Question 1

The eukaryotic cytoskeleton is made from filamentous proteins. The cytoskeleton provides
mechanical support to the cytoplasmic constituents. Cytoskeleton consists of three major
elements (Nature Education, 2014). Intermediate filaments are there to provide mechanical
strength and resistance to shear stress. Microtubules on the other hand determine the positions
of membrane-enclosed organelles and direct intracellular transport. Actin filaments determine
the shape of the cell's surface and are necessary for whole-cell locomotion. There is also a set
of accessory proteins called the motor proteins that either move organelles along the filaments
or move the filaments themselves (Alberts, et al., 2002).

Microtubules (the largest measuring 25nm)

Protein found: Tubulin which is a heteredimer of α-tubulin and β-tublin.

Forms of microtubule Filament: D and T form.

Microtubules are found in a star-like cytoplasmic array protruding from the center of an
interphase cell. They can speedily reorganize themselves to form a bipolar mitotic spindle
during cell division. They also form motile whips called cilia and flagella on the surface of the
cell. They can be tightly aligned bundles that serve as tracks for the transport of materials down
long neuronal axons (Alberts et al., 2002). In short, microtubules carry cargoes along the length
of nerve axons (British Society of Cell Biology, n.d).

In plants, microtubule re-aligns as they respond to chemical stimulation. Those near the root tip
are found at right angles to the direction of the growth of the root. If you move further back from
the root tip the microtubules rotate through a right angle and align themselves to become
parallel to the direction of root growth. Depolymerisation of microtubules causes cellulose to be
laid down in a disorganised way (British Society of Cell Biology, n.d). The root tip becomes a
mass of such cells and even though they expand they cannot elongate and the tissue grows in a
distorted manner. Chemicals such as colchicine inhibit polymerisation and hence stop the
production of microtubules. Some synthetic weedkillers produces microtubule depolymerisation
(British Society of Cell Biology, n.d).

How they function to facilitate movement

The speedy assembly and disassembly of microtubules can actually move vesicles and cell
organelles. Microtubules can locate them within the cell, hold them in position and re-locate
them. This happens for example during mitosis (British Society of Cell Biology, n.d).
Microtubules determine the positions of membrane-enclosed organelles and direct intracellular
transport (Alberts, et al, 2002). The heterodimers are linked together to form long strands of 13
protofilaments which are dynamic. They have a plus and minus end with the plus end
continuously polymerizing and the minus end depolymerizing. The plus end growly rapidly. The
other end is called the minus end and this end forms structures called microtubule organising
centers (MTOC). The main MTOC in a cell is known as the centrosome and this is located next
to the nucleus. Each of the tubulin subunits has a GTP/GDP binding site. The GTP bound to the
α-tubulin is ensnared and is by no means hydrolysed. The GTP bound to the β-tubulin can be
hydrolysed, which has a major impact on filament dynamics. The GTP in free tubulin is
hydrolysed very slowly but hydrolysis is speeded up once it is integrated into the microtubule
(Alberts, et al, 2002).

The T-Form is GTP bound and the D-Form is GDP bound. The D-form of the microtubule comes
apart more handily than the T-form under physiological conditions so the GTP-tubulin tends to
polymerise and the GDP-tubulin depolymerise. This means that T-form ends grow whereas D-
form ends shrink. The ratio of the two is controlled by the rate of hydrolysis of GTP.
Microtubule internal organisation and transport

Most cells are polarised and have one end that is structurally and/or functionally different from
the other end. A nerve cell is an example of a highly polarised cell. A cell’s polarity is in part
determined by the cytoskeleton and how it positions the organelles and other structures within
the cell. In a nerve cell all the microtubules in the axon face in the same direction, with the plus
ends all facing away from the cell body. In this way the polarity of the microtubules guides
components and other cellular material to the correct parts of the cell and can thus ensure that
specific compounds are brought to where they are needed. Microtubule motor proteins, another
type of microtubule accessory protein, carry vesicles, organelles and other cellular materials
along the microtubules.

Examples are the kinesins, myosin and dyneins which are known as movers of the cell.
Kinesins characteristically shift from the minus end of the microtubule to the plus carrying cargo
like organelles and vesicles. They tend to transport cargoes away from the area of the nucleus.
Kinesin-generated movements proceed by the same mechanism as myosin.

Dyneins are vital in sliding microtubules relative to one other during the beating of cilia and
flagella on the surfaces of some eukaryotic cells. The dyneins also have the ability to move
along microtubules but tend to move in the opposite direction to the kinesins, from the plus end
towards the minus end. There are two main groups of dynein: the cytoplasmic dyneins and the
ciliary dyneins (also called axonemal dyneins). The cytoplasmic dyneins are involved in
transporting membrane-bound organelles, chromosomes and mRNA towards the centrosome.
The ciliary dyneins are involved in driving the beating of cilia and flagella within the cell. Dynein
models travel toward the ‘minus’ end and hence transport cargoes along a microtubule to the
nucleus.

Micro Filaments (smallest measuring about 6nm)

Protein found in abundance: Actin

Actin filaments form many types of cell-surface projections. Some of these are dynamic
structures like the lamellipodia and filopodia. These are used by the cell to travel around territory
and pull themselves around. Others are stable structures such as the regular bundles of
stereocilia on the surface of hair cells in the inner ear, which tilt as rigid rods in response to
sound (Alberts, et al., 2002). Inside cells, actin filaments can also form either transient or stable
structures. The contractile ring, for example, assembles transiently to divide cells in two during
cytokinesis. The more stable arrays allow cells to brace themselves against an underlying
substratum and enable muscle to contract (Alberts, et al 2002).

Actin is essential for cell stability and morphogenesis. It is involved in many crucial processes,
such as cell division, endocytosis, and cell migration (Ibidi, n.d). Actin is available in two forms:
monomeric, globular G-actin and polymeric, filamentous F-actin. The monomeric G-actin is able
to polymerize. It is the polymerization of G-action that produces F-actin polymer filaments.
These microfilaments are an essential part of the cytoskeleton. They construct higher order
structures in cells like stress fibers, lamellipodia, and filopodia (Ibidi, n.d).
Intermediate Filaments (medium sized measuring 10nm).

Intermediate filaments line the inner face of the nuclear envelope. They then form a protective
cage for the cell's DNA. In the cytosol, they are wound around into strong cables that firmly hold
epithelial cell sheets together, help neuronal cells to extend long and robust axons, or allow us
to form tough appendages such as hair and fingernails (Alberts et al , 2002).

Intermediate filaments are made up of polymerized true fibrous subunit proteins. This is in sharp
contrast to the chains of globular subunits that make up microfilaments and microtubules. They
are cell/tissue-type specific. There are individual families of filament proteins for muscle
(desmins), neurons (neurofilaments), mesenchymally-derived cells like fibroblasts (vimentin),
epithelia (keratins), and glial cells (glial fibrillary acidic protein - GFAP). There are also a group
of three intermediate filament proteins that function in all cells as support for the nuclear
envelope and the distribution of chromatin. These are called lamins A, B, & C (The University of
Kansas, n.d).

Intermediate filaments are made up of long fibrous subunits of protein that are twined together
like the treads of a rope. They do not bind nucleotides and their congregation occurs
independent of the hydrolysis of ATP or GTP. Each subunit of an intermediate filament is an
elongated fibrous protein that has an N-terminal head and a C-terminal tail. Two subunits
associate together to form a stable dimer. Their C-terminal and N-terminal ends pointing in the
same direction. These dimers then relate to form a staggered tetramer, with both dimers
pointing in opposite directions. The tetramers then pack together, end to end, to form an
intermediate filament. Because the tetramer subunits have ends that are the same, the overall
intermediate filament structure does not have directionality.

Question 2

Kartagener syndrome

Kartagener syndrome is characterized by mirror-image reversal of internal organs including the

heart, liver, spleen and intestine. It has varying signs and symptoms which include neonatal
respiratory distress, frequent respiratory infections that can lead to severe lung damage, chronic
nasal congestion, and frequent sinus infections, recurrent middle ear infections, particularly in
early childhood, hearing loss, hydrocephalus and infertility. The cause is a mutation in a number
of genes that are inherited in an autosomal recessive manner. These genes encode proteins
that are important to the structure and function of cilia. Cilia are tiny, hair-like structures that are
found on the surface of cells in various parts of the body such as the lining of the airway, the
reproductive system, and other organs. The coordinated movement of cilia in wave-like motions
is significant to the standard functioning of certain organs and tissues. Mutations in these genes
cause the cilia to be either immotile or dysmotile. This is what leads to the many signs and
symptoms of Kartagener syndrome (NIH, 2015). There is no known cure for Kartagener
syndrome. Treatment is usually by treating signs and which includes airway clearance therapy
and antibiotics (NIH, 2015).

How protein defects results in disease symptoms (At molecular level).

Roughly all persons with Kartagener syndrome have ultrastructural deformities affecting
protein(s) in the outer and/or inner dynein arms. This gives cilia their motility, with roughly 38%
of these defects caused by mutations on two genes, DNAI1 and DNAH5. These genes code for
proteins found in the ciliary outer dynein arm (NIH, 2015).

Lou Gehrig's disease

Lou Gehrig's disease is a progressive motor neuron disease which results in problems with
muscle control and movement. Near the beginning, symptoms include muscle twitching,
cramping, stiffness, or weakness, slurred speech, and/or difficulty chewing or swallowing. With
time, patients become weaker and end up dependent on wheel chair. Death is eventual
resulting from respiratory failure within 2 to 10 years after the symptoms commence (NIH,
2018).
Majority of patients with Lou Gehrig's disease have a sporadic form of Lou Gehrig's disease.
The main cause is the interaction between genetic and environmental factors. About 10% of the
patients with Lou Gehrig’s disease have at least one relative with the disease and are said to
have a familial form of the disease (NIH, 2018). Familial Lou Gehrig’s disease is a result of
pathogenic variants in any one of several genes and the pattern of inheritance varies depending
on the gene involved. Diagnosis of Lou Gehrig's disease is based on symptoms which include
anxiety, weakness, muscle spasm, paralysis, muscle atrophy among a range of symptoms (NIH.
2018).
Nearly all familial cases are inherited in an autosomal dominant course. Less regularly, Lou
Gehrig’s disease is present at birth in an autosomal recessive manner with both copies of the
disease-causing gene having a mutation. In rare cases, Lou Gehrig’s disease is inherited in an
X-linked dominant manner. This occurs when the disease-causing gene is located on the X
chromosome. Females have 2 X chromosomes and having a mutation in one X chromosome is
still adequate to cause the condition. More often than not, males with an X-linked dominant form
of Lou Gehrig’s disease have more severe symptoms than females with the same form (NIH,
2018).

How protein defects results in Lou Gehrig’s disease symptoms.

Genes are the blueprint for making proteins and any change to the gene that code for a
particular protein can be fatal. For example, loss of function of the angiogenin protein due to
mutation in the gene is thought to be linked to some cases of Lou Gehrig’s disease. A gain of a
new and toxic function of the superoxide dismutase 1 (SOD1) protein is probably how mutation
of the SOD1 gene causes Lou Gehrig’s disease (NIH, 2018).

SOD1 is abundant enzyme within cells. It serves to keep cells safe from metabolic waste that
can do damage if not rendered harmless. It is clear that disease is not due to lack of function of
the protein, since deleting the gene in animal models does not cause Lou Gehrig’s disease.
Instead, it appears to take on some new toxic function, possibly related to an increase in the
tendency of mutant SOD1 molecules to aggregate and form clumps in motor neurons and
astrocytes (ALS Association, n.d).

Another protein that is probable is Ubiquilin-2 whose normal function is to aid degrade damaged
or defective proteins in the cell. It is likely that mutations in the gene interfere with this function
and may lead to accumulation of harmful material within the cell (ALS Association, n.d).

KIF5A is a microtubule-based motor protein involved in transport of protein cargo in the cell.
Kinesins play an important role in transport along a motor neuron axon (axonal transport), which
is crucial to motor neuron health. The mutations identified in KIF5A may cause disease by
interrupting axonal transport (ALS Association, n.d).

A mutation may also be damaging due to its effect on RNA. For instance, the C9ORF72 gene
may cause Lou Gehrig’s disease due to accumulations of RNA that occur when the gene is
mutated. FUS and TDP43 mutations may impair the normal processing of RNA from a wide
variety of genes, leading to Lou Gehrig’s disease (NIH, 2018).

References
Alberts, B et al (2002). Molecular Biology of the Cell. 4th edition. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK26862/ (5 April 2019).

ALS Association (n.d). Genetics. Available from: http://www.alsa.org/research/focus-

areas/genetics/ (5 April 2019).

British Society of Cell Biology (n.d). Cytoskeleton – the movers and shapers in the cell.
Available from: https://bscb.org/learning-resources/softcell-e-learning/cytoskeleton-the-movers-
and-shapers-in-the-cell/ (5 April 2019).

Ibidi (n.d). F-Actin—A Crucial Protein for Cellular Function and Motility. Available from:
https://ibidi.com/content/80-f-actin-a-crucial-protein (5 April 2019).

Nature Education (2014). Microtubules and Filaments. Available from:

https://www.nature.com/scitable/topicpage/microtubules-and-filaments-14052932 (5 April
2019).

NIH (2018) Amyotrophic lateral sclerosis. Available from:

https://rarediseases.info.nih.gov/diseases/5786/amyotrophic-lateral-sclerosis ( 4 April 2019).

NIH (2015). Kartagener syndrome. Available from:

https://rarediseases.info.nih.gov/diseases/6815/kartagener-syndrome (4 April 2019).

The University of Kansas (n.d). Introduction to the intermediate filaments. Available from:
http://classes.kumc.edu/som/cellbiology/cytoskeleton/intermediate/index.html (5 April 2019).