Cytoskeleton: Actin Dynamics, Nucleation, Motors, and Adhesions — Lecture Notes (Comprehensive)

Actin Filaments: Structure, Polarity, and Dynamics

The cytoskeleton comprises actin filaments, microtubules, and intermediate filaments; actin filaments are one of the three major cytoskeletal structures.
Actin is one of the most abundant proteins in the cell (roughly ~20–70 µM; up to ~1 mM in muscle cells).
G-actin (globular actin) is the monomer; F-actin (filament) is the polymer; G-actin monomers assemble into a two-stranded helical filament with a diameter of ~5–9 nm.
Filament polarity is defined by the two ends: the plus (barbed) end and the minus (pointed) end. G-actin monomers have polarity, which translates into filament polarity.
Actin monomer asymmetry defines filament polarity and influences assembly and disassembly dynamics.
The actin macrostructure varies within the cell; examples include stress fibers, filopodia, cell cortex, lamellipodia, contractile bundles, gel-like networks, dendritic networks, tight parallel bundles, etc.
Actin filaments can form different architectures within cells to fulfill diverse functions such as locomotion, shape maintenance, and division.
Actin filaments are regulated by accessory proteins that control nucleation, elongation, capping, severing, and crosslinking; the cytoskeleton is highly dynamic and remodels constantly.
In summary, actin cytoskeleton provides shape, mechanical strength, movement, and intracellular organization/transport; its assembly/disassembly is tightly regulated by nucleators, monomer availability, and motor proteins.

Actin Polymerization: Structure and Dynamics

Actin filaments are composed of polymerized actin monomers (G-actin) arranged in a two-stranded helical filament; diameter ~5–9 nm.
The actin filament exhibits polarity, with a plus (barbed) end and a minus (pointed) end.
Actin polymerization involves nucleation, elongation, and a steady-state phase.
The G-actin concentration and end-specific critical concentrations govern filament dynamics.
Key numeric facts:
- Filament diameter: ~5–9 nm
- Actin monomer concentration in most cells: ~20–70 µM (up to 1 mM in muscle cells)
- Filament length and network organization depend on local monomer availability and regulatory proteins
The critical concentration concept:
- The critical concentration (Cc) is the monomer concentration at which a filament neither grows nor shrinks.
- For a given end,
 $Cc = \frac{k{\text{off}}}{k_{\text{on}}}$
- The plus end and minus end can have different critical concentrations, denoted $Cc^+$ and $Cc^-$ , respectively.
- If the G-actin concentration [G-actin] is greater than the plus-end Cc, the plus end tends to grow; if [G-actin] is less than Cc^+, it tends to shrink.
- If [G-actin] is between Cc^+ and Cc^-, the filament may treadmill: the plus end grows while the minus end shrinks, maintaining a steady subunit number but with monomer exchange.
- Treadmilling condition:
 $Cc^+ < [G ext{-actin}] < Cc^-$
The actin polymerization cycle involves four steps (conceptual, as in the provided slides):
- Step 1: ATP-bound actin monomer binds to the filament end (often the plus end).
- Step 2: ATP bound to the incorporated monomer slowly hydrolyzes to ADP while bound in the filament, leaving ADP-actin.
- Step 3: ADP-bound actin dissociates from the filament ends more readily than ATP-bound actin, contributing to turnover.
- Step 4: ADP is exchanged for ATP on a monomer in solution (the G-actin pool replenishment), allowing ready incorporation again at the plus end.
- In formula form, the cycle can be summarized by:
  $\text{ATP-actin} \rightarrow \text{ADP-actin} \;(+\;\text{Pi}) \rightarrow \text{ADP-actin dissociation} \rightarrow \text{ATP-exchange on free monomer} \rightarrow \text{reincorporation}$
What happens if ATP-bound monomers are supplied faster than hydrolysis to ADP: the dynamics shift toward more rapid incorporation; the exact balance between addition and hydrolysis sets the growth rate.
The association and dissociation rates depend on kon (association rate constant) and koff (dissociation rate constant):
- Plus-end growth rate depends on kon and [G-actin]:
 $r+ = k{on}^{+} [G ext{-actin}] - k_{off}^{+}$
- Minus-end dissociation depends on koff at the minus end, often with distinct kinetics $k_{off}^{-}$ .
Nucleation is the rate-limiting step for actin filament polymerization (short lag phase before elongation).
Actin monomer availability regulates polymerization; two key regulators are:
- Thymosin: sequesters actin monomers, reducing binding to ends.
- Profilin: facilitates binding to the plus end and promotes addition of actin to the growing filament.

Nucleation: Formin

Formin proteins nucleate the formation of linear actin filaments and promote elongation.
Mechanism:
- Formin binds the profilin-ATP–F-actin complex, helping deliver actin monomers to the growing barbed end.
- Formin prevents binding of plus-end capping proteins, allowing extended elongation and formation of long F-actin stress fibers.
- Formin activity is activated by the small GTPase Rho.
Outcome: increased nucleation and sustained elongation of linear actin filaments, contributing to stress fiber formation.

Nucleation: Arp2/3 Complex and Actin Branching

Arp2/3 is a protein complex that nucleates new actin filaments as branches off existing filaments, creating a dendritic or branched network.
Arp2/3 forms a new filament at approximately a 70° angle relative to the mother filament.
Activation and function:
- The Arp2/3 complex is activated by specific nucleation-promoting factors (activating factors) and other cofactors.
- Once activated, Arp2/3 binds to a mother filament and initiates a daughter filament, creating a branched network that supports lamellipodial structures.
Role in cells: generates a dense, branched actin network critical for cell propulsion, endocytosis, and membrane protrusion.

Rho-family Small GTPases and Regulation

Key family: Rho, Rac, and Cdc42; these are molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state.
Regulation mechanisms:
- GEF (guanine exchange factor) activates GTPases by promoting GDP-to-GTP exchange.
- GAP (GTPase-activating protein) inactivates GTPases by accelerating GTP hydrolysis.
- GDI (GDP-dissociation inhibitor) prevents activation and recruitment to membranes.
General properties:
- Most small GTPases are off when GDP-bound and on when GTP-bound.
- GTP hydrolysis energy is not used to drive enzymatic reactions per se; instead, hydrolysis acts as a switch to regulate activity.
Roles in actin dynamics:
- Rho activates formin to nucleate linear F-actin.
- Rac and Cdc42 generally promote branched networks and filopodia, depending on context and effectors.
Regulation of Rho-family GTPases is essential for spatial and temporal control of actin assembly and cell morphology.

Actin-Based Force Generation and Motor Proteins

Actin polymerization can generate protrusive force against membranes, enabling cell motility and shape changes.
Myosin II as a primary actin-based motor protein:
- Structure: a dimer of dimers forming bipolar thick filaments.
- Mechanism: movement of myosin head groups toward the plus-ends of anti-parallel actin filaments drives contraction.
- The cycle involves ATP binding, hydrolysis, and product release that powers conformational changes and force generation.
In muscle cells, myosin II participates in sarcomere contraction where thick (myosin) and thin (actin) filaments interact to shorten the sarcomere.

Integrins and Focal Adhesions: Linking Cytoskeleton to ECM

Integrins: heterodimeric transmembrane receptors that bind extracellular matrix (ECM) and indirectly link to actin filaments inside the cell.
Linker proteins: vinculin, talin, tensin, α-actinin connect integrins to actin networks; these interactions help transmit contractile forces to the ECM.
Focal adhesions: clusters where multiple contractile actin bundles connect to clustered integrins, serving as hub for signaling and force transmission.
Important note: Integrin-mediated adhesion anchors cytoskeletal networks to the ECM, enabling cell migration, signaling, and stability.

Muscle Contraction: From The Neuromuscular Junction to The Sarcomere

Skeletal muscle structure:
- Long, multinucleated fibers composed of myofibrils.
- Myofibrils contain repeating units called sarcomeres, bounded by Z discs.
Key components:
- Actin (thin filaments) and myosin II (thick filaments) arranged in a contractile lattice.
- Cross-bridge cycling between myosin heads and actin filaments drives contraction.
Initiation and regulation of contraction:
1) Acetylcholine binds nicotinic receptor at the neuromuscular junction, triggering an action potential.
2) Voltage-gated Ca2+ channels release Ca2+ from the sarcoplasmic reticulum.
3) Ca2+ binds to troponin C, which allows tropomyosin to move away from myosin-binding sites on actin.
4) Myosin heads bind to actin, perform the power stroke, and cause sarcomere shortening.
The force-generating ATP cycle of myosin II underlies muscle contraction and is central to movement and force generation in non-muscle cells as well.

Listeria Monocytogenes, Vaccinia, and Actin-Based Propulsion

Certain opportunistic bacteria (e.g., Listeria monocytogenes) and some viruses hijack the host cell actin cytoskeleton to propel themselves through the cytoplasm.
Listeria utilizes actin polymerization at its surface to form comet tails that push the bacterium forward, enabling intercellular spread and invasion.
Vaccinia virus can also exploit actin-based motility for intracellular movement and spread.
These examples illustrate how actin polymerization can generate propulsion and enable intracellular movement of pathogens.

Summary: Key Concepts and Connections

Cytoskeleton provides shape, mechanical strength, motility, and intracellular organization/transport.
Actin is a highly dynamic polymer whose assembly is regulated by monomer availability and a suite of nucleators and regulatory proteins (Formin, Arp2/3, profilin, thymosin).
Formin promotes linear filament nucleation and elongation, activated by Rho GTPases; Arp2/3 generates branched networks, activated by specific factors.
Rho-family GTPases (Rho, Rac, Cdc42) act as molecular switches that regulate actin dynamics via GEFs, GAPs, and GDIs, coordinating cytoskeletal remodeling.
Actin dynamics produce force, driving processes such as cell migration, cytokinesis, and organelle positioning; myosin II converts chemical energy from ATP into mechanical work to generate contractile force.
Integrins and focal adhesions couple the actin cytoskeleton to the ECM, enabling traction and signaling necessary for cell movement and stability.
The actin cytoskeleton interacts with pathogens; actin-based motility is exploited by certain bacteria and viruses to move within cells.

Practice Exam Questions (with Answers)

Which cellular function is primarily associated with actin?
- A) Synthesis of DNA
- B) Energy production through glycolysis
- C) Cell division by meiosis
- D) Muscle contraction and cell movement
- E) Regulation of blood pressure
- Answer: D
What is the primary role of myosin in cells?
- A) Microtubule plus-end motor
- B) Microtubule minus-end motor
- C) Actin filament plus-end motor
- D) Actin filament minus-end motor
- Answer: C
What is the main function of the Arp2/3 complex in cellular processes?
- A) DNA replication
- B) ATP synthesis
- C) Cell division by mitosis
- D) Regulation of gene expression
- E) Nucleation of actin filaments in branching networks
- Answer: E
Which cell surface molecules play a key role in interacting with the extracellular matrix and facilitating adhesion to surrounding tissues?
- A) Myosin
- B) Rho GTPases
- C) Integrins
- D) Arp2/3
- Answer: C

References and Context

Slides derived from Alberts, Molecular Biology of the Cell, with emphasis on actin structure, dynamics, nucleation, and force generation; Formin and Arp2/3 as major nucleators; Rho-family regulation; Myosin II; Integrins and focal adhesions; and pathogenic actin-based motility (Listeria, vaccinia).