lecture 2

Importance of the Actin Cytoskeleton

  • The actin cytoskeleton is essential for normal cell function and motility

  • Defects in the actin cytoskeleton can lead to diseases such as:

    • Cardiomyopathies (Defect affecting the contractile apparatus of the heart)

    • Diseases of red blood cells that affect cytoskeletal components supporting the plasma membrane

    • Unregulated motility in metastatic cancer cells, allowing them to break away from their tissue of origin and migrate to new locations

Dynamics of Actin Filaments

  • Highly dynamic

  • Found in highly dynamic structures consisting of bundles or branched networks

  • Subject to constant depolymerization and repolymerization events

  • Can lead to changes in cell shape, locomotion, and intracellular movements

Actin Organization in Motile Cells

  • Microfilaments (actin) can organize into many different structures within a cell, each underlying a particular function and formed in response to a signalling cue

  • Disruption of the actin cytoskeleton with Latrunculin A abolishes cell motility, demonstrating the essential role of actin organization for cell crawling

Actin as the Building Block of Microfilaments

  • Basic building block of microfilaments

  • Can reversibly assemble into polarized filaments with functionally distinct ends

  • Comprising 10% of total cell protein in muscle cells and 1-5% in non-muscle cells

  • Different actin isoforms are found in various cell types, with distinct functions

Actin Filament Construction

  • Actin exists as a globular monomer (G-actin) and a filamentous polymer (F-actin)

  • G-actin contains a Mg2+ ion complexed to either ATP or ADP, and has a central cleft that allows for conformational changes

  • Actin monomers assemble into long, helical F-actin polymers, with all subunits pointing in the same direction, giving the filament polarity

  • One repeating unit of the F-actin filament consists of 28 subunits (14 in each strand) covering a distance of 72 nm

Actin Polymerization

  • Actin polymerization occurs in three stages: nucleation, elongation, and steady state (Actin polymerisation)

  • The critical concentration is the free G-actin monomer concentration required for polymerization to occur (Critical Concentration)

  • The two ends of an actin filament grow at different rates, with the "barbed" (+) end growing faster than the "pointed" (-) end

Actin Treadmilling

  • Powered by ATP hydrolysis, with ATP-G-actin adding preferentially to the (+) end and ADP-actin dissociating from the (-) end

  • The conformational change in actin during ATP hydrolysis promotes the disassociation of ADP-actin from the (-) end

  • Accelerated by the actin-binding proteins cofilin and profilin

Regulation of Actin Dynamics

  • Cofilin binds to ADP-actin and induces filament severing, increasing the availability of ADP-actin subunits for recharging by profilin

  • Profilin binds ADP-G-actin, enhancing the loss of ADP and promoting the formation of ATP-G-actin, which can then bind to the (+) end

  • Thymosin-β4 sequesters a reservoir of G-actin, preventing its polymerization until needed

  • Capping proteins, such as CapZ and tropomodulin, bind to the (+) and (-) ends of filaments, respectively, to regulate monomer addition and loss

  • Gelsolin can bind to the (+) end of filaments and sever them, generating new (-) ends that can disassemble

 

Actin-Binding Protein

Function

Cofilin

Binds ADP-actin, induces filament severing

Profilin

Binds ADP-G-actin, enhances loss of ADP and promotes ATP-G-actin formation

Thymosin-β4   

Sequesters a reservoir of G-actin, preventing polymerization

CapZ

Binds to (+) end, inhibits subunit addition and loss

Tropomodulin

Binds to (-) end, inhibits subunit addition and loss

Gelsolin

Binds to (+) end, severs filaments