Cytoskeleton

Question 1

What are three diseases resulting from defects in intermediate filaments? What is the reason for the
corresponding defects? (2) What linker protein can mimic some of these diseases and why?

Answer 1

1. ALS (Lou Gehrig’s disease) - abnormal accumulation of NFs in motor neurons
2. Epidermolysis bullosa simplex - mutant form of keratin
3. Progeria - defects in nuclear lamin

(2) Plectin

Question 2

How does the nuclear cytoskeleton interact with the cytoplasmic cytoskeleton? Hint: what linker proteins are involved? What factors influence the stability of MT and actin filaments?

Answer 2

KASH-domain - interacts with MTs, actin, and plectin (outer membrane)

SUN-domain - interacts with lamin and chromatin (inner membrane)

Question 3

What is a centrosome? Describe its behavior during interphase and mitosis. (3) What is a basal body?

Answer 3

(1)
- Close to nucleus in nonmitotic cells
- Consists of a pair of centrioles
- Where minus end of MT is embedded (plus end points outward)

(2)
Interphase: microtubule organizing centers (MTOCs)

Mitosis: organizes mitotic spindle

(3) Found at base of cilia; serves as organizing center for cilia

Question 4

How are MTs and IFs similar/different? Consider dynamics, nature of the subunits and accessory protein
interactions. Hint: how do respective motor proteins function?

Answer 4

MTs
- highly dynamic
- polarity: a-tubulin (minus end), b-tubulin (plus end)

IFs:
- subunits do not have polarity (same on both sides)
- Monomer has polarity
- Coiled-coiled dimer - has polarity
- Dimer + dimer –> tetramer (no polarity)

Both:

Question 5

(1) What happens during treadmilling? (2) What conditions keep AFs from altering their size during
treadmilling?

Answer 5

(1)

Actin monomers add to plus end at a rate faster than bound ATP can be hydrolyzed (plus end grows)

ATP is hydrolyzed faster than new monomers can be added (minus end)

Filament loses subunits from minus end at same time as it adds them to plus end

Filament undergoes a net addition of subunits at plus end while simultaneously losing subunits from minus end

(2)

Question 6

(1) Compare the structure and stability of cytoplasmic MTs with the complex MT structure found in cilia/
flagella, how do the latter function? (2) What accounts for the symptoms in Kartagener’s syndrome?

Answer 6

(1)
Cytoplasmic MT
- highly dynamic

Cilia/flagella
- Very stable
- No polymerization/depolymerization
- Stay polymerized

(2)
Kartagener’s syndrome:
- Increased susceptibility to bronchial infections (cilia unable to clear bacteria and debris from lungs)

• Problems with fertility
• Men: defects in ciliary dynein disables flagella that allow sperm to swim (immobile sperm)

Question 7

How can drugs affect the dynamics of MTs and AFs? How does this affect cell behavior?

Answer 7

MTs:
- Colchicine - drug that causes microtubule disassembly; ER and Golgi change location

Taxol - binds tightly to MTs and prevents them from losing subunits

Actin:
- Phalloidin - binds and stabilizes filaments against depolymerization

• Cytochalasin - caps filament plus ends, preventing polymerization there
• Latrunculin - binds actin monomers and prevents their polymerization

Question 8

What is the role (if any) of GTP and ATP in MT and AF dynamics and motor function?

Answer 8

GTP: involved in dynamic instability (MTs)

ATP:
- Involved in treadmilling (AFs)

• Involved in motor proteins (kinesin, dynein, myosin)

Question 9

Compare and contrast MT and actin structure/polymerization. Which is more flexible? Rigid?

Answer 9

MT:
- thickest (more rigid)
- dynamic instability (GTP)

Actin:
- thinnest (more flexible)
- treadmilling (ATP)

Both:
- Both have polarity (plus and minus end)
- Both polymerize
- Can be dynamic and stable
- Polymerization is faster on plus end than minus end

Question 10

What are lamellipodia and filopodia? How do they form? How is actin-based cell movement promoted?
Hint: what are the roles of formin, ARP and capping proteins? What is the role of actin-myosin contractions

Answer 10

Lamellipodia - help migrate (broad); grabs focal adhesions (drags cell forward)

Filopodia - wedge themselves between cells (spikes)

Question 11

What is the difference between myosin I and II?

Answer 11

Myosin I:
- present in all cell types
- Single head domain and tail
- nonmuscle myosin

Myosin II:
- two-headed myosin motor
- specialized form in muscle

Question 12

What subunits make up the three protein filaments (intermediate, microtubules, actin)?

Answer 12

Intermediate - fibrous proteins (provides cells with mechanical strength)

Microtubules - globular tubulin heterodimer (alpha-tubulin and beta-tubulin); organize cytoplasm; promotes cell movement through propulsion

Actin - globular actin (supports cell surface; promotes cell crawling)

Question 13

Describe the structure
of microtubules.

Answer 13

• Hollow cyndrical structure
• 13 parallel protofilaments (composed of aB-tubulin heterodimers)

Question 14

Describe intermediate filaments.

Answer 14

• Function: enable cells to withstand mechanical stress
• Toughest and most durable cytoskeletal filament

Question 15

What are desmosomes?

Answer 15

Cell-cell junctions that anchor intermediate filaments to plasma membrane

Question 16

Many of the intermediate strands are twisted together to provide tensile strength. What is tensile strength?

Answer 16

Ability to withstand tension without breaking

Question 17

What are the four classes of intermediate filaments. Which are cytoplasmic? Which are nuclear? Where are they found?

Answer 17

Cytoplasmic:
1. Keratin filaments - epithelial cells (e.g., epidermis)

2. Vimentin and vimentin-related filaments - connective-tissue cells, muscle cells, and glial cells
3. Neurofilaments - nerve cells (axons)

Nuclear:
4. Nuclear lamins - in all animal cells

Question 18

What is the difference between hemidesmosomes and desmosomes?

Answer 18

Hemidesmosomes - cell-matrix contact

Desmosomes - cell-cell contact

Question 19

(1) Describe the nuclear lamina. (2) How is the disassembly and reassembly of the nuclear lamina controlled?

Answer 19

(1)
- meshwork of intermediate filaments that supports nuclear envelope

• constructed from lamins

(2)
- Phosphorylation of lamins (breakdown)
- Dephosphorylation of lamins (assembly)

Question 20

What do linker proteins do?

Answer 20

• stabilize intermediate filaments
• connect cytoplasmic cytosekeleton to nuclear lamin and to chromatin

Question 21

What does plectin do?

Answer 21

Aids in bundling of intermediate filaments and links them to other cytoskeletal protein networks

Question 22

Accessory proteins such as plectin can cross-link intermediate filaments and connect them to what structures?

Answer 22

• Microtubules
• Actin filaments
• Adhesive structure in desmosomes

Question 23

What are examples of stable structures made from microtubules?

Answer 23

Cilia and flagella

Question 24

GTP hydrolysis occurs only within which subunit of the tubulin dimer?

Answer 24

Beta-tubulin

Question 25

What is the role of capping proteins?

Answer 25

Prevent MT growing out from centrosome from depolymerizing

Question 26

(1) What is dynamic instability? (2) How does it work?

Answer 26

(1) Switching between polymerization and depolymerization (growing and shrinking) (2) - Stems from intrinsic GTPase activity of tubulin dimers - End of rapdily growing microtubule is composed entirely of GTP-tubulin dimers (forms GTP cap)

Question 27

What causes MT to grow? To shrink?

Answer 27

Grow - Addition of GTP proceeds faster than GTP hydrolysis by dimers Shrink - Hydrolysis is faster than addition of new GTP-tubulin dimers

Question 28

What are microtubule-associated proteins?

Answer 28

Proteins that bind to microtubules

Question 29

Explain how cells can adjust microtubule instability depending on needs.

Answer 29

* MTs in differentiated cells tend to be relatively stable (e.g., nerve cells) * MTs are more dynamic in mitotic cells (e.g., stem cells)

Question 30

In most animal cells, for example, the tubules of the ________ reach almost to the edge of the cell, whereas the __________ is located in the cell interior, near the centrosome

Answer 30

ER; Golgi

Question 31

(1) What happens to the respective positions of these organelles (ER and Golgi) when cells are treated with cholchicine? (2) What happens when cholchicine is removed?

Answer 31

(1) ER and the Golgi apparatus change their location (2) organelles return to their original positions

Question 32

Compare and contrast Kinesin and dyneins.

Answer 32

Kinesins: - Travel towards plus end - Pull ER outward along microtubules Dynein: - Travel toward mins end - Pull Golgi along microtubules toward nucleus Both: - dimers made of two globular ATP- binding heads and a tail

Question 33

Compare the functions of cilia and flagella.

Answer 33

Cilia: - Single celled organisms - usually for motility (protists, like paramecium) - Multicellular systems - move extracellular fluid Flagella: - Singular - Used to propel entire cells (e.g., sperm) - Has very rapid whipping movement - Shares similar internal structure with cilia

Question 34

What is the 9 + 2 array?

Answer 34

MT arrays found in a cilium/flagellum consisting of 9 outer doublets and two central paired MTs (enclosed in an inner sheath)

Question 35

What accessory protein is important for movement of flagella?

Answer 35

Ciliary dynein (generates bending motion)

Question 36

(1) What are actin filaments? (2) Where are they found?

Answer 36

(1) Polymers of actin (2) Cell cortex

Question 37

Examples

Answer 37

- Stiff stable actin filaments are in microvilli (increases surface area) * NOT CILIA (microtubules) - Contractile bundles of AFs in cytoplasm (cell shape changes) - AFs form dynamic protrusions at leading edge of crawling cells * Chemotaxis * Lamellipodium - help migrate (broad); grabs focal adhesions (drags cell forward) * Fliopodium - wedge themselves between cells (spikes) - Important in cytokinesis * AFs form a contractile ring during cell division * Pershing model

Question 38

List and describe some actin-binding proteins.

Answer 38

Formin and ARP (actin-related protein) complex - promote actin polymerization Profilin - bind to actin monomers prevent them from polymerizing Capping protein (plus-end blocking) - prevents assembly and disassembly at plus end

Question 39

The action of profilin is similar to what drug?

Answer 39

Lactrunculin

Question 40

The action of capping (plus-end blocking) proteins is similar to what drug?

Answer 40

Cytochalasin

Question 41

What are general actin dynamics associated with cell crawling?

Answer 41

1. Cell send out protrusions at leading edge 2. Protrusions adhere to surface over which cell is crawling (e.g., interacting with focal contact) 3. Contraction at the rear of the cell then draws the body of the cell forward

Question 42

What causes the contraction in cell crawling?

Answer 42

Actin-myosin interaction

Question 43

What two structures are located at the cell's leading edge and promote cell crawling?

Answer 43

1. Lamellipodia 2. Filopodia

Question 44

Explain how actin-binding proteins influence the type of protrusions formed at the leading edge.

Answer 44

Actin related proteins (ARPs): - promote formation of branched AFs in lamellipodia - nucleate formation of new AFs and form complexes that bind side of existing AFs Filapodial protrusions depend on formin: * attaches to the growing plus end of AFs * promotes the addition of new monomers to form straight unbranched filaments

Answer 45

The AF web undergoes continual: * assembly at the leading edge (red) (capping proteins prevent further polymerizing) + disassembly in the back (pink) = Pushing the lamellipodium forward

Question 46

What is the role of the y-tubulin?

Answer 46

Form ring structures that serve as nucleation points for one MT

Question 47

What is the commonality of taxol and colchicine function?

Answer 47

Both induce mitotic arrest