16: The Cytoskeleton Flashcards
(38 cards)
What are the three families of protein filaments related to the cytoskeleton?
Actin filaments
Microtubules
Intermediate filaments
What are the main roles of actin filaments?
Determine the shape of the cell’s surface.
Whole-cell locomotion.
Drive the pinching of one cell into two.
What are the main roles of mictotubules?
Determine the position of membrane-enclosed organelles.
Direct intracellular transport.
Form the mitotic spindle.
What is the main role of intermediate filaments?
Provide mechanical strength.
Where are actin filaments most highly concentrated?
Give some examples of different actin filaments
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
Where in the cell does one find microtubules?
Give some examples of different types
In a cytoplasmic array that extends to the cell periphery.
Examples:
- Mitotic spindle
- Cilia
Where in the cell does one find intermediate filaments?
Give some examples of functions
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.
How are cytoskeleton subunits assembled?
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.
Describe the actin subunit and polymer
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
What is filament nucleation and polymerization?
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
How is actin nucleation regulated?
Key words: formins, Arp 2/3, cofilin
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.
Why do the plus ends of actin and tubulin filaments grow faster than the minus ends?
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
How do the critical concentration and the rate constants for assembly/disassembly of subunits to actin change when the nucleotide is hydrolyzed?
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
What is filament treadmilling?
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
How is actin filament extension regulated by thymosin and profilin?
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.
What is dynamic instability and in which cytoskeleton filament does it predominate?
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
How are myosin and myosin filaments built?
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.
How does myosin generate muscle force?
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:
- Attached (no ATP bound)
- Released (ATP-binding => conf. change)
- Cocked (ATP-cleft closes, ATP hydrolysis->ADP+Pi bound, movement in lever arm=> 5 nm movement)
- Force-generating (Pi released as head binds tightly to actin => power stroke, ADP off, return to the start of the cycle)
What is a myofibril?
Key words: size, contractile units
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)
What are sarcomeres?
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)
What is sarcomere shortening? What happens to the thick/thin filaments during the process?
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)
How are the sarcomere filaments organized?
Length, spacing, accessory proteins, etc.
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.
Which ATP-consuming processes are related to muscle contraction?
- Filament sliding, driven by the ATPase of the myosin motor domain (on head)
- Ca2+ pumping driven by the Ca2+ pump (Ca2+-ATPase)
How is muscle activity regulated by Ca2+ and its accessory proteins?
Keywords: T tubules, SR, ATP, accessory proteins
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
