BIO 212 Topic 22: The Cytoskeleton—Actin Filaments

Actin filaments

• form long microfilaments

• 7-8 nm, smaller than the width of the PM

• often bundle together and associate with other proteins

• no single point of origin; originates at lots of locations around the cell where there are connections between cells and other cells (cell-cell junctions, such as cadherins), and cells and ECM (cell-ECM junctions, such as integrins)

Actin abundance

• found in all eukaryotic cells, and some prokaryotes also have actin-like filaments

• most abundant proteins in animals, composing about 5% of the total protein in a typical animal cell (very impressive, considering the average cell has about 10-20,000 types of proteins)

• 2nd most abundant protein on Earth, after RUBISCO

Actin monomers

• noncovalently attached G-actin (globular actin) monomers

• very average; lumpy spherical/globular chape, secondary structure has lots of alpha helices and loops, some beta sheets, average size (375 AAs, 42 kDa)

• has an ATP-binding cleft where the nucleotide fits

• structural polarity; the side with the binding cleft and the side without

• G-actin has intrinsic ATPase activity

Actin family

• 6 genes in humans

• beta and gamma actin are expressed in nonmuscle cells, where they allow cells to have contractile activity associated with moving the cell around

• beta and four different alpha actins are expressed in muscle cells

• isoforms are sorted into different structures

Microfilament structure

• composed of F-actin (filamentous/polymerized actin)

• 2-stranded helix, like 2 strings of beads wrapped around each other

• 7-8 nm diameter

• structural polarity; + end is at the ATP-binding cleft, and - end is the other end (unlike microtubules, actin filamnets have no gamma TuRC, and the - end can be exposed, so the addition and removal of monomers is possible from either end)

F-actin assembly

• G actin can add to or dissociate from either end if not bound to anything, but the + end has an affinity for new monomers that is about 5-10 times the - end affinity (+ end is favored for addition)

• G actin (high-affinity state, polymerized) undergoes hydrolysis, resulting in a conformational change; ADP-actin is in the low-affinity state, and the monomer can dissociate

• Wave of ATP hydrolysis after the monomers are added to the filament

Factors affecting F-actin assembly

• concentration of G-actin; nucleates and elongates more quickly as concentration increases

• association of proteins which help or inhibit nucleation and elongation

Nucleation

• since there is no gamma TuRC, the actin filament must start itself

• tiny fiber/starting filament called the nucleus serves as the base for further addition

• the creation of the nucleus is slow, but can be speeded by proteins which act as the base

Elongation

• much faster than nucleation, but can be prevented by things such as the protein protillin

Decreased [G-actin]

• relatively high concentration: net addition to both ends; filament grows

• relatively low concentration: net addition to the + end, net loss from the - end—filament grows in the direction of the + end but shrinks in the - end due to difference in affinity

• 0.1 micromolar: + end addition equals - end loss; filament begins treadmilling (like the tread on the treadmill, actin monomers are added to the + end as fast as they are lost from the - end

Phalloidin

• toxin from death cap mushroom

• freezes cell movement/whole cell motility by altering actin disassembly—prevents F-actin depolymerization

Cytochalasin

• freezes whole cell motility by preventing ATP-actin addition to the + end

Cytosolic [G-actin]

• ~ 50-100 micromolar—is a very high concentration of proteins, but only ½ of the total cellular actin can be found in microfilaments

• there are very few “free” G-actins due to monomer sequestering proteins (e.g. protillin) which prevent addition into filaments

• actin polymerization and filaments are highly regulated

Actin binding proteins

• over 60 families

• filament nucleating

• monomer sequestering

• end-blocking/capping

• monomer polymerizing

• depolymerizing

• bundling

• filament-severing

• cross-linking

• a lot of the dynamics for actin come from other proteins, compared to microtubules, which are responsible for their own dynamics

Non-muscle actin skeleton

• very dynamic

• concentrated at the leading edge of motile cells; lots of F-actin anchored at the plasma membrane

• tail/trailing end must break connections to retract the back end (simply rips off the cytoplasm if not quick enough)

• underlying actin cortex

Leading edge (motility)

• as actin filaments polymerize, they push the membrane outward and form characteristic structures

• filopodia (finger-feet) form F-actin bundles that poke at the plasma membrane and extend dramatically outward

• lamellipodia (sheets-feet) cover a broad area and are formed of an F-actin meshwork

• focal contacts/adhesions—actin filaments bind directly to integrins, which bind to the ECM/substrate and pull the cell forward

• F-actin polymerization drives plasma membrane extension

Stress fibers

• traverse the cell

• attach by adaptor proteins to form focal contacts/adhesions with the integrin transmembrane receptors

• put foot down; contract actin-myosin complexes and move the cell body forward

• extensive branching is facilitated by proteins

Arp 2/3 complex

• nucleates F-actin stress fibers (?)

• nucleates at the - end; + end faces and grows outward

Cell movement

• myosin-actin contraction at the edge of the cell body provides the force to pull the cell along

• myosins have their ends bound to actin filaments and walk along them to pull them/contract

Actin-dependent motor proteins

• look like really skinny and long kinesins, with a very long coiled coil central region/tail and two heads

• myosins

Myosins

• over 40 different kinds in humans

• great variety in non-muscle cells

• non-muscle (often non-conventional) and muscle (often conventional) forms

• 2 identical motor heads; release of inorganic phosphate after ATP hydrolysis strengthens myosin’s binding affinity to actin, resulting in a “power stroke” that pulls the actin filament along

Myosin II

• “conventional myosin”

• very long tails

• non-muscle and muscle forms

• Heads have ATPases and bind actin—change conformation, throwing the head forward and binding actin —> ATP hydrolysis —> power stroke

  • Head-over-head movement

• tails: double-helical rod/coiled coil which interact with cargo and are very long (can be 150 nm)

• Movement toward the + end of the actin filament

Myosin II structure

• head and tail is composed of two intertwined heavy chains (dimer), which is associated with an essential light chain and a variety of regulatory light chains

Contractile bundles (non-muscle)

• bundles are transient

• contractile elements are much less static and often include “unconventional myosins,” which may have much shorter tails and act more like kinesins

Contractile bundles (muscle)

• bundles are highly stable

• myosin forms bipolar thick filaments with the tails associating to keep the filament together, and heads facing in opposite directions

• highly stable; the myosins are not what moves; highly organized and static structure

• myosin heads bind with high processivity to actin and pull actin toward them, resulting in contraction (thick filaments pull actin toward the M line/closer together, so the sarcomere contracts)

Bipolar myosin filament

• muscle

• myosins associate in alternating orientations/directions to contract toward the M line/center of the sarcomere

Sarcomere structure

• I band—light appearance, non-overlapping actin

• A band—dark appearance, overlap of actin and myosin filaments

• H-zone: region in the center with non-overlapping myosin filaments; adjacent to the - end of the thin/actin filaments

• M line: center of sarcomere

• Z-line: bounds of the sarcomere; adjacent to the + end of the thin/actin filaments

Unconventional myosins

• at least 17 different classes

• structure: single-headed (except myosin V), short-tailed, and highly variable

Unconventional myosin functions

• moving vesicles locally in the periphery of the cell

• sliding filaments past other filaments along the plasma membrane; contractions are responsible for cell cortex movement

• moving chloroplasts to the places in the cell with optimal light intensity (plants)—not as involved in the transport of organelles in non-plant cells (microtubules)

Local vesicle movement

• unconventional myosins are responsible for local vesicular movement on F-actin in the peripheral cortical regions of the cell (which are very rich in meshworks of actin filaments)

• vesicles are transferred off from the microtubule kinesins and onto the actin myosins

• microtubules are like long-distance transport (ex: shipment of my package from Hong Kong to New Jersey or something via freight boat)

• actin/myosin are more short-distance/local transport (ex: transport of my package from, like, the Moon Township fedex sorting center to my house via truck)