Cytoskeleton

Question 1

What structure is in red? What structure is in green?

Answer 1

Red = F-actin

Green = microtubules

Question 2

Complete the attached table.

Answer 2

GTP alpha/beta tubulin heterodimer

+ end

Yes

GTPase

Kinesins, Dyneins

MAPs

Stiff, hollow center

25nm diameter

All eukaryotes

Support, intracellular transport, cell organization

Question 3

Complete the information in the table for intermediate filaments

Answer 3

Various globular proteins

Internal

No

None

None

Plakins

Tough, ropelike

10nm

Animals

Structural support

Question 4

Complete the following table for actin.

Answer 4

ATP-actin monomers

+ end

Yes

ATPase

Myosins

Actin-binding proteins

8nm

All eukaryotes

Motility, contractility

Question 5

What are the 4 key features of the cytoskeleton?

Answer 5

1. Structure and support
2. Intracellular transport
3. Contractility and motility
4. Spatial organization

Question 6

Describe the structure of an individual microtubule

Answer 6

• A heterodimer of alpha and beta subunits
• Alpha and beta subunits both form alpha helices so they come together as a coiled coil to form a microtubule
• In a cross-section of microtubule, there are 13 subunits visible
• Globular protein

Question 7

Describe the dynamic nature of a microtubule.

Answer 7

New subunits can be added to the positive end such that it grows some, becomes unsable and degrades some, and then extends some more

Question 8

What causes dynamic instability in microtubules?

Answer 8

Once new alpha/beta subunits are added to the + end of microtubule, they hydrolyze their GTP to GDP + Pi, releasing energy. Some of this energy does into deforming the tubulin subunit, causing it to be most stable in a slightly curved state. This creates excess pressure on the microtubule as new subunits are added up stream of this happening and the microtubule is made unstable and collapses spontaneously.

Question 9

Describe the structure of a centriole.

Answer 9

A centriole is made up of 9 clusters of microtubules. Each cluster consists of an A, B, and C tubule. The A tubule (closest to center) has 13 subunits, while B and C each have 10 subunits. They are connected to the one another by proteins that form a web like structure inside the centriole.

Question 10

What is the centrosome?

Answer 10

The microtubule organizing center - all microtubules have their (-) ends attached to the centrosome. The centrosome consists of 2 centrioles.

Question 11

What is the purpose of gamma tubulin?

Answer 11

Gamma tubulin is found only near the (-) end of the microtubule. It is added to proteins that anchor the microtubule to the MTOC and it provides a place to initiate polymerization of the microtubule using alpha and beta tubulin.

Question 12

Kinesins

• Where do they transport cargo?
• Is their motion processive (never detaches from microtubule)?
• What do they use to power their motion?
• What is the purpose of the heavy chain?
• What is the purpose of the light chain?

Answer 12

• Toward (+) end of microtubule
• Yes
• GTP
• Catalytic core, binds to microtubule and provides motion
• Confers specifiticy for cargo that binds to kinesin

Question 13

Describe how Kinesins work.

Answer 13

Head has GDP bound. Head recognizes microtubule via brownian motion (random). The head binds to the microtubule and releases GPD and binds GTP. This causes it to anchor to the microtubule. This gives the other head the chance to attach to the microtubule also. Once both heads bound, one hydrolyzes GTP to GDP + Pi, head dissociates and the released energy is used to catapult the head forward along the microtubule. Process repeats.

Question 14

Dynein

• In what direction do they transport cargo?
• Do they require energy?
• Describe their mechanism of movement.

Answer 14

• Toward the (-) end
• Yes - they are also GTPases
• Their mechanism is the same as Kinesin

Question 15

Are cellular contents always carried by only 1 kinesin or dynein?

Answer 15

No - some transport cargo require multiple kinesins and dyneins

Question 16

Cilia

• What regulates the “beating” of cilia?
• Describe the action of motile (secondary) cilia?

Answer 16

• Ca2+
• There is a power stroke and a recovery stroke. During power stroke, cilia propel things like particles (respiratory) and food (intestines) forward and then flop back to their starting position in the recovery stroke.

Question 17

Label this image.

Answer 17

Question 18

Why are the dynein arms so important in secondary cilia?

Answer 18

They are bound to the microtubule duplexes and they provide the force for movement of the cilia (GTPase activity) by causing the microtubule duplexes to slide past one another (attached between adjacent A and B tubules). Doing so causes the cilia to curve b/c the are fixed at the bottom by the basal body so they curve in a direction such that, in the attached image for example, the cilia would “beat” from 12 to 6 if this were a clock.

Question 19

Label this image.

Answer 19

A - Innner dynein arm

B - Outer dynein arm

C - Interdoublet bridge

D - Central microtubule

E - Radial Spoke

F - B Tubule

G - Outer Doublet

H - A tubule

I - Central Sheath

J - Plasma membrane

Question 20

Kartagener syndrome is a disorder of motile cilia. It is also known as primary cilia dyskinesia, where the primary does not mean “primary cilia”, but rather that the cilia dysfunction is the primary cause of disease.

• What structure in the cilia is altered that leads to this dysfunction?
• What symptoms would you expect patients with this disease to have?

Answer 20

• Autosomal recessive inheritance of mutant allele that encode for components of outer dynein arm of cilia
• Lung infections, chronic sinusitis

Question 21

Describe how intermediate filaments are formed.

Answer 21

Single IF monomer subunit (alpha helical structure) associates with another subunit to form dimer. 2 dimers associate to form tetramer. Tetramers (fibrils) associate to form fiber.

Question 22

Do intermediate filaments have a + or - end?

Answer 22

No

Question 23

What are 4 important types of intermediate filaments we should know?

Answer 23

Keratin

Glial fibrillary acidic protein

Neurofilament proteins

Lamin proteins

Question 24

What do lamin proteins do?

Answer 24

They give structure and support to the nucleus and it is believed that they help organize the chromatin in the nucleus so that gene rich chromatin is organized in a way that is conducive for transcription and gene-poor chromatin is buried inside the nucleus away from transcription machinery.

Question 26

**Actin** * What are the monomers of actin? * How do the monomers associate with one another to form actin?

Answer 26

* Alpha (contracile motion in muscle), beta (cytoskeleton) and gamma * Actin is made of **A SINGLE TYPE OF MONOMER** that bind to one another and twist around eachother

Question 27

**Beta Actin** This type of actin is found in the cytoskeleton and is able to polymerize to push against plasma membrane. This pushing force places an equal and opposite compression force on the actin fibers. Actin is not good in compression, so how can this be possible?

Answer 27

When many actin filaments bundle together and associate with one another they are able to acheive some compressive strength

Question 28

Describe how actin fibers grow.

Answer 28

Actin has a (+) and (-) end. Unlike microtubules, actin can polyermize and degrade on both ends. However the rate of polymerization is much faster at the + end and slower at the - end. The rate of degradation is faster at the - end and slower at the + end.

Question 29

**Myosin** * What is bound to the myosin heads? * Compare the mechanism of myosin movement to that of kinesin and dynein.

Answer 29

* ATP * Although the overall mechanism is similar, myosin uses ATP whereas kinesin and dynein use GTP for motion. Also, myosin is not processive, meaning it does not stay bound to the actin during motion.

Question 30

Explain this figure.

Answer 30

Step 1 - ATP binds to myosin head, myosin head dissociates from actin Step 2 - ATP splits into ADP + Pi, both remain bound to myosin, no energy release yet Step 3 - Change in ATP allows myosin head to bind to actin Step 4 - Release Pi --\> provides energy for powerstroke to drag actin Step 5 - release ADP

Question 31

Explain this figure.

Answer 31

1. Profilin is bound to ATP actin, inhibits nucleation 2/3. Proteins (Arp2/3) form nucleation complex allowing for elongation of filament 4. Beta actin monomers join to one another to elgonate the filament 5. Elongating (+) end of actin pushes membrane 6. Other proteins can cap the growing actin filament to prevent further growth of the + end 7. Once actin monomers bind in filament, they hydrolyze ATP to yield bound ADP and a free Pi. ADP-actin has a lower affinity for monomers than ATP-actin, so (-) end begins to depolymerize. 8. Cofilin severs ADP-actin filament 10. Profilin facilitates exchange of ADP for ATP to reproduce ATP actin

Question 32

**Plectin** * What is plectin? * What is its role in the cell?

Answer 32

* A cytoskeletal crosslinker and signaling scaffold that affects mechanical as well as dynamic properties of cytoskeleton * It forms networks and bridges between different cellular structures