16: The Cytoskeleton

Question 1

What are the three families of protein filaments related to the cytoskeleton?

Answer 1

Actin filaments
Microtubules
Intermediate filaments

Question 2

What are the main roles of actin filaments?

Answer 2

Determine the shape of the cell’s surface.
Whole-cell locomotion.
Drive the pinching of one cell into two.

Question 3

What are the main roles of mictotubules?

Answer 3

Determine the position of membrane-enclosed organelles.
Direct intracellular transport.
Form the mitotic spindle.

Question 4

What is the main role of intermediate filaments?

Answer 4

Provide mechanical strength.

Question 5

Where are actin filaments most highly concentrated?

Give some examples of different actin filaments

Answer 5

In the cortex, just beneath the plasma membrane.

Examples:

• Lamellopodia and filopodia
• Stereocilia (on the surface of hair cells, tilt in response to sound)
• Microvilli

Question 6

Where in the cell does one find microtubules?

Give some examples of different types

Answer 6

In a cytoplasmic array that extends to the cell periphery.

Examples:

• Mitotic spindle
• Cilia

Question 7

Where in the cell does one find intermediate filaments?

Give some examples of functions

Answer 7

Line the inner face of the nuclear envelope.
In cytosol: twisted into strong cables that can hold cell sheets together, and help nerve cells to extend long and robust axons.
Allow us to form tough appendages e.g., hair and fingernails.

Question 8

How are cytoskeleton subunits assembled?

Answer 8

Small subunits are (helical) assembled using a combination of end-to-end and side-to-side protein contacts (noncovalent).
Small subunits rapid diffuse in the cytosol => rapid structural reorganizations.

Actin and tubulin subunits - compact and globular.
Head-to-head binding of asymmetrical subunits => points in same direction polarity.

Intermediate filaments - smaller subunits that are elongated and fibrous. Symmetrical subunits.

Question 9

Describe the actin subunit and polymer

Answer 9

Subunit: G-actin (globular)
375 aa ppt
Carries a tightly associated molecule of ATP or ADP (hydrolysis).
Structural polarity

Polymer: F-actin (filamentous)
2 parallel protofilaments
Polymerization- subunits linked head-to-tail by noncovalent bonds
Minus end (with nucleotide-binding clefts) grows more slowly

Question 10

What is filament nucleation and polymerization?

Answer 10

Nucleation: Rate-limiting step
Assembly of subunits into an initial aggregate (nucleus, trimer) that is stabilized by multiple subunit-subunit contacts. Can afterward elongate rapidly by the addition or more subunits.

Induced by changes in salt concentration or temperature

Polymerization:
The rate of filament assembly depends on the concentration of the free subunit
At the Critical Concentration (Cc) rate of subunit addition = rate of subunit loss. No net polymerization.

LAG PHASE: time taken for filament nucleation
GROWTH PHASE: subunit addition to exposed ends
EQUILIBRIUM PHASE: no net change in polymer

Question 11

How is actin nucleation regulated?

Key words: formins, Arp 2/3, cofilin

Answer 11

Arp = Actin Related Proteins

• Nucleates actin filament at the minus end => rapid elongation at the plus end.
• Two binding sites allow filament network formation: one for actin, one for the side of an actin filament.
• Inactive until binding to an activation factor.
• Nucleation at 70 degrees relative to original filament.
• If Arp2/3 stays bound to nucleation start (minus end) => stabilization of the filament, no adding/removal at - end.

Formins:

• Mediate nucleation of straight/unbranched filaments ➔ controlled nucleation/de novo formation of actin
  filaments.
• Remain associated with plus end of microfilament and facilitate addition of actin monomers ➔ increased elongation rate

Depolymerization:
Cofilin
- Facilitates filament breakdown through interactions
with F-actin (ADP-bound) and creation of mechanical stress.

Question 12

Why do the plus ends of actin and tubulin filaments grow faster than the minus ends?

Answer 12

The conformation of the free subunit as it enters the polymer fits differently at the two ends.
Must be changed at the minus end.

K_off and K_on are different but the ratio K_off/_on is the same. Critical concentration (C_c) same at both ends
C>Cc both ends grow
C

Question 13

How do the critical concentration and the rate constants for assembly/disassembly of subunits to actin change when the nucleotide is hydrolyzed?

Answer 13

Hydrolyzation => storage of more energy in the polymer.
=> more negative free-energy change for dissociation of a subunit from the D form than for the T-form.

=> Cc(D) > Cc(T)
At certain concentrations, the D-form polymers will shrink while the T-form polymers grow.

Subunit concentration above Cc for both forms: grow
Below: shrink

Question 14

What is filament treadmilling?

Answer 14

Occurs at filaments that are asymmetrical.
At intermediate concentration of subunits
=> rate of subunit addition is faster than nucleotide hydrolysis at the + end. Opposite at the - end.

At particular concentrations, the growth at + end will balance the shrinkage at - end
=> subunits cycle between free and filamentous states.
No net change in length

Predominate in actin filaments

Question 15

How is actin filament extension regulated by thymosin and profilin?

Answer 15

Thymosin binding prevents actin monomer from association with plus ends of the filament.

Profilin binding prevents binding to minus end of filament. ➔ selection for binding to growing end.
- Activated by phosphorylation or binding to inositol phospholipids.

P and T compete with each other for binding to local actin monomers thus regulating filament extension.

Question 16

What is dynamic instability and in which cytoskeleton filament does it predominate?

Answer 16

Alternations between periods of slow growth and a period of rapid disassembly at a uniform free subunit concentration.

Microtubules depolymerize about 100 times faster from an end containing GDP-tubulin.
GTP cap favors growth (straight protofilaments)
If it is lost => depolymerization (curved protofilaments)

Growth -> shrinkage: catastrophe
Shrinkage -> growth: rescue

Question 17

How are myosin and myosin filaments built?

Answer 17

Myosin:
Elongated protein with two heavy chains and two copies of each of two light chains.
H-chains have head domains at their N-terminals with force-generating machinery.
Light chains bind close to heads.

Long aa. seq forms extended coil-coil that mediates heavy-chain dimerization. Bundles itself with tails of other myosin molecules -> myosin filaments.
- Several hundred myosin heads, oriented in opposite
directions at the two ends of the filament.
- Central “bare zone” free of head domains.

Question 18

How does myosin generate muscle force?

Answer 18

Each head binds and hydrolyzes ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament by conformational changes.
- At the base of the lever arm: a helix connects movements at the ATP-binding cleft in the head to rotations in a converter domain
=> movement, ~5 nm, muscle contraction
- Changes in conformation of myosin are coupled to changing in its binding affinity for actin => release/reattach

Steps:

1. Attached (no ATP bound)
2. Released (ATP-binding => conf. change)
3. Cocked (ATP-cleft closes, ATP hydrolysis->ADP+Pi bound, movement in lever arm=> 5 nm movement)
4. Force-generating (Pi released as head binds tightly to actin => power stroke, ADP off, return to the start of the cycle)

Question 19

What is a myofibril?

Key words: size, contractile units

Answer 19

Basic contractile elements of the muscle cell.
Cylindrical structure, 1-2 μm in diameter, often the length of a muscle cell.
Long, repeated chain of contractile units; sarcomeres (2.2 μm long)

Question 20

What are sarcomeres?

Answer 20

Contractile units of myofibrils
Composed of parallel and partly overlapping thin/thick filaments.
Thin filaments: actin and associated filaments attached at their + end to a Z disc at each end of the sarcomere.
Capped - end extends towards middle of sarcomere, overlapping with thick filaments.
Thick filaments: bipolar assemblies of myosin II.

Adjacent myosin II are linked by proteins at the M line (midline)

Question 21

What is sarcomere shortening? What happens to the thick/thin filaments during the process?

Answer 21

Caused by the myosin (thick) filaments sliding past the actin (thin) filaments, with no change in the length of the filaments.
=> muscle contraction.

The individual myosin motor heads spend only a small fraction of the ATP cycle time bound to the actin filament (force-generating)

Question 22

How are the sarcomere filaments organized?

Length, spacing, accessory proteins, etc.

Answer 22

Z-disc: CapZ and α-actinin

• Caps the filaments => prevents depolymerization
• Holds filaments together

Nebulin:

• Enormous size, actin-binding motifs
• Influence length
• Stretches from Z disc towards the minus ends (capped and stabilized by tropomodulin.

Titin:

• Long template protein
• Positions th ethick filaments midway between the Z discs.
• Springlike, allows the muscle fiber to recover after being stretched.

Question 23

Which ATP-consuming processes are related to muscle contraction?

Answer 23

• Filament sliding, driven by the ATPase of the myosin motor domain (on head)
• Ca2+ pumping driven by the Ca2+ pump (Ca2+-ATPase)

Question 24

How is muscle activity regulated by Ca2+ and its accessory proteins?
Keywords: T tubules, SR, ATP, accessory proteins

Answer 24

Action potential arrives at the neuromuscular junction.
=> Membrane potential change on the plasma membrane of the muscle cells, including on the T tubules (transverse tubules, invaginations)
=> Ca2+ release via voltage-gated Ca2+-channels in the T tubules. Connected to Ca2+ release channels of the sarcoplasmic reticulum (SR) => Ca2+ release from SR.

Rapid pumping back of Ca2+ into SR by Ca2+ ATPase (ATP-dependent Ca2+ pump).
=> steady state

Important accessory proteins:

• Tropomyosin: binds groove of the actin filament
• Troponin: complex of 3 proteins
  
  • T = tropomyosin binding
  • I = inhibitory
  • C = Ca2+-binding
• Binding of troponin I-T to tropomyosin => inhibition, no force-generation => resting
• Troponin C => troponin I released from actin

Question 25

How is myosin II activation regulated in non-muscle and smooth muscle cells?

Answer 25

The Ca2+ sensor calmodulin activates myosin light chain kinase (MLCK) which leads to myosin light chain
phosphorylation and contraction.
Dephosphorylation => dissociation of myosin head from actin filament -> inactive.

Events occur relatively slowly. Myosin II hydrolyzes ATP ~10 times more slowly than skeletal muscle myosin.

Question 26

Describe the structure of a microtubule

Answer 26

Polymers of tubulin (protofilaments):
- Heterodimer formed from two closely related globular proteins α-tubulin and β-tubulin (aa. bound together by noncovalent bonds).
- Each α and β monomer has a binding site for one GTP molecule.
On α: never hydrolyzed
On β: GTP or GDP
- Subunits point in the same direction => polarity.
α-tubulins exposed at - end.
β-tubulins exposed at + end

• Hollow cylinder built by 13 parallel protofilaments.
  αβ-tubulin heterodimers stacked head-to-tail and
  folded into a tube.
• Multiple contacts within the lattice hold the subunits in place => most addition/loss of subunits at the ends

Question 27

How can drugs inhibit microtubule functions? Why are these chemicals useful as anti-cancer drugs?

Answer 27

Can impair polymerization (taxol) or depolymerization (colchicine, nocodazole) to kill dividing cells, such as cancer cells. Also toxic to rapidly dividing normal cells e.g., hair follicles.
Microtubule dynamics are crucial for the correct function of the mitotic spindle.

Question 28

Briefly describe the nucleation of microtubules

Answer 28

Occurs in a microtubule-organization center (MTOC) with a high concentration of γ-tubulin.
Depends on the γ-tubulin ring complex (γ-TuRC) assembled by accessory proteins. Serves as a template that creates a microtubule with 13 protofilaments.
Nucleates - end of a microtubule.

Question 29

Can you describe the kinesins?

Answer 29

Conventional kinesins (kinesin I):

• 2 light and 2 heavy chains
• HCs mediate tubulin binding and contain ATPase domains
• LCs required for organelle/cargo attachment

Small movements at the nucleotide-binding site regulate the docking and undocking of the motor head to a linker region => movement, ~8nm (5 μm/sec)

Most of them walk towards the plus end of microtubules (motor domain at N-terminal). (Exception example: Kinesin 14- motor domain at C-terminal)

Kinesin 5 can form a bipolar motor complex that slides oppositely oriented microtubules past each other (similar to thick filaments of myosin II).

Kinesin 13 – lost typical motor activity and promotes
depolymerization

Question 30

Can you describe the dyneins?

Answer 30

Minus-end directed.
Largest and fastest (14 μm/sec) molecular motors.
One, two, or three HCs (incl. motor domain).
Variable number of intermediate, light-intermediate, and light chains.

Two major types: cytoplasmic and axonemal.
Cytoplasmic:
- Golgi localization, vesicle trafficking
- Centrosome positioning
- Mitotic spindle construction
Axonemal:
- Intraflagellar transport.

Couples ATP hydrolysis to microtubule binding/unbinding and a force-generating conformational change.

Question 31

Can you describe the intermediate filament structure`?

Answer 31

Elongated proteins with a conserved central α-helical domain containing ~40 heptad repeat motifs.
Form extended coiled-coil structure with another monomer => dimers.

Tetrameric subunit composed of 2 dimers pointing in the opposite direction => non-polar
No nucleotide binding site.

Lateral packaging of 8 parallel protofilaments (made up of tetramers) => filament

• Each filament: 32 individual α-helical coils
• Strong lateral hydrophobic interactions => difficult to break

Plectin (plakin protein family) links IFs to microtubules, actin filaments, and myosin II.

Question 32

What characterizes keratins?

Functions, structure, etc.

Answer 32

• Diverse intermediate filament family.
• Expressed in highly specific patterns related to epithelial type and stage of cellular differentiation.
• Important for mechanical stability and tissue integrity.
• Expression is altered in disease => can be used as a diagnostic tool

Structure:
- One filament consists of a type I (acidic) and a type II
(neutral/basic) chain, that form initially heterodimers, which in turn give rise to tetrameric subunits.
- Networks are held together by (tough) disulfide bonds.

Filaments in each cell are indirectly connected by desmosomes (cell-cell contacts)

Question 33

Which three activities are involved in cell migration?

Hint: P, A, T

Answer 33

Protrusion:
Plasma memb. is pushed out at the front of the cell.
Attachment:
Actin cytoskeleton connects across the memb. to the substratum.
Traction:
The bulk of the trailing cytoplasm is drawn forward.

Question 34

What are filopodia and lamellipodia?

Answer 34

Protrusive structures.

Filopodia:

• Migrating growth cones of neurons.
• One-dimensional

Lamellipodia:

• Formed by epithelial cells and fibroblasts.
• Two-dimensional
• Unidirectional motion maintained by unidirectional treadmilling.
• Actin filaments oriented with their + ends facing forward. - ends are attached to the side of other actin filaments by Arp 2/3 complexes.
• Cofilin and actin behind the leading edge => depolymerization

Question 35

How can cells pull themselves forward?

Key words: leading edge, rear, nucleation, myosin

Answer 35

Advance of leading edge:
Protrusion of the membrane must be followed by adhesion to the substratum at the front.
Cell body follows by:
coupling of myosin contraction with de-adhesion at the rear of the cell.

Filament nucleation (regulated by Arp 2/3) and PLUS end polymerization occurs at the leading edge- pushing the membrane forward.

• ATP-hydrolysis in the filament (T-form to D-form actin)
• Cofilin preferentially binds to actin filaments containing D-form actin. Cofilin destabilizes the filaments predisposing them to depolymerization.

Myosin II:

• Contractile force required for retracting the trailing edge.
• Can pull actin filaments at the rear in a new direction.

Question 36

Which protein family can locally regulate the actin cytoskeleton by extracellular signals?

Answer 36

Rho protein family: Cdc43, Rac, Rho.
Monomeric GTPases, activated by guanine nucleotide exchange factors (GEFs)

Cdc42 => actin polymerization and bundling to form filopodia.
Rac => actin polymerization at the cell periphery -> sheetlike lamelipodial extensions.
Rho => bundling of actin filaments with myosin II filaments into stress fibers and the clustering of integrins and associated proteins to form focal adhesions.

Question 37

Which protein family can locally regulate the actin cytoskeleton by extracellular signals?

Answer 37

Rho protein family: Cdc43, Rac, Rho.
Monomeric GTPases, activated by guanine nucleotide exchange factors (GEFs)

Cdc42 => actin polymerization and bundling to form filopodia.
Rac => activate Arp2/3 complex, actin polymerization at the cell periphery -> sheetlike lamelipodial extensions.
Rho => bundling of actin filaments with myosin II filaments into stress fibers and the clustering of integrins and associated proteins to form focal adhesions.

Question 38

What is the role of stathmin relative to microtubules?

Answer 38

Destabilization.
Binds to two tubulin heterodimers and prevents their addition to the ends of microtubules.
Decreases the concentration of tubulin subunits that are available for polymerization.

Phosphorylation inhibits interaction.