Bending Sidearms

Several important cytoplasmic movements occur due to bending of contractile proteins attached along rigid cytoskeletal filaments. In muscle, arrays of parallel, interdigitating "thin" filaments (actin) interact with "thick" filaments (myosin). Tiny sidearms or crossbridges ("myosin heads") attached to the myosin thick filaments extend across a gap of about 13 nanometers and "cyclically row like banks of tiny oars" to move the filaments relative to each other. The energy from hydrolysis of ATP drives the conformational changes which causes the myosin molecules to curl. The precise utilization of ATP hydrolysis energy to cause contractility and other conformational changes remains poorly understood, but will be discussed in Chapter 6.

Each myosin filament carries about 500 heads, each of which cycles about 5 times per second. There is some evidence of cooperativity in that once a myosin head has detached, it is carried along by the action of other myosin heads along the thick filament. The myosin head rowing is initiated and coordinated by waves of calcium ion released from a reservoir (the sarcoplasmic reticulum) triggered by membrane electrical activity. Rises in calcium ion causes an actin-bound regulatory protein called troponin to shift its position and allow actin-myosin ratcheting.

Many MT related activities generate force, locomotion and movement of vesicles and other material; axoplasmic transport is one well studied example (Lasek, 1981; Ochs, 1982). Parallel MT within axons are polarized with their fast growing "plus-ends" distal from the cell body facing the synapse. Force generating sidearms occur about every 16-18 nanometers along MT lengths. These contractile crossbridges generate directional movement of material along MT by undergoing a sequence of conformational changes involving attachment of crossbridges to vesicles, ATP dependent force generation by the crossbridges, and detachment of crossbridges from vesicles. Detachment occurs only at "plus" ends near synapses. The process is similar to rowing of myosin heads to slide actin and myosin filaments past each other and shorten muscle fibers. MT based dynein activities, however, are far more variable, flexible, and interactive than the repetitive nanoscale events in muscle. For example, in MT dependent axoplasmic transport different material is simultaneously transported in the opposite direction, from the synapse to the cell center. This "retrograde" axoplasmic transport is thought to provide feedback to the protein synthesis machinery as to what enzymes or material are required, and/or to allow "recycling" of some molecules (Figure 5.25).

Robert Allen (1985) was among the first to suspect that MT and MAPS served as intracellular conveyor belts. He and his colleagues studied isolated MT and MT fragments which, with .available biochemical energy in the form of ATP, "glide" along glass cover slips at velocities of 150 to 450 nanometers per second. The velocity is independent of MT fragment length, occurs essentially in a straight path, and is in the direction of retrograde axoplasmic transport. The straight paths of gliding MT segments suggest that the force generating enzymes are deployed in linear, rather than helical paths along the MT, and that the stroke that causes gliding is parallel to the MT with a spacing interval of about 17 nanometers. Reducing the available ATP concentration slows the gliding speed significantly, but does not affect the number or behavior of gliding MT. Gliding MT almost never interact when they cross paths, and when the forward progress of a gliding MT is blocked by an obstacle, it "fishtails" slowly from side to side through a series of serpentine maneuvers. Allen and colleagues concluded that the forces observed in their slithering free MT as well as in axoplasmic transport and ciliary bending are due to "force generating enzymes" directly attached to MT. Dynein, which functions to cause binding in cilia and flagella, is one MT force generating enzyme and kinesin is another motor for organelle transport along microtubules. Latex beads coated with kinesin translocate along microtubules similar to organelles, although at a slower velocity. Purifled kinesin can increase the frequency of axoplasmic organelle movement along purified MT.

Allen and colleagues proposed the "backstroke hypothesis" which states that the force generating enzyme (dynein or kinesin) makes an elliptical stroke which imparts some force in both directions. Allen's dynein backstroke model is capable of carrying vesicles in opposite directions simultaneously through sufficiently separated pathways so that they seldom collide. Further, the motion generated can be continuous, not interrupted by cycles of attachment and detachment. The mechanical cycle of each sidearm includes a radial stroke that moves vesicles in the anterograde direction toward the microtubule plus end. This part of the cycle also causes isolated MT to glide "retrograde." The return stroke is tangential to the MT surface and transports the larger organelles in a retrograde pathway, and propels gliding MT toward their. "anterograde" plus end.

The mechanisms by which the force generating protein arms may use ATP energy to contract will be discussed in Chapter 6. Even less well understood is the signaling and communication which orchestrates contractile activities of rows of arms spatially arrayed on MT lattices. Collective communication among MT lattice subunits (solitons, coherent excitations, lattice vibrations) could explain this orchestration.

The backstroke model is currently favored more than another model: microstreams. Shimizu and Haken (1983) had proposed a dynamic cooperativity of cytoskeletal elements which generated hydrodynamic microstreams conveying cellular materials. They specifically focused on actin-myosin interactions to generate these microstreams. New techniques such as Nanovideo Microscopy developed by Marc DeBrabander and colleagues (1986) at Janssen Pharmaceutica Research Laboratories in Belgium show direct tracking of immunolabeled particles along MT, questioning the significance of microstreams. Particles are seen to travel in opposite directions along the same MT, passing each other like two railroad trains on adjacent tracks. Microstreaming does not appear dominant in axoplasmic transport, but could be important in other phenomena. The primary site of coordinated transport and its complex orchestration appears to rest solely in the province of MT.

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