phosphate group
Only associate with RTKs not GPLRs

Outline the events involved in a growth-factor/RTKmediated pathway. Using the example outlined in class,
explain why expression of cyclin/CDK is an appropriate
outcome for such a pathway.

Signal Transduction Growth Signaling Pathway via Ras:


1. Epidermal Growth Factor acts as ligand or primary
messenger and binds to receptor, RTK
2. Binding of growth factors to individual RTK polypeptides
cause them to dimerize and autophosphorylating the
OH group on the tyrosine residues on eachother,
activating the complex
3. Activated RTKs transduce cascade to adaptor molecules
(or secondary messenger)
a. Message transduced by a series of conformational
changes
4. Secondary messenger interacts with Ras (G-protein)
5. Ras switches out GDP for GTP and becomes activated
a. Ras changes conformation when it binds with
secondary messenger, thereby changing affinity for
nucleotides (GDP for GTP)
6. Phosphorylation cascade occurs where is message is
transduced down the rest of the cascade by
phosphorylation of one molecules triggering the
phosphorylation of another molecule
7. A molecules activated by phosphorylation and enters the
nucleus , where it causes changes in gene expression
8. The products of the pathway are Cyclin and CDK
a. The intended message of this pathway is mitosis or
cell division/growth
b. Cyclin and CDK regulate the progression of the cell
through the cell cycle

c. The primary messenger was a growth factor, so cell


would eventually be triggered to go through mitosis
6.

Explain how cytosolic calcium levels can be regulated by


either a GPLR-mediated pathway or a RTK-mediated pathway.
IP3 mediated release of calcium can involve:
GPLR pathway
RTK pathway
o Involves activation of phospholipase C
Triggers IP3 release, which triggers calcium
release in the cytoplasm
EGF

Example of signal integration in which different receptors


activate the same pathway
o Could be a redundancy issue to ensure the pathway gets
activated
If one pathway is faulty or doesnt get activated,
there is another pathway there to ensure other
pathway gets activated
7.

Diagram the insulin/glucagon system used in our bodies to


maintain blood glucose homeostasis.
Normal Fasting Blood Glucose Level: 70-110 mg of
glucose per 100 mL of blood
Insulin: peptide hormone, involved in uptake of glucose
(to be stored as glycogen)
Glucagon : peptide (very small) hormone involved in
triggering the breakdown of glycogen and release of
free glucose into the blood

In Hyperglycemic State: Glucose levels above normal range


Body detects increased glucose levels
Signal sent to Beta Cells in Pancreas to become activated and
produce and secrete insulin
Insulin released into bloodstream and binds to receptors on
different cells, causing them to take up glucose
o Also binds to receptors on liver, causing it to take up
glucose and store it as glycogen
Glucose is eventually cleared out of the bloodstream, bringing
blood glucose levels down to homeostatic level

In Hypoglycemic State: Glucose levels below normal range


Signals sent to pancreas and Alpha Cells, which are stimulated
and produce and secrete glucagon
Glucagon binds to cells on liver, and triggers breakdown of
glycogen and release of it into the bloodstream
Continues until blood glucose levels rise back to homeostatic
level

8.

Systematize the signal-transduction pathway involved


in insulin-mediated uptake of glucose and glycogenesis.
Explain how defects in this pathway may lead to diabetes.

Insulin-mediated regulation of Blood Glucose Levels:


1. Insulin acts as a ligand and binds to RTK receptor
a. Binding of insulin to each polypeptide of RTK causes them
to dimerize and autophosphorylate
2. Binding of R-L recruits in molecule of IRS (Insulin Receptor
Substrate)
a. IRS is a target of the RTK, which will phosphorylate the IRS
b. IRS also involved in transducing cell division pathway
involving Ras
3. IRS activates PI-3 Kinase
4. PI-3 Kinase targets PIP2 (lipid in PM) and phosphorylates it,
forming PIP3
5. PIP3 recruits protein kinases, which leads to phosphorylation and
activation of Akt
6. PIP3 activates Akt
7. Akt causes production and activation of GLUT (Glucose
Transporter)
8. GLUT gets put into the membrane of the hepatocyte and
facilitates entry of glucose into the cell (and out of the
bloodstream)
9. Akt also activates Glycogen Synthase (by phosphorylation)
a. Glycogen synthase synthesizes glycogen
9.

Define diabetes. What are the long-term effects of


diabetes. Differentiate between Type I and Type II diabetes.

Diabetes: a result of high blood glucose levels


2 Types:
o Type 1 Diabetes : Insulin Dependent (Insufficient Insulin)
Results from defect in beta cells of pancreas
Beta cells being destroyed by the body (autoimmune
disorder), cells attacking beta cells, and therefore, no
insulin is being produced
There is no insulin ligand present to bind to receptor
to trigger uptake of glucose
Glucose levels remain chronically high
o Type 2 Diabetes : Insulin Independent (Insulin
Resistance)
Body is producing enough insulin to begin with, but
signal transduction cascade is not responding
properly
Defect in cells ability to recognize and process
insulin message
Blood Glucose Levels remain chronically high
Long term effects of Diabetes:
Can lead to heart disease, kidney failure, and blindness
4/22 - lec 23:
10.
Describe in detail the steps taken by insulin from gene
expression to secretion from beta cells of the pancreas, all the
way to the binding of insulin to its receptor on target cells.
Explain why not all cells respond to insulin.
Insulin goes through the Co-Translational Import Pathway
It is a protein secreted from the beta cells of the pancreas
It is a vesicle that goes through the Endomembrane System
Inside the Golgi, it goes through post-translational processing
o Signaling sequence gets cleaved off and becomes
proinsulin
o Different chains held together by disulfide bridges
fully functional insulin
Once it leaves the beta cells, it travels to its target cells, like the live,
and binds to receptors on these cells, causing them to take up glucose
and store them as glycogen
11.

List the functions of the cytoskeleton.

1. Organization:
a. Spatial organization of cellular contents
b. Like cytoplasmic and nuclear (genetic) materials
2. Cell Shape
a. Provides mechanical support to the cell and nucleus
b. Scaffolding that helps generate maintain and
maintain cell shape
3. Motility:
a. Movement of the cell itself (cell crawling)
b. Intracellular movement of structures
4. Cell Division:
a. Manages chromosomes (Microtubules bind to and
help separate them)
b. Cytokinesis (Microfilaments actually divide the cell
itself)
5. Regulation:
a. Transmits mechanical signals from environment
i. Like adhesion molecules, either directly or
through adaptor molecules
b. Intracellular movement of molecules in signaling
12.
Recall the three different elements that make up the
cytoskeleton and describe their main functions.
1. Microtubules:
a. Spatial organization in cytoplasm
b. Intracellular transport (motor proteins)
2. Microfilaments:
a. Forming and maintaining cell shape
b. Cell locomotion (specific to crawling cells)
3. Intermediate Filaments:
a. Mechanical strength
b. Nucleus shape ( and organizing chromosomes)
13.

Describe the roles of cytoskeletal accessory proteins.

The cytoskeletal accessory proteins:


Link cytoskeletal elements to each other or to other
cellular components
Regulate assembly/disassembly of elements
14.
List the functions specific to microtubules
(MTs). Differentiate between cytoplasmic MTs and axonemal
MTs.
Microtubules:

15.

Cytoplasmic MTs : dynamic MTs


o Cell organization (contents of cytoplasm)
o Intracellular movement (tracks for motor proteins)
Vesicles and organelles
Chromosomes
Axonemal MTs : static MTs
o axoneme is a motility structure
o Components of motility structures
Flagella, Cilia
Describe in detail the structure of MTs.

Microtubule Structure:
a single MT is a hollowed out tube made out of
protofilaments
each MT is 25 nm in diameter (largest of the
elements)
protofilaments made up of dimers of the protein
tubulin
beta tubulin and alpha tubulin form tubulin
heterodimers
tubulin heterodimers come together to form
protofilaments, which come together to form hallow
tube
oriented in a specific polarity
o beta subunits face plus(+) end
o alpha subunits face minus (-) end
Tubulin Heterodimer :
alpha and beta tubulin both bind to nucleotide GTP
only beta tubulin can hydrolyze GTP to form GDP
(GTPase activity) (and can exchange GDP for GTP)
alpha tubulin is permanently bound to GTP
o alpha and beta tubulin held together by non-covalent
interactions
o structural polarity to MT because subunits in each
protofilament all point in the same direction
Cytoplasmic MTs
singlet arrangement of protofilaments
hollow ring comprised of 13 parallel protofilaments
Axonemal MTs
Doublet arrangement
Singlet ring with partial ring of 10 protofilaments

o Triplet arrangement
Singlet ring with 2 partial ring (10 protofilaments
each)
16.

Explain the model of MT assembly.

MT Assembly :
Start off with population of tubulin dimers (alpha and beta)
Link together to form short fragments : oligomers
Oligos come together to form longer strands :
protofilament
Protofilaments line up laterally to form sheet of
protofilaments
Protofilament sheet folds in on itself to form hollowed
out tube
Hollowed out tube grows from either by the addition
of free tubulin dimers in the cytoplasm to form fully
functional Microtubule
Growth of MT starts out really slow with free dimers
in the cytoplasm
Growth gets rapid once the dimers start linking to
form protofilaments
17.
Define 'critical concentration' as related to MTs. Explain the
effects of the critical concentration on MT assembly. Explain
how critical concentration is related to the polarized assembly
of MTs
Critical Concentration :
The concentration of free tubulin in the cytoplasm at which
rate of tubulin subunit addition is approximately equal to
the rate of subunit loss
MTs tend to grow when tubulin concentration
exceeds Cc and depolymerize when tubulin
concentration falls below Cc
Overall length change is constant
Reached during the plateau phase
Point where growth slows down dramatically
and subunits and coming off and being added
Cc differs for plus end and minus end of MT, which is
why is there is a difference in rates of growth and
disassembly
Plus end grows much faster than minus end
( elongates faster)

Growth Rates reflect differences in Cc requirements


Plus (+) end: lower Cc
Minus (-) end : higher Cc
o Minus end will reach Cc earlier than
plus end, as it does so at a higher
concentration of free tubulin
o Treadmilling: simultaneous
polymerizing (at plus end) and
depolymerizing (at minus end) within one
MT
o Once Cc is reached at plus end (at a
lower concentration of free tublin),
catastrophe event can occur
Rapid depolymerization at + end
Growth/Shrinkage determined by critical
concentration

18.
Describe the process of 'dynamic instability' and the role
played by GTP in this process.
MTs are continually changing and are very dynamic, at any
given time, some MTs are growing while others are shrinking
Dynamic Instability:
Model to explain constantly changing MT behavior
In a given population of MTs:
Some polymerize and grow while others are
depolymerizing and shrinking
Polymerization may continue for some undefined
period of time
MT may suddenly shrink rapidly (catastrophe)
Can also shrink partially and recommence
growing (rescue)
Can also completely depolymerize and growth
does not resume
Cc extremely sensitive to environment
immediately surrounding that end of MT
Regulated by GTP Cap:
Free tubulin is in the GTP form
Tublin dimers (bound to GTP) added to the of MT during
polymerization
Eventually GTP within tubulin dimer is hydrolyzed to GDP
Beta subunit hydrolyzes GTP when it is a part of MT
GTP hydrolysis destabilizes MT structure

Events occur at both ends of MT (more activity at the plus


end)
At High Tubulin Concentration:
GTP-tubulin subunits get added to MT
If Growth Rate > Rate of GTP Hydrolysis
As subunits get incorporated into MT structure, more
subunits will get added
Beta tubulin will hydrolyze GTP to GDP, but rate of
growth greater than GTP hydrolysis
GTP Cap on the end of the MT
o Stabilizes MT and promotes further
growth
As growth continues, rate of growth slows down because amount
of free tubulin in the cytoplasm decreases as growth occurs
At Low Tubulin Concentration:
Growth Rate <Rate of GTP Hydrolysis
GTP cap is lost, GTP hydrolysis catches up with end of
MT
MT becomes unstable and favors depolymerization
When tubulin dimer polymerizes, forms straight protofilaments
(with beta subunit bound to GTP)
As beta subunits hydrolyze GTP to GDP, GDP changes
conformation of beta subunit
Interferes with interactions between individual
heterodimers
Causes protofilament to become a slightly curved
structure
When straight, protofilaments can form a lot of
IMFs in the hollow tube
However, when curved, that disrupts the IMF
between protofilaments, which destabilizes
the molecule
o Depolymerization occurs
19.
Discuss the evidence which indicates that dynamic
instability is required for proper MT function.
MTs always changing and are dynamic (growth and shrinkage)
If fluctuations are prevented, cells are no longer viable
Dynamic Instability is crucial for proper MT function
Taxol: binds to MTs and stabilizes them
Prevents depolymerization

Result: arrests cell cycle and cell undergoes


Apoptosis
Colchine: binds to tubulin dimer
Prevents polymerization
Result: prevents mitosis
20.
Explain the function of MTOCs. Describe the structure of
the centrosome and, if present, the centrioles.
Microtubule Organization Center (MTOC) :
Nucleation sites for MTs
Site where MT assembly is initiated
Also serve to anchor and organize MTs
Interphase Cells:
MTOC is focal point
Most of cytoplasmic MT emanate from MTOCS
Dividing Cells:
MTOC organize MTs to form spindle apparatus
Control MT orientation
Contain Gamma Tubulin Ring Complexes
Serve as nucleating sites for MT within MTOC
Binds to the minus (-) end of most MTs
Plus (+) end grows outward from gamma- TuRC
Centrosomes : MTOC in animal cells
Contain centrioles (made up of MTs)
2 symmetrical barrel shaped structures
2 centrioles in one centrosome, organized at
right angles to one another
role unclear
made up of Triplet MT
9 triplets arranged in a conformation to make
up a centriole
Pericentriolar Material
Surrounding centriole
Gel-like proteinaceous matrix
Includes gamma-TuRC (nucleation sites for MTs)
4/24 - lec 24:
21.
Explain how MTOCs are involved in cell
shape/differentiation, especially in polar cells.
As cell goes through growth/differentiation patterns, placements
of MTOCs

important for how a cell differentiates


Polar Cells : ends of cells are structurally and/or functionally
different
Example: Neuron, that has axon and dendrites (structurally
different at each end)
Example: Epithelial Cells, Apical end and basal end are
structurally different, apical end is ciliated

Polarization of Polar Cells determined by underlying MT


arrangement:
EX: Neuron
Axonal MTs
Controlled by MTOC (centrosome)
Axonal MTs drive process of growth
Uniform parallel orientation of MTs in axons
o All anchored in centrosome
o Minus end in centrosome, since they are
bound to gamma-TuRC
o Grow out in plus direction
Dendrites
Not determined by MTOC (centrosome)
Do not contain MTOCs
Dendrites MTs are mixed polarity
Not parallel, woven together in different
orientations
Underlying MT arrangement gives rise to
branching patterns
22.

Explain the various roles of MAPs.

Microtubule-associated Proteins (MAPs)


Functions:
Link MTs to other cellular components (PM, vesicles,
other cytoskeletal elements)
Stabilization of MTs (like in motility structures)
Destabilization of MTs (need for dynamic instability)
Severing Proteins
Motor Proteins

23.
Describe the role of Tau and how it is correlated with
different neurodegenerative diseases.
Tau: (MAP)
found in axons of nerve cells
binds to axonal MTs
stabilizes MTs
promotes MT polymerization/growth
present at the beginning of development, helping
neuron develop properly and keep it stabilized
induces MTs to start polymerizing and stabilizing to
form long outgrowths
play a role in development of neuron polarity
To regulate activity of Tau, kinase phosphorylates Tau
Once Tau is phosphorylated, it changes its conformation so
it can no longer associate with MTs
No longer binds to MTs, so MT is destabilized and
start to depolymerize
To regulate Tau in the opposite direction:
In order to allow phosphorylated Tau to rebind to M,
action of phosphatases are needed to
dephosphorylate Tau and change conformation back
to a state where it can interact with MT and stabilize
them.
PHF: paired helical filament
Insoluble, stable aggregate of Tau
Role of Tau in Neurodegeneration:
Tau has been implicated in neurodegenerative diseases,
such as Alzhemiers disease
Hyperphosphorylation of Tau disable Tau from being
able to bind to MTs
when it cannot bind, MTs become destabilized and
breakdown
when Tau is phosphorylated, can bind to one another
and form filaments
some sort of signaling occurs, making it
hyperphosphorylated, which forms larger aggregates
and forms PHF
PHF: play a role in breakdown of cell
function
o Cytotoxic filaments , impair functions
of neurons and eventually cause their

death, leading to neurodegenerative


disorders
24.

Describe the structure and functions of microfilaments.

Microfilaments

Function:
Underlie the PM and form cell cortex
Stabilize cell shape
Involved in muscle contraction
If a cell can crawl, cell locomotion , which is MFbased motion
MFs involved in cytokinesis
During cell division, when cell splits into two
Cytoplasmic Streaming: mainly plant cells
Cytoplasmic contents move around the cells,
which can function to assist diffusion rates
Structure:
Similar to MTs
7 nm in diameter : smallest of the three
elements
polymers of the protein : actin
2 forms:
G-actin (globular)
o Free form of actin
o Isnt polymerized into a MF
F-actin (Filamentous)
o Polymerized form of actin
o When actin is in filament

25.
Compare and contrast microfilament assembly to that of
MTs.
MFs have polarity, similar to MT: has +/- ends
+ end grows faster than end
G-actin (free actin) bound to ATP
similar to GTP in MTs
ATP is eventually hydrolyzed to ADP in MF polymer (when in
filamentous form)
ATP cap to MF
o Actin can reversibly bind ATP and ADP,

o Can hydrolyze ATP to ADP (Hydrolase activity): break


apart phosphate bonds
o Hydrolase activity causes conformational change which will
destabilize overall structure of MF and lead to its
depolymerization
26.
Explain how microfilaments are regulated and organized by
their associated proteins.
Regulated by actin-binding proteins
Severing proteins
Bundling proteins
Cross-linking proteins
Capping proteins
Anchoring Proteins
Proteins that stabilize unbound form
o Regulate if they can bind to filament or not
Model of Regulation of MF Organization:

Cell growth and cell locomotion generated by action


of MFs
MFs move in different directions and branch to push
cell in a particular way
o Regulated by associated proteins
A MF polymerizes in a certain direction and once it
gets to proteins associated with PM, interacts with
them and pushes PM in that direction and continues
to polymerize
MF branch at particular points wit defined angles
o Branching regulated by grouping of proteins
Arp 2/3 : actin related proteins
WASP
These two bind and form
complex that allow branching
for MFs
Complex necessary to initiate
branching of filaments
Complex formed is like a new nucleation
site that MFs continue polymerizing from
WASP is needed to initiate to complex
with Arp 2/3 to initiate branching of MFs

Cytoskeleton linked to PM
Integral membrane proteins associated with cytoskeletal
elements, like MFs
In this way, MFs linked to PM membrane proteins can push
PM in direction to generate cell locomotion
27.
Explain the role of WASP. Explain how a defective WASP can
lead to the symptoms associated with Wiskott-Aldrich
Syndrome.
WASP: Wiskott Aldrich Syndrome Protein
Wherever there is actin, you see WASP , it is associated with MFs
Regulates actin polymerization in Hematopoietic Cells (RBCs)
Hematopoiesis : developmental process by which immature
precursor cells develop into mature blood cells

start off with hematopoietic stem cell (HSC)


o get precursor cells
o from one line of stem cells, can generate any of the blood
cells depending on which type of signals they receive.
Genetic plasticity of cells

Wiskott-Aldrich Syndrome: genetic disorder due to defect in


WAS gene that gives rise to defective WASP
Symptoms:
o Prone to bruising
o Prone to eczema (inflamed skin disorder)
o Susceptible to pneumonia and other infectious diseases
o More susceptible to developing lymphomas (cancer of
WBC)
If WASP is defective, will not form complex with Arp 2/3, which
will not allow the MFs to branch properly to form the proper cell
shape
o EXAMPLE: Improper platelet cannot wont be able to bind
properly to others to clot, or can clot too much, making
person more prone to bruising
o Improperly formed WBC, cant crawl or fight infections
properly
WAS macrophages have a defect in polarization
o With WAS, when WBC exposed to chemo attractant , WBC
crawls in all directions
Ability to polarize in one direction is impaired
because their underlying branching is defective

28.
Describe the potential effects of drugs that disrupt
microfilaments.
Drugs that affect MFs will affect the viability of the cell
- they prevent MFs from being able to polymerize/depolymerize or
branch, affecting viability of the cell
29.
Describe the structure and functions of intermediate
filaments (IFs).
Intermediate Filaments :
- Function:
o Structural support
Tension bearing cables
o Nuclear Lamina Scaffolding
Underly nuclear envelope
Provide structural support for the nucleus
Nuclear lamina : network of Ifs beneath inner
layer of nuclear envelope
o Can also provide organization for chromatin in nucleus
- Structure :
o 8-12 nm in diameter : in between diameters of MTs
and MFs
o all share same structure
o Single Subunit: helical protein (N terminus and C
terminus )
o Dimer: single subunits interact and coil around one
another with other subunits to form dimer
o Staggered Tetramer: 2 dimers interact with eachother in
a staggered, antiparallel way
C terminus of one associated with N terminus of the
other
o IF Subunit: Tetramers interact with one another to form
subunit that will be used to construct IFs
Like tough ropes
o 2 tetramers pack together; form 8 protofilaments
o 8 Protofilaments all wind around one another to give IF:
perfect for absorbing tension
30.
Explain how and why cytoplasmic IFs can be used in
tissue/cell-typing and why nuclear IFs cannot.
Cytoplasmic IF Proteins are Cell/Tissue Specific :
- Many different proteins can be used to make IFs
o Many different IFs can therefore be found in
cytoplasm of cells

o Different depending on cell type


Except Nuclear Lamins
Found underlying nuclear envelope of all
cell types
Cytoplasmic IFs are the only cytoskeletal element
that can be used to ID specific cell type
o MTs and MFs and Nuclear IFs (lamins) are all
made up of the same protein
o Cant distinguish cell types from these elements
Can use Cytoplasmic IFs for tumor diagnosis to determine
where a tumor originated from
Identify site of origin based on IF identity (protein profile)

31.
Provide an overview of the cytoskeleton. Recognize the
cytoskeleton as a complex, dynamic system with a high level
of interaction taking place among the three elements.
Interactive Cytoskeleton Network:
- all elements are linked together
- no one elements alone could provide shape or strength to
cell
- interact and linked with one another through accessory
proteins
- depend on each other to properly function and support the
cell
o requires large group of accessory proteins linking
filaments to one another and to other cell
components
32.
Describe the different levels of cell movement.
Cell Movement:
- Intracellular Movement:
o refers to movement of contents within the cell
o motor proteins transporting cargo along MTs
o cytoskeleton acts as a track and provides machinery
for movement of contents within the cell
- Movement of Cell Itself :
o Motility Structures:
Cilia and Flagella (MT-based)
Crawling Cells (Cell Locomotion) : MFbased

33.
Recall the types of microtubule-associated motor proteins
and describe in general their overall structural/functional
domains.
Motor Proteins:
- type of accessory protein (MAP)
- have ATP hydrolase activity (bind to and hydrolyze ATP)
- use energy from ATP hydrolysis to transport cargo along
cytoskeleton tracks (MTs or MFs)
- travel in a single direction
o move to + end or end of an elements
- motor proteins only associated with MTs and MFs because
there is no polarity associated with IFs, proteins wouldnt
know what direction to go
o need elements that have directionality
34.
Recall the direction of movement associated with each
motor protein with respect the the +/- ends of MTs.
Kynesin: move towards plus end of MTs
Dyneins : move toward minus end of MTs
-

Both have 2 globular ATP-binding heads and a tail


o Heads can hydrolyze ATP (ATPase activity) and
interact with MTs
o Interaction is Stereospecific
Will attach to MT only if pointing in the
correct direction
o Tails interact with cargo
o 2 globular heads bind to ATP, hydrolyze it, and
hydrolysis causes conformational change that swivels
the foot (head) forward
this happens again in the opposite foot
Protein will physically walk along MT track
Note: Dyneins would move cargo towards the end of MT linked
to gamma tubulin, as gamma tubulin ring complexes embed
the minus end of the MT, and Dyneins move toward the minus
end.

35.
Explain how the cell contents are organized by the
cytoskeleton. Generate a picture to describe how this
organization changes as the cell grows/develops.
MTs Organize the Cell :
- During cell growth/development, there is a particular
organization for the organelles
o Organelles organized by the MTs
- During cell growth/development
o Motor proteins associated with MTs bind to organelles
They pull organelles out as cell grows
Pull ER outward
Pull Golgi inward
36.
Describe how vesicle-based movement occurs in the cell.
Specifically describe how vesicles are transported between the
components of the endomembrane system.
MT Based Vesicle Transport:
- there is directionality to vesicle transport between different
compartments and to the PM
- if MTs all arranged so all plus end would point toward the
PM, Kynesins would be carrying vesicles to PM to be
secreted (move toward plus end)
- If things are being brought into the cell through
endocytosis, Dyneins would carry vesicles into the
body of the cell
37.
Explain why motor proteins are necessary. Given the
diffusion coefficient constant and the equation t = x2/2D,
calculate the time of diffusion for a given molecule or vesicle.
Motor proteins interact with cytoskeletal elements to produce
motion at the molecular level. They are necessary to help
vesicles and molecules move into and out of the cell . Much
faster than simple diffusion
Time of Diffusion: t=x2 /2D
D for a vesicle=5*10-8
Note: 1 cm= 104 um
Rate of Transport for Motor Proteins : 2 um/sec
- for axonemal dyneins: 14 um/sec

38.
Compare and contrast the structure and function of cilia
and flagella.
MT-Based Cell Motility : Involves AXONEMAL MT
Cilia:
- made up of Axonemal MTs
- number per cell: one to many cilia per cell
- Size (Length): 2-10 um long
- Mechanism of movement: Oar-like motion
Flagella
- made up of axonemal MTs
- Number per cell: one to few flagella per cell
- Size (Length): 10-200 um long
- Mechanism of movement: wave-like motion
Note: Prokaryotes have flagella, but they are
structurally different that eukaryotic flagella (proks do
not have cytoskeletal elements)

4/29 - lec 25 - LAST DAY!!!


39.
List and describe the three types of cilia found in
eukaroytic cells. Provide an example of each to support your
description.
Cilia:
- Motility: so cells can swim, fallopian tube and tracheal
- Sensory: detect signals from environment
- Nodal: involved in embryogenesis (type of sensory)
Sensory Cilia :
- transmit sensory information to the cell
- Example: Detecting Odors
o Inhalation of odors into nasal cavity
o Odorant molecules cross over surface of Olfactory
Epithelium
Epithelial cells lining nasal cavity
Within epithelial layer, there are specialized
neurons called olfactory sensory neurons
In epithelial layer, OSNs have axons and
dendrites

Within DENDRITES of these cells, cilia branch


off dendrites and have odorant receptors
Cilia extend into lumen of nasal cavity,
where odorant molecules are detected by
receptors on cilia
Cilia have odorant receptors that are
capable of binding to odorant molecules
o Once odorant molecules bind,
initiates signal transduction
cascade (Olfactory signaling
cascade)
o Causes depolarization of
membrane of neuron and
signal gets transmitted to
olfactory bulb, where neurons
are present to transmit signal
to the brain
Nodal Cilia :
o Provide positional information to the cell
o also detect information outside the cell, but are very
specific
o like in a developing embryo, embryogenesis
o nodal cilia provide positional information to
embryonic cells to know where they are relative to
other cells
required for proper development
nodal cilia extend from surface of these cells
and as embryonic fluid travels over the surface
of the cell, it is being moved by nodal cilia
which provides positional information to get
proper embryogenesis

40.
Describe the ultrastructural details of cilia and flagella.
Both cilia and flagella made out of Axonemal MTs
- at the base of cilia/flagella is basal body
o basal body similar to centriole
o has triplet MT arrangement
consists of 9 sets of triplet MTs
- basal body contains nucleating MTs that extend out
- as MTs extend, they go through Transition Zone (slightly
different MT arrangement)
- Once MT gets up to axoneme , has 9+2 arrangement
o 9 doublets forming outer ring with a pair of
single MTs inside
- MTs associated with many accessory proteins

o Help generate movement and maintain structure of


axoneme
o Bind at regular points along length of MT
o Some serve to X-link bundles
o Some generate forces to cause bending
(particularly dyneins)
41.
Explain how the axonemal components interact to
generate movement in cilia and flagella.
Dynein-Mediated Bending of Axoneme :
- Dynein= motor protein that binds to MT (moves toward
minus end)
- Specific dyneins found in axoneme
o Axonemal dyneins : have very rapid rates of
movement: 14 um/sec
- Experimental Evidence:
o Shows that dyneins are the motor proteins
cause movement of MT in axoneme
o Experiment used isolated MT doublet from sperm
flagellum
Added ATP
Also isolated associated dyneins
Axonemal dynein attached by its tail to MT,
while other end interacts with adjacent MT
Cargo is essentially the other MT
Dyneins will hydrolyze ATP, and in
isolation, will cause MT to slide past
one another
In isolated doublet MTs: dynein
produces MT sliding
o In intact flagellum
Accessory proteins crosslink MT
Cant move past one another
MT doublets tied to each other by
flexible accessory proteins
o They can slide a little bit, but will
go right back
o Crosslinking prevents them
being able to fully slide past one
another
Dyneins hydrolyzing ATP will cause
structure to bend because dyneins

cant walk along, are permanently


bound to MT
In intact flagellum, action of dynein
causes bending rather than sliding
Movement of cilia/flagella
produced by bending of core,
which causes movement of whole
axonemal structure

42.
Describe the structure and function of the microfilamentassociated motor proteins. How do they compare to MT-based
motor proteins?
Actin = Microfilament
Actin-based Cell Locomotion = Crawling Cells
- for cells that lack cilia/flagella
- based on action of MFs (actin)
- MFs have many different accessory proteins and motor
proteins
o Motor proteins associated with MFs : Myosins
Have domains that bind to MF (actin) that
can hydrolyze ATP
Have linker region in between
Move in a directional way (+/-) ends
MF-associated proteins similar to MT-based motor proteins
- both have directionality
- both have ATPase activity
43.
Describe the general structure and function(s) of the cell
cortex.
Cell Cortex :
- made of MFs
- what gives a cell its shape
- MT determine cell shape as cell goes through
differentiation
- MFs maintain cell shape
- Cell cortex rearranges MFs and gives rise to cell
locomotion
- Meshwork of actin filaments underlying PM
o Also includes spectrin network
Network of proteins linking PM to MFs

Held together by actin-binding proteins to create strong,


strong, supportive structure
Linked to PM by various accessory proteins
o Ankyrin : interacts with spectrin and MFs to
form network (actin-binding proteins

44.
Create a detailed model outlining the subcellular events
involved in cell locomotion.
Cell Crawling:
- cell that lacks cilia and flagella
- action of MFs
- includes cell with network of MFs just underneath and
connected to PM
o cell will undergo rearrangements to push cell
forward and retract rear of cell to move forward
- Cell will crawl in direction of stimulus
o Extends PM in that direction, pushing forward due
to action of MFs polymerizing in that direction
o Lamellipodium : extension of PM
- Events:
1. Leading edge extends via polymerization of actin
filaments at its tip (forming lamellipodium)
2. New adhesions, anchored by actin, form on the
undersurface of the lamellipodium
Still adhering at other end of the cell
3. The trailing edge (tail) of the cell detaches, and is
drawn forward by the contraction of the cell body
45.
Describe the arrangement of MFs in a cell during cell
locomotion. Are the MFs arranged the same throughout the
crawling cell?
In the leading edge (lamellipodium)
- actin MFs polymerizing in the direction of movement,
pushing PM in that direction
- accessory proteins linking MFs to PM
o proteins crossing through PM and adhering to
whatever cell is crawling across
Cell Locomotion
- as cell crawls forward, forms initial lamellipodium
o small extensions in lamellipodia called filopodia

In leading edge (lamellipodium) and filopodia, MFs


all pointing in the same direction, pushing PM
forward
o Parallel arrangement of MFs (parallel
Bundle)
In cell body, typical cell cortex arrangement of
MFs to maintain structure of cell
o Criss-cross arrangement of MFs (Gel)
In trailing edge, cell is releasing adhesion sites
and propelling cell forward, and thick fibers form
o Stress fibers
o Antiparallel arrangement of MFs (Contractile
Bundles)
Pulling cell while adhesion sites are still
attached and when cell releases cell
adhesion sites, cell coils forward

46.
List and describe the various levels of organization in a
multicellular organism.
Cell Systems in Multicellular Organisms
- cells interact on different levels
- cells interact with eachother to form tissues
- tissues interact to form organs
- organs interact to form organ systems
- organ systems function together to make sure organism
functions in homeostatic capacity
47.

Define a tissue.

Tissue: an organized collection of cells

48.
List and describe the structure and function(s) of the four
major tissue types found in animals.
4 major tissue types in animals:
1. Epithelial
Cell layer that lines cavities and surfaces
of body structures
Lies on top of connective tissue
Connected to those tissues by basement
membrane (basal lamina)

Example: lining of small intestine, tracheal


lining
2. Muscle
Contractile tissue
Produce force and motion
Skeletal, cardiac, smooth
3. Nervous
Main component of nervous system
Main function is communication and
coordination
Brain, spinal cord, nerves
Sensory input, control of muscles and glands,
homeostasis, mental activity
4. Connective
Provides structure and support to
organism
Enormously varied
Tendons, bone, adipose, blood

49.
Describe the structure and function of endothelial tissue.
Endothelial Tissue:
- specific type of tissue found in lining of
cardiovascular system (lining of BV)
- considered a type of epithelial tissue
o even though they have different embryonic origins
- seen in vasodilation
o sent signals to overlaying smooth muscle cell to
relax muscle and to cause BV to dilate
50.

Explain the basic events necessary for tissue formation.

Tissue Formation
1. Cell Recognition
2. Cell Adhesion
Either direct attachment through
transmembrane adhesion proteins
Or through communication of Extracellular
matrix
Like bone (molecules outside the cell)
Important during development and
embryogenesis
Has to start immediately for cells
undergoing embryogenesis

Have to have immediate


expression of proteins or secretion
of extracellular matrix molecules
In order for development to occur
properly and cells attach to form
larger organisms
3. Continued Communication
in order to properly coordinate functions
Describe ways that cells can adhere to one another.

51.

52.

Either direct attachment through


transmembrane adhesion proteins , which
allow interaction of cells
Or through communication of Extracellular
matrix
Like bone (molecules outside the cell)

Why is cell adhesion important?

Important during development and


embryogenesis
Has to start immediately for cells
undergoing embryogenesis
Have to have immediate
expression of proteins or secretion
of extracellular matrix molecules
In order for development to occur
properly and cells attach to form
larger organisms

53.
Differentiate between homophilic and heterophilic
interactions of cell adhesion proteins.
Homophilic Interactions: cell adhesion protein interacts
with identical protein
Heterophilic Interactions: interacts with different
proteins
Cell Adhesion Proteins: all transmembrane proteins

54.
List and describe the four categories of cell adhesion
proteins discussed in class (CAMs, cadherins, selectins,
integrins). Explain how each type of adhesion protein mediates
cell adhesion
Cell Adhesion Proteins
- all are transmembrane proteins
- all have some type of extracellular domain
o interacts with:
another cell adhesion protein
or an extracellular matrix component
- all have cytoplasmic domain
o interacts directly with cytoskeletal elements
o interacts indirectly through adaptor proteins
o may facilitate signaling pathway
1. CAMs (Cell Adhesion Molecules)
Have extracellular domains with loops
Loops generated by disulfide bridges
Must have cysteine residues to generate
disulfide bridges
Interact in a homophilic manner
2. Cadherins (Calcium-dependent adhesion
molecules)
In extracellular domain, have regions that bind
calcium
Helps generate structure
Transmembrane domain: anchors protein to PM
Cytoplasmic domain has cytoskeletal
interaction
Interact in hemophilic manner
P-Cadherin and E-Cadherin are
inversely expressed
Shows hemophilic interaction
3. Selectins (type of lectin, or carbohydrate
binding protein)
Have extracellular domains with different
carbohydrate binding domains
Bind to carbohydrates expressed in
another cell type
Cytoplasmic domain interacts with
cytoskeleton
Different types:

L-selectin: expressed on leukocyte


(WBC)
E-selectin: expressed on endothelial
cell
P selectin: expressed on platelets
and endothelial cells
Platelets was where it was first
identified
Bind to carbohydrates
Interact with glycoproteins : proteins
with carbohydrates (sugar group)
bound to it
interact with proteins in heterophilic manner
4. Integrins
Expressed in cells that will interact with
eachother through the extracellular
matrix
Integrins are the main way cells adhere to the
ECM
Can also interact with other cell adhesion
proteins
Form dimer molecules
Have binding domain that interacts with
ECM
Have transmembrane segment
Have cytoplasmic segment that interacts
with cytoskeleton
Interacts with proteins in heterophilic
manner
Can interact with ICAM: intercellular
adhesion molecules

55.
Describe the evidence suggesting that cadherins are
important for preferential cell aggregation during
embryogenesis.
Important during vertebrate embryogenesis
- Helps segregate cells during tissue
formation/differentiation (when expressed in embryonic
cells)
- Cells expressing cadherin preferentially bind in
homophilic manner
o P-Cadherin and E-Cadherin are inversely
expressed
Shows hemophilic interaction

When a mixture of cells are transfected with different


cadherins
o Over time, will sort into masses of same cadherin
cell types
o All cells expressing one type of cadherin interact
and segregate away from cells expressing other
type of cadherin

56.
Outline and describe the events involved in leukocyte
extravasation (e.g. What allows a WBC to roll along the
endothelial cells? What stops it from rolling? Why is it leaving
the blood vessel...these are just a few examples of questions).
Leukocyte Endothelial Cell Adhesions:
- leukocytes roll along endothelial cells
o cells adhere to one another
o mediated by selectins
leukocyte: L-selectin
Endothelial Cells: E-selectin and P-selectin
o Signals received by leukocyte (from endothelial
cells)
Cause expression and activation of
integrins
o Signals received by endothelial cells
Endothelila cells express ICAM
o Rolling of leukocyte along endothelial cells
STOPS when integrins on leukocyte adhere
to ICAM on endothelial cells
o Further signaling events cause cell cortex of
leukocyte to rearrange
Action of MFs
Allow WBC to leave BV
o WBC crawls in the direction of stimulus
Like a site of inflammation