Myosin Bound Vesicles Are Carried Along Actin Filaments

Among the many movements exhibited by cells, vesicle translocation has been one of the most fascinating to cell biologists. In early studies of the cytoplasm, researchers found that certain particles, now known to be membrane-bounded vesicles, moved in straight lines within the cytosol, sometimes stopping and then resuming movement, at times after changing direction. This type of behavior could not be caused by diffusion, because the movement was clearly not random. Therefore, researchers reasoned, there must be tracks, most likely actin filaments or microtubules, along which the particles travel, as well as some type of motor to power the movement.

the coverslip, as in Figure 19-17. When the optical trap is turned on, it captures the filament, through the bead, and holds the filament to the surface of a myosin-coated coverslip. The force exerted by a single myosin molecule on an actin filament is measured from the force needed to hold the bead in the optical trap. A computer-controlled electronic feedback system keeps the bead centered in the trap, and myosin-generated movement of the bead is counteracted by the opposing force of the trap. The distance traveled by the actin filament is measured from the displacement of the bead in the trap.

In the sliding-filament assay, walking of the myosin head along an actin filament causes the filament to move because the myosin tail is immobilized. In cells, however, the situation is often reversed: when part of an extensive network, actin filaments are largely immobile, whereas myosin is free to move. In this case, if the tail of a myosin molecule binds to the membrane of a vesicle and the head walks along a filament, the vesicle will be carried along as "cargo." Here, we present evidence that some myosins, including myosins I, V, and VI, do just that. We also consider the related process of cytoplasmic streaming, which is most likely powered by myosin XI.

Vesicle Trafficking (Myosins I, V, and VI) Findings from studies with amebas provided the initial clues that myosin I participates in vesicle transport. Indeed the first myosin I molecule to be identified and characterized was from these organisms; subsequently, the cDNA sequences of three myosin I genes were identified in Acanthameba, a common soil ameba. Using antibodies specific for each myosin I isoform, researchers found that the isoforms are localized to different membrane structures in the cell. For example, myosin IA is associated with small cytoplasmic vesicles. Myosin IC, in contrast, is found at the plasma membrane and at the contractile vacuole, a vesicle that regulates the osmolarity of the cytosol by fusing with the plasma membrane. The introduction of antibodies against myosin IC into a living ameba prevents transport of the vacuole to the membrane; as a result, the vacuole expands uncontrollably, eventually bursting the cell. In addition, myosin I in animal cells serves as a membrane-microfilament linkage in microvilli, another example of a membrane-associated function.

Several types of evidence suggest that myosin V also participates in the intracellular transport of membrane-bounded vesicles. For example, mutations in the myosin V gene in yeast disrupt protein secretion and lead to an accumulation of vesicles in the cytoplasm. Vertebrate brain tissue is rich in myosin V, which is concentrated on Golgi stacks. This association with membranes is consistent with the effects of myosin V mutations in mice. Such mutations are associated with defects in synaptic transmission and eventually cause death from seizures. Myosin VI also is implicated in membrane trafficking of vesicles.

Unlike myosin in a thick filament where multiple heads interact with the same actin filament, cytoplasmic myosins work alone in carrying their membrane cargos. How do these myosins move without dissociating from the filament? The answer lies in the duty ratio, the fraction of time spent attached to the filament during the ATPase cycle. Myosins with a high duty ratio, such as myosins V and VI, are bound to actin filaments for most of the ATP cycle. Consequently, these myosins process or move along a filament for considerable distances with little danger of falling off.

▲ FIGURE 19-19 Cytoplasmic streaming in cylindrical giant algae. (a) The center of a Nitella cell is filled with a single large water-filled vacuole, which is surrounded by a layer of moving cytoplasm (indicated by blue arrows). A nonmoving layer of cortical cytoplasm filled with chloroplasts lies just under the plasma membrane (enlarged lower figure). On the inner side of this layer are bundles of stationary actin filaments (red), all oriented with the same polarity. A motor protein (blue dots), most likely myosin XI,

Cytoplasmic Streaming (Myosin XI) In large, cylindrical green algae such as Nitella and Chara, cytosol flows rapidly, at a rate approaching 4.5 mm/min, in an endless loop around the inner circumference of the cell (Figure 19-19). This cy-toplasmic streaming is a principal mechanism for distributing cellular metabolites, especially in large cells such as plant cells and amebas. This type of movement probably represents an exaggerated version of the smaller-scale movements exhibited during the transport of membrane vesicles.

Close inspection of objects caught in the flowing cy-tosol, such as the endoplasmic reticulum (ER) and other membrane-bounded vesicles, show that the velocity of streaming increases from the cell center (zero velocity) to the cell periphery. This gradient in the rate of flow is most easily explained if the motor generating the flow lies at the membrane. In electron micrographs, bundles of actin filaments can be seen aligned along the length of the cell, lying across chloroplasts embedded at the membrane. Attached to the actin bundles are vesicles of the ER network. The bulk cytosol is propelled by myosin attached to parts of the ER lying along the stationary actin filaments. The flow carries parts of the endoplasmic reticulum (ER) along the actin filaments. The movement of the ER network propels the entire viscous cytoplasm, including organelles that are enmeshed in the ER network. (b) An electron micrograph of the cortical cytoplasm shows a large vesicle connected to an underlying bundle of actin filaments. This vesicle, which is part of the ER network, contacts the stationary actin filaments and moves along them by a myosin motor protein. [Part (b) from B. Kachar.]

rate of the cytosol in Nitella is at least 15 times as fast as the movement produced by any other myosin. This evidence and other evidence suggest that cytoplasmic streaming is powered by myosin XI, one of the fastest moving myosins.

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