The Cytoskeleton

Question 1

What are the three major elements of the cytoskeleton?

Answer 1

1) Actin Filaments
2) Microtubules
3) Intermediate Filaments

Question 2

Actin Filaments Function

Answer 2

Actin filaments are the smallest of the three cytoskeletal filaments, it is 5-9nm in diameter. They are responsible in changing the cell shape and thus are primarily located underneath the plasma membrane and referred to as cortical actin. They are involved in cell adhesion via actin-based adhesion junctions as well as involved in cell polarization. Because of their close proximity to the cell membrane they are also involved in phagocytosis. Finally they are involved in muscle contractions. Actin can also form a lamellipodium which is involved in cell migration.

Actin primarily functions in cell migration, cell shape, muscle contraction, and phagocytosis. Sometimes called microfilaments because they are the smallest.

Question 3

Actin Filament Cellular Location and Structure

Answer 3

It is usually located near the cell membrane (except in a muscle contractile apparatus). Localized primarily beneath the plasma membrane and control its shape and movement.

Actin is made up of soluble actin monomers that have a bound ATP. The monomers are globular proteins and they are asymmetrical, the monomers have a plus and minus end. The monomers assemble to form a protofilament and then two protofilaments come together to form a twisted helix called F-actin. The actin is rather flexible. Also, the monomers, over time will hydrolyze the ATP to ADP and can thus dissociate. There are many accessory proteins that bind to actin and regulate its equilibrium and polymerization/depolymerization. The nucleation is the rate limiting step (the point where it forms a oligomer that can then be stable). Thus cells use these performed oligomers to speed up the polymerization of actin filaments. This WILL NOT change the critical concentration, it will simply allow it to get there quicker. Actin is treadmilling, meaning it can have subunits added and removed from both ends.

Question 4

Microtubule Function

Answer 4

Microtubules have 3 main functions:

1) Positioning of Organelles: Organelles have motor proteins that allow them to move around on the microtubules. The microtubules are polar so the motor proteins have specificity to direction. Two important organelles are the golgi and ER
2) Intracellular Transport: First off, the microtubules are essential for the formation of the mitotic spindle to get the separation of chromosomes during mitosis. They are also important in vesicular transport along the ER. They are key for anterograde and retrograde transport because they are polar.
3) Cell motility: Cilia and Flagella are microtubule based structures.

Question 5

Microtubule Cellular Location and Structure

Answer 5

These are located throughout the cell as a network for transport.

The microtubules are made up of alpha/beta tubulin subunits and thus it is a heterodimer subunit. These subunits bind to GTP instead of ATP like the actin monomers. It is also polar, the alpha subunit is negative and towards the centrosome. The positive beta subunit is dynamic end in the lumen. The subunits combine to form a protofilament and then 13 protofilaments come together to form a microtubule. The microtubule is hollow and thus the microtubule is stiff and brittle. The filaments form from the base upwards, with the base in the MTOC. The minus end in the MTOC is completely capped and stable where the plus end can have subunits added to it or removed from it. Here you see dynamic instability. They can have a “GTP Cap” which protects it from being depolymerized.

Question 6

Intermediate Filament Function

Answer 6

4 Functions:

1) Mechanical Strength: Help in cell and tissue strength and integrity. They are very prominent in tissue with a lot of stress like the heart, skin, etc.
2) Cell adhesion: They are involved in cell-cell (desmosomes) and cell-matrix (hemidesmosomes) adhesion.
3) Axon diameter and strength: They are prominent in nerves to keep them strong and stable since they are so spread out.
4) Nuclear Lamins: They help give the nucleus its integrity

Question 7

Intermediate Filaments Cellular Location and Structure

Answer 7

Located throughout the cell.

It is composed of tetramers which are composed of 2 coiled-coil dimers. They are rope-like and have high physical strength. Also, they are non-polar, meaning both ends are exactly the same. Because it is nonpolar, it is not involved with motor proteins because of this nonpolar nature, things would not have specificity for their directionality. 8 of the tetramers associate to form the intermediate filament in a rope-like structure and there are many lateral interactions that give it its strength.

Question 8

Describe the subunits that make up the actin microfilaments

Answer 8

The subunits that make up actin are in the form of a polar monomer. Due to the polarity, the monomer is asymmetrical. The monomers are bound to ATP and thus can then be added to a forming chain of actin. Accessory proteins help regulate its assembly and disassembly. When it forms an actin filament, the ATP bound to the monomer can be hydrolyzed to ADP and now the monomer can disassemble from the actin filament.

Question 9

Understand and Describe the kinetics of actin filament assembly

Answer 9

One of the obstacles that a cell must overcome is that the initiation of polymerization (nucleation) is slow and thus rate-limiting. Cells utilize preformed oligomers of actin subunits to initiate rapid filament polymerization.

When actin subunits begin to form an oligomer, initially it is not a very stable structure. So initially there is a lag phase or delay until they reach a more stable structure. This is a rate-limiting step because it is slow. Once oligomers are established you get very rapid formation of actin polymers. There is then a stable structure for the actin to grab to for the growth or elongation phase and as the filament lengthens, it actually begins to deplete the free actin in the cytosol and polymerization begins to slow down until it reaches a steady state where it reaches a constant equilibrium length. This is the critical concentration of actin when the filament is neither growing nor shrinking. On and off rate of actin to filament are equal and this is the critical concentration.

You will still have the addition of actin to the + end and loss of actin from the – end. The – end is less dynamic. Because the loss and gain from these ends are equal though you are at a critical concentration

• The lag –> If you add a preformed oligomere it can give rapid polymerization of actin. It does NOT CHANGE THE CRITICAL CONCENTRATION, IT JUST GETS THERE QUICKER
• There are proteins similar to these oligomeres that will rapidly trigger actin polymerization when and where it wants it
• ATP bound monomer is always free floating and then binds to the polymerizing actin.

Question 10

Describe the importance of nucleotide (ATP) hydrolysis in actin filament dynamics and treadmilling

Answer 10

When actin monomers are in their ATP-bound state, they will remain bound to the actin filament. However, when it becomes ADP-bound, the actin monomer can dissociate from the filament. The addition of subunits to the minus end is relatively slow when compared to the plus end, so the plus end will have more GTP-bound subunits when compared to the minus end. This results in the treadmilling because the minus end will lose subunits and the plus end will bind new subunits. The minus end will have a much higher critical concentration because it required more subunits to be present in order to polymerize. This difference in critical concentration results in the treadmilling.

Question 11

Describe the global cytoskeletal rearrangements regulated by Rho family proteins.

Answer 11

• There is a family of GTP proteins of GTPases called the Rho family that give global regulation of the actin filaments.
• G-proteins are biochemical switches. In the GTP state they are on, in the GDP state they are off.
• You have GTP forms of Rho families that cause very global and dramatic changes in actin cytoskeleton
• If you activate Rho in cells, you get a very robust amount of antiparallel stress fibers that allow the cell to hang on to its surroundings and migrate if necessary
• In contrast, if you have Rac, you get Lamellipodia and membrane ruffles. In culture these cells send up a curtain or ruffle in the migrating edge
• Then there is Cdc42 which creates filopodia or small bundles of actin. You can also see microspikes or spiky actin protrusions
• Just know that the Rho family causes global actin rearrangements

Question 12

Describe the basis for the toxic physiological effect of actin-specific drugs

Answer 12

If a drug targets actin, it is targeting all healthy cells with actin as well as muscle cells, including the heart. If you interfere with the actin, it will cause the heart to not function properly and ultimately result in death.

Question 13

Spectrin

Answer 13

It is a tetramer and is a very long, flexible molecule with actin-binding domains at both ends and will bind to different actin filaments. Because it is long and flexible, the actins will be assembled in different orientations relative to one another. This helps form the gel-like or mesh-like structure

Question 14

Fimbrin

Answer 14

It is a monomer. involved in filapodia because it binds actin in the same direction and very close together. You will get a very tight bundle of actin filaments with fimbrin

Question 15

Alpha Actinin

Answer 15

It is a dimer. Here you have the actin binding domains orientated in opposite directions so it will give you antiparallel organization. This is also a little space in there which allow for motor proteins like myosins to get in there between the actin filaments.

Question 16

Filamin

Answer 16

It is a dimer and it binds actin and orients them at different angles to each other. It will generate a type of web of actin filaments

Question 17

Describe the molecular characteristics of actin based molecular proteins.

Answer 17

Myosin is the actin motor protein. Majority of the myosins are plus end directed, meaning they will move toward the plus end of the actin. Some are minus end though.

• Know that this is good for high speed movement and each head is good for the powerstroke
• Know actin mutations cause muscle problems
• The features of myosin to remember are that the heads actin independently and the heads work via an independent motion
• You can think of it as rowing –> Myosin heads only touch actin very briefly and then move out of the way for another actin to come in
• Start in Rigor State –> Myosin head stuck to actin in absence of ATP
• Then ATP comes in and binds to myosin head, this causes dissociation of myosin from actin
• Hydrolysis of ATP to ADP +Pi on myosin head cocks the myosin head
• release of inorganic phosphate causes head to bind to actin
• Release of ADP results in powerstroke
• Actin is one of the most evolutionarily conserved proteins  This is because a mutation anywhere in actin almost always cause some defect in muscle function
• Myosin is a large coiled-coil region and a head region. Within the head region there are these light chains that give you the force generation.
• Myosin is dimer with two identical motor heads that act independently
• Each head has a catalytic core with a lever arm
• Coiled-coil chain keeps them together
• The large coiled-coils are just structural.
• The tails for myosin will interact with each other and form what is called a bipolar thick filament. They are oriented in opposite directions too.
• There are hundreds of myosin heads that are exposed on the head of the bipolar thick filament and they are able to contact actin and move along actin.
• Myosin contacts actin for a very short period of time and moves itself a little bit along actin
• Because there are hundreds of myosin heads though, it can move along actin very fast. Each head acts independently as well.

Question 18

Describe the overall structure of Myosin II in the bipolar thick filament found in muscle

Answer 18

• Myosin is a large coiled-coil region and a head region. Within the head region there are these light chains that give you the force generation.
• Myosin is dimer with two identical motor heads that act independently
• Each head has a catalytic core with a lever arm
• Coiled-coil chain keeps them together
• The large coiled-coils are just structural.
• The tails for myosin will interact with each other and form what is called a bipolar thick filament. They are oriented in opposite directions too.
• There are hundreds of myosin heads that are exposed on the head of the bipolar thick filament and they are able to contact actin and move along actin.
• Myosin contacts actin for a very short period of time and moves itself a little bit along actin
• Because there are hundreds of myosin heads though, it can move along actin very fast. Each head acts independently as well.

Question 19

Describe the subunits that make up microtubule filaments

Answer 19

Alpha/Beta tubulin dimers are the soluble subunits for microtubules.
-Soluble subunit form is a dimer between alpha and beta tubulin
-Microtubules bind to GTP instead of ATP like actin.
-The tubulin is bound to GTP in the soluble form
-It has a lot of other similarities to actin though
-The dimer is polar –> Plus end is different from the minus end.
-They form together, head to tail into protofilaments and 13 of the protofilaments form a tube with a lumen
(interior)
-The microtubule is actually a tube that is hollow in the middle and this structure is much larger and stiff and brittle
-The protofilaments don’t form by themselves, they form from the base upwards, with the minus end at the base in the MTOC or centrosome up to the plus end.
-Plus end is the more dynamic end and this is the end where the dimers add
-The minus end is burried into the MTOC (microtubule organizing center)
-Minus end of microtubule is completely capped and stable and thus get no loss or addition at this end. All of the loss and addition is at the plus end

Question 20

Understand and describe the kinetics of microtubule assembly

Answer 20

• Analogous to actin, the triphosphate in the filaments which in this case is GTP, can become hydrolyzed which will tend to destabilize the microtubules
• You have microtubule with a minus end that is completely protected and capped within the MTOC
• At the plus end you have the addition of GTP bound tubulin dimers
• Over time the GTP becomes hydrolyzed to GDP and if this “T-Cap” or GTP Cap is lost you now have a GDP bound plus end which destabilizes the plus end and causes rapid depolymerization of the plus end and is called a catastrophy event. –> rapid depolymerization
• Then you get a rescue, in which a GTP bound tubulin is added again and get rapid growth again
• This cycles over and over again –> Microtubules have dynamic instability which is where you have from the plus end polymerization and depolymerization constantly occuring. This is different from actin in which you have treadmilling occurring and this occurs at both the plus and minus end in actin
• When GTP cap is gone, you get depolymerization
• Many, but not all, microtubules grow from the centrosome

Question 21

Describe the importance of nucleotide (GTP) hydrolysis in microtubule filament dynamics and dynamic instability

Answer 21

The GTP forms a GTP-Cap at the plus end of the microtubule. If the GTP is hydrolyzed to GDP, it results in destabilization of the microtubule at the plus end and you get this dynamic stability in which depolymerization will occur at the plus end. The minus end is never effected because it is in the MTOC and thus us capped.

Question 22

Explain why some microtubule specific drugs are useful for cancer chemotherapy

Answer 22

• These are very clinically useful, unlike actin drugs
• This is because microtubules are so important for mitosis and can thus treat cancer this way
• There are side-effects though associated with these medications though because the only sort of specialization is cell proliferation and thus they will attack these cells, but many cells in the body too are affected by this because they rapidly undergo mitosis like hair cells
• Toxicity results such as hair loss and diarrhea but these tend to be reversed

Question 23

Describe the structural composition and function of microtubule-based astral arrays, cilia and flagella

Answer 23

• Know the structural differences
• They allow cells to swim or allow fluid to move past cells in a certain direction
• This is an Axoneme and motor proteins associated with them are Axonemal Dyneins which are minus end motor proteins
• The structure is very recognizable –> 9 microtubule doublets, with two microtubules in the center
• The doublets have a complete microtubule and then a partial B microtubule attached to that
• Then in the middle there are two complete microtubules.
• 9 + 2 or 9 doublets around 2 singlets
• This is a characteristic structure of the cilia and flagella
• Flagella Do an even wave-like type of motion
• Cilia are a whip-like motion

Basal Bodies similar to centrioles inside centrosomes.

• The cilia and flagella are anchored in basal bodies which are similar to centrioles
• They have 9 triplets of microtubules with no interior of center microtubule
• Found at the base and are nucleating structures for cilia and flagella
• Can be distinguished by knowing where you are in the cell. Centrioles are found near the nucleus where the Basal Bodies are found near the plasma membrane
• Have to know where you are in the cell to know if it is a basal body or centriole
• Kartagener’s Syndrome or primary ciliary dyskinesia (Ciliary Dynein)
• Results in infertility (males sperm (flagella) and female fallopian tubes (cilia)
• Respiratory/sinus infections because cilia cannot beat properly
• Situs Inversus (organ position is inverted)

Question 24

What are the two microtubule motor proteins?

Answer 24

1) Kinesins and Kinesin related proteins

2) Dynein

Question 25

Kinesin

Answer 25

Both kinesins and dyneins are involved in chromosome segregation.

• There are motor proteins that move along microtubules.
• There are kinesins and Kinesin related proteins that move toward the plus end –> They are directional
• Tail end binds to cargo that needs to be moved
• Kinesins thus move the ER towards the plasma membrane

Question 26

Dynein

Answer 26

Both kinesins and dyneins are involved in chromosome segregation.

• There are motor proteins that move along microtubules.
• The Dyneins move towards the minus end of the microtubules
• Dyneins are involved in vesicle transport. They move stuff back towards the nucleus where the Kinesins move things towards the outside of the cell
• Axonemal: Special class of dynein motors that are involved in cilia and flagella motion

Question 27

Describe the molecular characteristics of microtubule-based molecular motors

Answer 27

• Here know that the dimer head worked together.
• They are dimers with a long coiled-coil tail. Each head remains attached 50% of the time so it sort of walks on the microtubule. They remain attached to the microtubule for a long period of time.
• The have a lot of processivity means that they go slow and steady. Can remain attached for a long distance. This is good for vesicular transport, etc.
• It is Hand-over-Hand movement (or walking)
• It is a dimer with two identical motor heads
• They start with ADP bound. It will bind to microtubule and then lose ADP and get ATP. This throws the other head forward. Then the the trailing head hydrolyzes ATP to ADP to be released while the leading head now binds ATP to throw it forward. This process continues and the two heads work together.
• It is like a multi-lane highway where traffic can go in either direction

Question 28

Compare and contrast the functions of actin and microtubule motor proteins

Answer 28

Actin motor proteins, myosin, act independently of one another where the microtubule motor proteins, kinesin and dyneins, function together to get a walking like motion instead of the myosin rowing like motion. Both utilize ATP and function to move along the cytoskeletal filament. The microtubule motor proteins, however, can move in both the + and - direction and, they remain bound about 50% of the time where myosin is unidirectional and most only move toward the plus end. They are also only associated with actin for a short period of time.

Question 29

Describe the subunits that make up intermediate filaments

Answer 29

A tetramer (two coiled-coil dimers) is the soluble subunit for Intermediate Filaments.

• Main thing with these is they are nonpolar –> The front and backs are the same –> No plus or minus end.
• Soluble subunit is a tetramer
• So you have an individual intermediate filament protein which is a long rope-like structure with a globular amino and carboxy-terminus and this long center domain allows it to interact as a dimer to form a coiled-coil dimer.
• Two of these then interact to form a tetramer
• The tetramer is symmetrical –> Left and right sides are identical so when they go to assemble into a filament
• 8 tetramers can associate together and are twisted into a rope like structure called the intermediate filament
• There are many, many lateral interactions which gives it a lot of strength
• There is cross-linking too.
• The strength is due to all of this lateral interactions
• No motor proteins associated with them and this is probably because it is nonpolar so the motor protein cannot distinguish one end from another end

Question 30

Describe how the structure of intermediate filaments provides high tensile

Answer 30

The intermediate filaments are composed of tetramer subunits that come together in which 8 tetramers can associate and are twisted into a rope like structure called the intermediate filament. Then, there are many lateral interactions which give it a lot of strength as well as cross-linking. It is basically forming a cable which is very high strength

Know that there are proteins that bind to intermediate filaments that help them bind.

• There are proteins that bind to them and organize them
• They can get bundled into very large tonofilaments
• There are some proteins that bind to multiple cytoskeletal filaments like plectin  binds all three

Question 31

Describe why the tissue specific expression pattern of intermediate filaments is useful in tumor diagnosis

Answer 31

The primary tumor will come from somewhere. So if there is a secondary tumor that forms somewhere else, you can look at the intermediate filaments present and this will tell you where these tumor cells originated from.

• Just know cytokeratins are in epithelial
• Most important feature of intermediate filaments is their diversity
• It has very tissue-specific expression
• They are useful for tumor diagnosis
• Epithelial intermediate filaments have cytokeratins in epithelial cells and epithelial appendages like hair and nails
• Axonal intermediate filaments have neurofilament proteins or in neurons