Unit 6

Question 1

Cytoskeleton

Answer 1

System of dynamic protein filaments in eukaryotic cells and gives shape, support, and allows it to organize its organelles and move them

Question 2

3 types of cytoskeleton filaments and general functions

Answer 2

1) Intermediate filaments - mechanical strength (absorbs mechanical strain) and cell shape
2) Microtubules - determine positions of organelles, vesicles transport, and spindle formation, cilia and flagella motility
3) Actin filaments - determine shape of the cell’s surface, cell locomotion, and cytokinesis

Question 3

Know general location of the three filaments

Answer 3

Draw it

Question 4

Location of microtubules and actin in cells

Answer 4

MTs - abundant around nucleus, radiating around cell. Involved in transport of organelles and vesicles

Actin - in cortex under plasma membrane and supports the cell. Determines cell shape and helps with cell movement

Question 5

Give an example of an intermediate filament and the role they play in the cell

Answer 5

Nuclear lamina is made up of intermediate filaments (lamins). They are involved in the disintegration (via phosphorylation of lamins) and re-fusion of the nuclear envelope (via dephosphorylation of lamins). It provides support for the nuclear envelope and is a source of attachment for interphase chromosomes

If they did not function properly, the nucleus could not maintain its shape

Question 6

Intermediate filament structure depends on…

Answer 6

1) twisting of coiled-coil dimers. Alpha helices wrap around each other minimizes exposure of hydrophobic amino acid side chains to aq. env.
2) lateral staggered bundling
3) twisting of bundles into a “rope”

Make sure to know specific steps and visualize it

Question 7

Why don’t motor proteins use IFs as a substrate?

Answer 7

Motor proteins cannot use IFs because they lack structural polarity. Polarity is important for the motor proteins to move in a certain direction and orient themselves. IFs have no difference in ends so motor proteins cannot orient themselves

Question 8

How do IFs differ in different tissues?

Answer 8

They are made of different types of proteins. The N and C domains on either side of the central domain differ in seq., size, and fxns. Allows for functional diversity. However, the central domain is highly conserved

This can be used for cancer diagnosis and where they originated because N and C domains are very different among different tissue cells,

Question 9

Which cell type would have the highest density of IFs in their cytoplasm? What would happen if the IFs could not function in this type of cell?

Answer 9

Skin epithelial cells - this is because IFs are prominent in the cytoplasm of cells that are subject to mechanical stress

If they could not fxn properly, the cells would not be able to hold together and the cells would rupture. This is because IFs hold epithelial sheets together

Question 10

What would happen if there was a mutation in the IFs?

Answer 10

It would affect the cell’s ability to resist externally applied force

Question 11

Desmososomes

Answer 11

They join IFs of adjacent epithelial cells

Question 12

Intermediate filaments in epthelia

Answer 12

Forms a strong network in the cytosol that links indirectly to neighboring cells

Question 13

Hemidesmosomes

Answer 13

Anchors IFs in a cell to the basal lamina

Question 14

IF assembly/structure

Answer 14

Two monomers pair and form a coiled-coil structure and the alpha helical domains look like rope. They bond via hydrophobic interactions, while the hydrophilic AA side is exposed. The two dimers interact with another coiled-coil dimer to make a tetramer, where the two dimers have their C and N are antiparallel. Then 8 strands stack together and twist like rope

Question 15

Microtubule structure

Answer 15

Hollow tubes made up of alpha and beta tubulin dimers, which both bind GTP, but a-tubulin cannot hydrolyze its GTP since it is an intrinsic part of the subunit structure, unlike b-tubulin. Microtubules have structural polarity

Question 16

Rate of addition/removal of tubulin subunits

Answer 16

• end is the slow growing end because the polymer does not readily bind b-tubulin since it is not the right conformation. + end is the fast growing end and readily binds a-tubulin. Adding new subunits leads to a conformational change that increases the binding for more subunits

Question 17

Microtubules in vitro

Answer 17

Cytoskeletal polymerization, which is nucleation, has to be initiated to create MT polymers. This is a slow process, but it is spontaneous

Question 18

Critical concentration

Answer 18

The concentration of tubulin subunits at which MT growth rate is at an eq’m, where growth rate = assemble rate. No net growth or shrinkage

Question 19

Treadmilling

Answer 19

Polymer maintains length despite subunits being added at the + end and dissociated from the - end

Question 20

How does GTP bound to b-tubulin influence MT growth? This is called dynamic instablility

Answer 20

GTP bound to the b-tubulin is added onto the + end. Once incorporated into the polymer, the GTP is hydrolyzed to GDP

Question 21

Formation of the GTP cap

Answer 21

If GTP hydrolysis happens more slowly than MT assembly, the GTP cap forms on the + end. This occurs in conditions with a high concentration of free tubulin

Question 22

Purpose of GTP cap. What would happen if [tubulin] decreases?

Answer 22

Protects the MT from shrinking. If polymerization slows, all the GTP will be converted to GDP and the GTP cap disappears. Tubulin-GDP has a lower affinity for the tubulin polymer so the MT unravels – rapid shrinking

Question 23

How does the conformation change of GTP to GDP affect the MTs?

Answer 23

GTP hydrolysis changes subunit conformation into a curved shape. This does not pack nicely and begins MT begins to unravel. This is called catastrophe. Rescue during the GDP-GTP exchange, where the b-tubulin is GTP bound again

Question 24

Increased activated tubulin (GTP-bound) subunit pool leads to…

Answer 24

Increased incorporation into MT polymer, leads to depletion of the activated tubulin subunit pool

Question 25

Decreased activated tubulin (GTP-bound) subunit pool leads to…

Answer 25

decreased incorporation into MT polymer, coupled with GTP to GDP hydrolysis and increased disassembly leads to increased free tubulin. This increases monomer pool results in exchange of GDP with GTP and build up of the activated tubulin pool and renewed incorporation into MT

Question 26

How does the concentration of activated tubulin (GTP-bound) dimers affect assembly/disassembly?

Answer 26

Above Cc, assembly exceeds disassembly. below Cc, disassembly exceeds assembly

Question 27

3 functions of MTs in the cell

Answer 27

1) Organelle positioning
2) Vesicle trafficking
3) Spindle formation

Question 28

Microtubule Organizing Centers (MTOCs)

Answer 28

These are nucleation sites (made of gamma tubulin) that microtubules grow from. The most common one is the centrosome

Question 29

What causes a MT to stop growing and to rapidly shrink/depolymerize

Answer 29

1) Concentration of free GTP-tubulin in the sol dec
2) Rate of addition of GTP-tubulin to the MT is lower than the hydrolysis of GTP
5) Presence of GDP-tubulin dec the stability of the MT polymer

Question 30

Are the + ends of the MT always** shrinking/growing?

Answer 30

No - some MTs that have been capped are stabilized at the membrane, therefore, do not grow/shrink

Question 31

Significance of MT stabilization at the cell membrane

Answer 31

Helps generate cell polarity, which is correlated to the asymmetric shape of the cell and is important for the distribution of organelles and vesicles

Question 32

Why are MTs important in nerve cells?

Answer 32

MTs maintain its polarity. It extends throughout the nerve cell and allows for motor proteins to move cargo across the axon. Kinesin moves towards the + end (away from the centrosome and towards edge of cell) and dynein moves towards the - end (towards the centrosome or other MTOC)

Question 33

Motor proteins and how they move

Answer 33

Its “head” region associates with the tracks of the MT and the tail region binds to cargo. They have two head domains that bind ATP and its hydrolysis causes a conformational change that drives the movement of the motor protein across the MT

This helps position organelles in the cell

Question 34

How MTs position the ER and Golgi, and what would happen to the Golgi is the MTs depolymerized

Answer 34

Kinesin is responsible for positioning ER so it can spread throughout the cell and dynein is responsible for positioning the Golgi. If the cell was exposed to Nocodazole, the Golgi fragments would be found throughout the cell

Question 35

Actin filaments in vitro

Answer 35

Nucleation is a slow process. Similar to tubulin, such that is undergoes dynamic instability and subunits are activated by the binding of ATP, but actin has structural differences

Question 36

Actin monomers and filaments dynamic eq’m in vitro

Answer 36

Monomer is G-actin (globular actin) filament is F-actin (filamentous actin). When assembling, the - end has low ATP-bound monomer binding and requires a higher Cc of actin monomers (favours disassembly), but the + end has high binding and a lower Cc of actin monomer is required (favours assembly)

Question 37

Actin in vivo functions

Answer 37

Provides structural stability

Involved in vesicle traffic and organelle position in plants (in animals cells, MTs do this fxn)

Movement of cell

Question 38

What controls the behavior of F-actin polymers in cells?

Answer 38

Actin-binding proteins (ABPs). They bind to free actin subunits

Question 39

Thymosin

Answer 39

An actin sequestering protein (a type of ABP?) that binds to actin and does not allow the G-actin subunits to bind to the filament

Question 40

Nucleation sites for actin

Answer 40

No equivalent to MTOC, so actin is nucleated at multiple sites with the Arp2/3 (actin-related protein) complex. It nucleates branched actin arrays. It is branched because it is how the complex most efficiently nucleates filaments

Question 41

Actin and cell movement

Answer 41

Formation of actin network at the plasma membrane pushes the membrane forward and allows cell to move. The + end (which is at the edge of the cell) becomes protected by capping proteins and the - end is severed and disassembled

Question 42

Overall movement of the cell

Answer 42

Branched actin polymerization in the lamellipodium pushed the membrane forward and the contractile bundles of actin and myosin provide traction. The rear contraction helps the cell detach to move

Question 43

Myosins

Answer 43

They are actin-dependent motor proteins. Myosin-1 facilitates vesicle and microfilament movements. Myosin-II is involved in muscle contractions as they form thick filaments in muscle tissue. Myosins hydrolyze ATP to induce conformational changes and allows the head to move along the actin filament towards the + end