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Flashcards in Cytoskeleton Deck (30):
1

How does cytoskeleton make cells move?

A cell receives a signal on the other side of where the large filamentous polymer.

The large filamentous polymer, they are non covalent, quickly degrades to smaller protein subunits and then reassemble on the side of the signal.

The reassembling of primarily actin filaments creates a leading edge which protrudes (known as lamellipodium) and then anchors onto the substrate it is crawling on. Then myosin II will contract the tail end so the cell moves.

2

Describe intermediate filaments.

Primary role is protection from mechanical stress, they are stress absorbers, and at the junctions between cells and with the ECM. no known associated motors.

Their stability is controlled by phosphorylation and they surround the nucleus extending to the cell periphery.

Other functions include cytoarchitecture in the axon, cell migration and movement, and signal transduction.

Basic structure is a coiled coil that forms antiparallel tetramers which 8 tetramers go on to form the intermediate filament.

Examples: keratins, neurofilaments, nuclear lamins.

3

Describe actin and actin filaments.

Actin monomers bound With ATP which is hydrolyzed to ADP when it is in the filament. It has a polarity with a plus end and a minus end. The filament is helical and actin binding proteins modify the dynamics and higher order assemblies.

4

Describe tubulin an microtubules.

Microtubules are polymers of alphabeta tubulin. The alpha and beta both bound GDP or GTP, (in this case we only talk about the Beta tubulin on top because the alpha is not going to let the GDP be exchanged.

Microtubules are important for vesicular transport, forming mitotic spindle, in cilia and flagella, basal bodies and centrioles.

5

Describe microtubule and actin polymerization.

They are both assembled from globular proteins via condensation polymerization reactions.

Nucleotide hydrolysis will lag behind the addition of monomers so an ATP or GTP cap will form at the growing end.
On the minus side, there is no lag and instead, the Monomers are dissociating faster than addition and on the plus side, more is added than lost.

Energy of hydrolysis is not needed for polymerization, instead the nucleotide at the plus end determines the stability.

Dynamics and state of assembly are regulated by actin/microtubule binding proteins,

ACTIN:
nucleation is the rate limiting step, ATP actin is preferentially added to the barbed end.

Microtubules:
Dynamic instability: rapid transitions between growth and shrinking. Sometimes there is loss of the GTP cap and the polymer rapidly shrinks (catastrophe), with rescue the cap is reinstated and rapidly grows.

6

Describe the various roles of cytoskeletal binding proteins.

Depolymerizing proteins will bind to monomers sequestering them.

They can cap short filaments from a severed long filament and prevent group.

Or they can serve as linkers to form a vast variety of structures.

They can also severe the protein. And stablize or destablize plus or minus ends.

Example tau, a microtubule associated protein, they cross link microtubules together. In Alzheimers, tau forms neurofibrillary tangles.

Example + tip proteins, the bind and track with the + end of a growing microtubule so there can be communication with the periphery. They essentially stabilize it.

7

Describe primary cilia

Its non motile and most cells have them.

Usually one per cell and they serve as sensory protrusions.

They are structurally different from motile cilia with a lack of a central doublet and dynein arms.

8

What is the centrosome

Its the microtubule organization center. It contains the centriole pair in addition to pericentriolar material.

It contains a gamma tubulin ring which nucleates the + end of protofilaments and caps the minus ends.

As a result + end is always oriented toward the periphery.

Centrioles duplicate at the beginning of S phase.

9

Phalloidin

Colchicine

Taxol

Phalloidin - binds and stabilizes actin filaments. Found in the Angel of Death

Colchicine - depolymerizes microtubules

Taxol - binds an stabilizes microtubules, good for cancer because that means it can't destablize as it does for forming spindle fibers.

10

What are examples of cell migration?

Neural development - pathfinding for neurons

Chemotaxis- migration of neutrophils to infection sites
-a neutrophil has actin polymerization at the leading edge and myosin II dependent contraction at the other end.

Repair and remolding - migration of cells to repair wounds

Tissue formation

Cancer metastasis.

11

Describe actin polymerization in regards to movement.

First of all elongation at the barbed ends. And you need alot of actin filaments to generate the force needed. So you nucleate more actin filaments. Or you sever existing ones to create more.
Or form branches from existing filaments.

Arp2/3 complex - it nucleates filaments from the sides of actin, making complex branched structures and is activated by Arp 2/3

-neutrophil migration, wound healing, metastasis, bacterial infections,

12

How are motor proteins and actin related to mitosis?

Kinesins and dynein are involved in spindle assembly, chromsomal alignment and segregation.

Actin and myosin II are in the contractile ring and cleave the cell.

13

Describe how the cytoskeleton plays a role in morphogenesis

A sheet of epithelial cells will feature an adhesion belt, the terminal web, + ends are associated at each junction so the terminal web has actin filaments going in both directions. Myosin II makes the belt contract and this causes the folding of the sheet (formation of neural tube)

14

Generally describe the three classes of motors.

Myosins move along actin filaments, generally to the plus end.

Dyneins move towards the minus end on microtubules

Kinesins move towards the plus end on microtubules.

All three of them are ATPases.
All three of them can only move in one direction
Vesicles and organelles can move on microtubules and actin filaments in two directions so they feature multiple motor proteins.

All three motors turn chemical energy from the binding or hydrolysis of ATP into an intramolecular conformational change which becomes mechanical movement.

15

Describe the actin motor myosin.

They have two heavy chains with two light chains.

They are generally plus end directed.

The globular head domain contains the ATPase and the heavy chain + light chain (at the base of the head)

The tail is a variable domain, it contains a coil coil for dimerization or to membrane or a vesicle.

Most light chains are camodulin so they can be regulated by calicum. The heavy chain is what binds to actin.

Binding to actin or microtubules accelerate the rate limiting step of releasing inorganic phosphate.

16

Describe the microtubule based motor: Kinesins

smaller than myosin, the head domain contains the ATPase. The sequence of the motor head is different obviously since it binds to a different molecule.

The heavy chain has the ATPase and the microtubule binding site.

The neck region towards the C terminus just after the motor domain determines polarity.

The stalk region - is like the tail region of the myosin. When its a coiled coil it can dimerize and form filaments.

The tail region is what binds to the target or binds light chains, specific kinesins lacking stalk regions will bind cargo.

17

Describe dynein

It is the minus end directed, and is 6 domains.

It is found in ciliary/flagellar arms and is necessary for beating.

The tail binds cargo or another microtubule like in cilia. The microtubule binding site is at the coil coil stalk. The conformational change from hydrolysis would have to be translated up the stalk and lead to the movement of one alpha beta tubulin.

For dynein, the microtubule binding site is remote from the ATPase domain.

18

What is primary ciliary dyskeneisa (PCD)

aka Kartagener syndrome

It is due to a mutation in the heavy chain of dynein. So essentially a lack of dynein in the right location, it is in the basal bodies and centrosomes but not in the cilia.

The non motile cilia leads to infertility and chronic infections of the respiratory tract. Also associated with situs inversus (the organs are on opposite sides of the body)>

19

Describe the mechanism of movement via myosin II (a filament forming myosin)

When ATP is bound, the myosin is loosely associated with the actin and can come off. When the inorganic phosphate is released from the ATP (rate limiting step) that allows the myosin to bind with strong affinity towards the plus end of the actin. So when Phosphate is released there is a power stroke which is the movement up the actin.

Actin acts like a GEF, exchanging ATP with ADP so it is not forever tightly associated. When it is replaced with ATP, the myosin head comes apart.

Both hydrolysis and ATP binding result in conformational changes.

In myosin only one head is associated and does the moving.

20

Describe the kinesin mechanism??

It is similar to the myosin mechanism. THe binding of ATP to the head lowers binding affinity for microtubules. The ATPase activity releases the inorganic phosphate, the rate limiting step, and it will bind with higher affinity to MT.

Binding to MT accelerates product release and conversion from a weak to strong binding.

21

How is the motor and cargo relationship specified?

It is all in the tail region. They can interact directly with specific cargo or associate with adaptor proteins, scaffold proteins. For the most part heads are conserved by the tails are what differ.

22

Describe myosin II and myosin V

Myosin II are bipolar filaments, when they move, they will contract the muscles by contracting the muscle fibers. It is also the role player in cytokinesis, and folding of cell sheets.

For the transport of vesicles, they will travel from cell body to the periphery, going from microtubules to the actin filaments. This will involve dynein, kinesin and myosin V.

Myosin V carries cargo on their tail and is designed for long range transport because they take longer steps than other myosins.

Its neck region has calmodulins so without calcium and cargo binding, it will not be active and remain in the folded conformation.

23

Describe characteristics of intracellular transport (typical in a neuron)

Vesicles/organelles/proteins are transported from the ER to the cell periphery and back.

Transport can occur on + end of a microtubule by a plus tip protein, as the microtubule grows, the + tip will stabilize the + end of the microtubule.

Vesicles/organelles, travel in both directions, and is dependent on motor proteins

For this reason, a vesicle and organelle typically have more than one motor protein that allows bidirectional and bifilament (actin and microtubule

24

Describe the transport of pigment granules and the diseased state.

Pigments are packaged into vesicles called melanosomes and their distribution depends on transport on microtubules and actin filaments.

IN skin, projections go into the apical surface from the basal surface and transport melanosomes to the periphery. This will go from microtubules to myosin V acting actin transport. The melanosomes are bound to Rab protein which is bound to melanophilin which is bound to the tail region of myosin V. Rab GTPases targets myosins to the specific intracellular membranes.

A mouse lacking myosin V will have no pigment because no melanosomes are delivered to the periphery.

So MUTATIONS in myosin V, melanophilin or Rab27a will cause lack of pigmentation.

25

There are many different kinesin families in the genome? Why is that

It is because of variable tail domains.

Motor domains are HIGHLY conserved. Only tail domains are variable.

26

How do cytoskeletal motors move?

Via the hydrolysis of ATP only.

(they are ATPases)

The power stroke occurs when products of hydrolysis are released.

27

Myosin Dan is a plus end directed actin associated motor. However it can move in both directions, how can it move to the minus end?

Minus end directed movement is passive.

it is a result of varying kinetics at the two ends of the actin filament.

28

There are many different kinesin families in the genome? Why is that

It is because of variable tail domains.

Motor domains are HIGHLY conserved. Only tail domains are variable.

29

How do cytoskeletal motors move?

Via the hydrolysis of ATP only.

(they are ATPases)

The power stroke occurs when products of hydrolysis are released.

30

Myosin Dan is a plus end directed actin associated motor. However it can move in both directions, how can it move to the minus end?

Minus end directed movement is passive.

it is a result of varying kinetics at the two ends of the actin filament.