Premed · Premed · Cell Biology
Lecture 16: The Cytoskeleton: Actin Filaments
Cell Biology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the structure, assembly, and polarity of actin filaments
- Explain the treadmilling and dynamic behavior of actin filaments
- Describe the roles of key actin-binding proteins in filament regulation
- Explain how actin is organized into different cellular structures
- Discuss the role of Rho family GTPases in regulating the actin cytoskeleton
Lecture Content
I. Actin Filament Structure and Properties
Actin is one of the most abundant proteins in eukaryotic cells, constituting approximately 5 to 10 percent of total cellular protein. G-actin (globular actin) is a 42 kDa monomer that binds one molecule of ATP (or ADP) and one divalent cation (Mg2+ or Ca2+). Mammalian cells express several isoforms: alpha-actin in muscle, and beta- and gamma-actin in non-muscle (cytoplasmic) cells.
F-actin (filamentous actin) is the polymerized form, consisting of two intertwined helical strands forming a right-handed double helix approximately 7 nm in diameter (the thinnest of the three cytoskeletal filaments) with a helical repeat every 37 nm (approximately 13 subunits per half-turn). A defining feature of actin filaments is their structural polarity: all subunits are oriented in the same direction, creating two distinct ends. The plus end (barbed end) grows faster and is the preferred site of monomer addition, while the minus end (pointed end) grows more slowly and is the preferred site of monomer loss. This polarity can be visualized by decorating filaments with the S1 fragment of myosin, which creates a characteristic arrowhead pattern.
ATP hydrolysis is intimately linked to filament dynamics. G-actin-ATP adds preferentially to the plus end. After incorporation, ATP is hydrolyzed to ADP plus inorganic phosphate (Pi) with a characteristic delay. Subsequent release of Pi converts the subunit to ADP-actin, which is less stable in the filament. This creates an ATP cap at the growing plus end and an ADP-actin core toward the minus end, a structural asymmetry that drives the phenomenon of treadmilling.
II. Actin Polymerization and Treadmilling
In vitro, actin polymerization proceeds through three phases. The nucleation (lag) phase is rate-limiting and involves the formation of a stable actin trimer that serves as a nucleus for filament growth. The elongation phase follows, with rapid addition of monomers to both ends (faster at the plus end). At steady state, the rate of monomer addition at the plus end equals the rate of monomer loss at the minus end.
The critical concentration (Cc) is the free G-actin concentration at which the polymerization rate exactly equals the depolymerization rate. Because the kinetics differ at each end, the Cc at the plus end (approximately 0.1 micromolar) is lower than the Cc at the minus end (approximately 0.8 micromolar). When the free G-actin concentration falls between these two values, net addition occurs at the plus end while net loss occurs at the minus end. This is treadmilling, a phenomenon in which subunits effectively flow through the filament from the plus end to the minus end. Treadmilling is crucial for cell motility and the dynamic remodeling of actin-based structures, and in living cells, it is greatly accelerated by actin-binding proteins.
Several drugs targeting actin are important research tools. Cytochalasin D caps the plus end and prevents polymerization. Latrunculin sequesters G-actin monomers. Phalloidin binds and stabilizes F-actin, preventing depolymerization; fluorescently labeled phalloidin is widely used to stain F-actin in microscopy. Jasplakinolide stabilizes actin filaments and promotes polymerization.
<image>Actin filament structure and treadmilling. Panel A: G-actin monomer showing ATP-binding cleft, and the polymerization of G-actin into F-actin double helix with plus (barbed) and minus (pointed) ends labeled. Diameter 7 nm and helical repeat 37 nm indicated. Panel B: Treadmilling at steady state — ATP-actin monomers (green) adding at the plus end, ATP hydrolysis and Pi release converting subunits to ADP-actin (red) through the filament core, ADP-actin dissociating from the minus end. Arrows show the net flux of subunits through the filament. Panel C: Graph of actin polymerization in vitro showing three phases: lag (nucleation), elongation, and steady state.</image>
III. Actin-Binding Proteins
Hundreds of actin-binding proteins regulate filament dynamics, organization, and function in the cell. Nucleation factors overcome the kinetic barrier to filament initiation. The Arp2/3 complex, activated by WASP/WAVE family nucleation-promoting factors, binds the side of an existing filament and nucleates a new branch at a characteristic 70-degree angle, creating the dense branched networks that drive lamellipodia formation and endocytic actin patches. Formins (such as mDia) nucleate and elongate unbranched filaments, processively moving with the growing plus end while acting as a "leaky cap." Formins drive the formation of filopodia, stress fibers, and the contractile ring during cytokinesis.
Capping proteins control filament length. CapZ caps the plus end, preventing both addition and loss of subunits. Tropomodulin caps the minus end and is important for stabilizing actin in the muscle sarcomere.
Severing and depolymerization factors promote filament turnover. Cofilin/ADF binds ADP-actin subunits in the filament, induces a twist change that severs the filament, and promotes depolymerization from the minus end. By generating new free ends, cofilin works in concert with Arp2/3 to increase filament turnover. Cofilin activity is regulated by phosphorylation: LIMK phosphorylation inactivates it, while the phosphatase Slingshot activates it. Gelsolin is a Ca2+-activated severing protein that also caps the newly created plus ends.
Monomer-binding proteins regulate the pool of available G-actin. Profilin binds G-actin-ADP and promotes the exchange of ADP for ATP, delivering ATP-actin to the plus end (especially to formins) while inhibiting spontaneous nucleation. Thymosin beta-4 sequesters G-actin monomers and serves as the major actin buffer in cells.
Cross-linking and bundling proteins organize filaments into higher-order structures. Filamin cross-links filaments into orthogonal gel-like networks. Alpha-actinin bundles filaments into loose parallel arrays found in stress fibers and sarcomeres. Fimbrin/plastin tightly bundles filaments in microvilli and filopodia. Fascin bundles filaments specifically in filopodia.
IV. Actin-Based Cellular Structures
The diverse actin-binding proteins described above organize actin filaments into distinct cellular structures, each with a specialized function. Lamellipodia are broad, flat, sheet-like protrusions at the leading edge of migrating cells, composed of dense branched actin networks generated by Arp2/3. They push the membrane forward during cell migration. Filopodia are thin, finger-like protrusions containing parallel bundles of actin filaments nucleated by formins and bundled by fascin. They serve sensory and exploratory functions and help cells adhere to substrates.
Stress fibers are thick contractile bundles of actin filaments and myosin II, anchored to focal adhesions at cell-substrate contacts. They generate contractile force and are important for cell shape and mechanotransduction. The contractile ring, assembled during cytokinesis from actin and myosin II, constricts at the cleavage furrow to divide the cell in two.
Microvilli are finger-like projections on the apical surface of epithelial cells, with a core of parallel actin bundles cross-linked by fimbrin and villin. They dramatically increase the surface area available for absorption, as in the intestinal epithelium. The cell cortex is a thin actin meshwork beneath the plasma membrane, linked to the membrane by ERM proteins (ezrin, radixin, and moesin). It determines cell shape and mechanical properties.
<image>Actin-based cellular structures. Panel A: Migrating cell showing lamellipodia at the leading edge (zoom inset: branched Arp2/3-mediated actin network), filopodia extending beyond the lamellipodium (zoom inset: parallel bundled actin filaments with fascin cross-links), and stress fibers spanning the cell body (zoom inset: alternating actin and myosin II filaments connected to focal adhesions at the substrate). Panel B: Intestinal epithelial cell with microvilli on the apical surface (zoom inset: parallel actin core with fimbrin/villin cross-links, attached to the terminal web at the base). Panel C: Contractile ring during cytokinesis showing actin and myosin II ring at the cleavage furrow.</image>
V. Rho Family GTPases: Regulators of the Actin Cytoskeleton
The Rho family GTPases are small monomeric GTPases of the Ras superfamily that act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. They are regulated by GEFs (which activate by promoting GDP-to-GTP exchange), GAPs (which inactivate by stimulating GTP hydrolysis), and GDIs (which sequester the inactive GDP-bound form in the cytoplasm).
Three major Rho GTPases control distinct aspects of actin organization. RhoA promotes the formation of stress fibers and focal adhesions by activating ROCK (Rho-associated kinase), which phosphorylates myosin light chain to stimulate contractility, and mDia (a formin) for unbranched actin polymerization. Rac1 drives lamellipodia formation by activating the WAVE complex, which in turn activates Arp2/3 to generate branched actin networks and membrane ruffling. Cdc42 promotes filopodia formation by activating WASP, which activates Arp2/3 for actin polymerization at filopodial tips, and also activates formins. Cdc42 additionally plays a central role in establishing cell polarity through the Par6-Par3-aPKC polarity complex.
These GTPases engage in extensive crosstalk: Cdc42 can activate Rac, and Rac and Rho can reciprocally inhibit each other. Rho GTPases are also important targets of bacterial virulence factors: C3 transferase from Clostridium botulinum ADP-ribosylates and inactivates Rho, while CNF1 from E. coli activates Rho, Rac, and Cdc42.
VI. Actin in Disease
Several diseases highlight the biological importance of actin regulation. Listeria monocytogenes is an intracellular pathogen that hijacks the host actin cytoskeleton. Its surface protein ActA mimics WASP to activate Arp2/3, generating an actin "comet tail" that propels the bacterium through the cytoplasm and into neighboring cells. Wiskott-Aldrich syndrome, an X-linked disorder caused by mutations in the WASP gene, produces defective actin regulation in hematopoietic cells, leading to immunodeficiency, thrombocytopenia, and eczema. In cancer, actin dynamics are fundamental to metastatic invasion. Invadopodia, which are actin-rich protrusions that degrade the extracellular matrix through Arp2/3 and cortactin-dependent mechanisms, are key structures in tumor cell invasion. ROCK inhibitors and actin-targeting drugs are being explored as anti-metastatic agents.
