Cell Bio Exam 3

Actin

A cytoskeletal helical polymer 7nm in diameter, made up of globular monomers, and organized into bundles and networks. Found abundantly in G or F form in the cytoplasm, plasma membrane, and cell junctions. Locomotion, cell shape changes, cytokinesis, and adhesion functions.

G-Actin (Globular Actin)

Actin monomer molecule. Is split into two lobes with a cleft in the middle for binding ATP/ADP complexed with Mg2+. This cleft, being on only one side of the molecule, gives the monomer intrinsic polarity.

F-Actin (Filamentous Actin)

Helical polymer of G-actins held together with hydrophobic and ionic interactions. All monomers in a filament are oriented the same way, with each cleft end bound to the non-cleft end of the next, making it a polar polymer.

Actin Polymerization (in vitro)

Initiated by adding Mg2+ and/or K+ salts to pure G-actin. If salt removed, depolymerization occurs. 3 steps: nucleation, elongation, steady state.

Actin Polymerization Monitoring

Light scattering (more from polymers than monomers), fluorescent pyrene-actin that gets brighter as F-actin, other fluorescence, electron microscopy, sedimentation analysis (F sediments more readily because larger).

Nucleation (Actin)

Formation of a stable “seed”/“nucleus” of three monomers. Dimers are unstable, causing lag phase when no nucleating factors are added.

Actin Critical Concentration (Cc)

When the rate of assembly equals the rate of dissociation of polymers, or when the concentrations of G-actin and F-actin are in equilibrium. Rate is increased by increasing monomer concentration and vice versa.

Actin Treadmilling

In presence of ATP, polymerization elongates the filament until monomer concentration falls between the two ends’ Cc’s, then begins. Actin filaments grow at the + end and shrink at the - end, causing a flux of G-actin “through” the filament. Requires ATP hydrolysis.

Steady State (Actin)

When there is active treadmilling but no net filament growth or shrinkage.

Actin-Binding Proteins (ABPs)

Profilin, cofilin,

Profilin

Binds actin monomers to increase rate of polymerization (speeds elongation).

Cofilin

A severing protein that severs actin filaments, creating more minus ends and leading to increased actin depolymerization (filament falls apart).

Actin Sequestering Proteins

Thymosin beta4, profilin, cofilin.

Thymosin beta4

An actin sequestering protein that is abundant in cells and is the primary monomer sequestering protein.

Profilin

An actin sequestering protein that promotes G-actin to exchange ADP for ATP. Binds g-actin’s + end, inhibiting nucleation of pure G-actin and promoting actin polymerization into existing filaments. Competes with Thymosin beta4, controlling filament growth.

Cofilin

Interacts with and severs ADP-actin filaments, leading to increased - end depolymerization or growth from new + ends.

Actin Nucleating Proteins

Formin, tandem actin monomer-binding proteins, Arp2/3 and WASP complex. Accelerate the initial kinetics of polymerization. Control cell shape, movement, and division during health and disease.

Formins

An actin nucleator family (15 types) that generate long, unbranched filaments. Dimerize and facilitate + end growth while remaining attached.

Tandem Actin Monomer-Binding Proteins

Nucleate unbranched filaments. May cooperate with formins, and remain at the - end during filament assembly.

Arp2/3 Complex

The only actin nucleator known to produce branched filaments (sit at branch point, holding new on old). Work in conjunction with WASP family proteins. Neither alone is a strong nucleator, but together they cause formation of a new actin filament. New filament is capped at the - end.

Phalloidin

Derived from the “death cap” mushroom. Binds at the interface of filament subunits and prevents depolymerization.

Cytochalasin

Fungal alkyloid that binds to the + end of actin filaments, capping them and preventing elongation. Filaments depolymerize due to Cc at the + end reaching that of the - end. Effects are reversible.

Latrunculin

Derived from a sea sponge. Binds to actin monomers, sequestering them so they are unable to polymerize into filaments. Causes rapid disassembly of filaments. Effects are reversible.

Microtubules

Polymers of tubulin subunits arranged in cylindrical filaments ~24nm in diameter. Are polar, with + ends that grow faster than the - ends. Composed of 11-15 linear protofilaments. Hollow structures with outer ridges for binding to other proteins.

Tubulin Heterodimers

The subunits of microtubules. Consists of one alpha-tubulin and one beta-tubulin, which never come apart under physiological conditions.

Alpha-Tubulin

One subunit of tubulin heterodimers. Binds GTP irreversibly. Forms a ring around the - end.

Beta-Tubulin

One subunit of tubulin heterodimers. Binds GTP. Can hydrolyze GTP to GDP, and exchange GDP for GTP. Forms a ring around the + end.

MT Protofilaments

Interact laterally to form MT cylinder. Composed of alternating alpha and beta heterodimers. Each has intrinsic polarity, so when aligned with the same orientation, the MT itself is polar.

MT Organizing Center (MTOC)/Centrosome

Minus ends of MTs are usually capped and embedded in this.

MT Assembly Lag Phase

The period when nucleation is taking place. Once enough nuclei are formed, elongation occurs rapidly.

Microtubule Polymerization (Steps)

Nucleation, elongation, steady state.

Nucleation

Formation of a stable seed of oligomers.

Oligomers

Small polymers. Only a few heterodimer subunits.

Steady State

When the free tubulin concentration reaches Cc. There is no net increase or decrease in the amount of microtubules.

MT Critical Concentration (Cc)

When the concentration of tubulin is in equilibrium with microtubules.