The potential functional diversity of closely related myosin isoforms found in eukaryotic cells is not yet understood in detail. We have previously provided evidence from functional knockouts of Neuro-2A neuroblastoma cells that myosin IIB is essential for neurite outgrowth. Here we investigate the role of non-muscle myosin IIA in the same cell line. We show that suppression of myosin IIA transcript and protein expression, brought about through exposure to isoform-specific antisense oligonucleotides, caused a rearrangement of the actin cytoskeleton and loss of cell adhesion. This also led to disruption of focal contacts, as evidenced by coincident reduction in paxillin and vinculin immunofluorescence, but did not diminish transcript expression. All effects were fully reversible. Before myosin IIA antisense-induced detachment, neurite outgrowth remained unaffected. By contrast, antisense oligonucleotides directed against myosin IIB transcripts had no effect on adhesion but severely attenuated neurite outgrowth. We infer that the two main isoforms of neuronal conventional myosin, myosins IIA and IIB, have separate but linked functions during neuronal adhesion and neurite outgrowth.

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actinassociated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.Filopodial extension, growth cone motility, and neurite outgrowth are important characteristics of developing neurons, their combined action playing an essential role in the formation of the nervous system during development and in axonal regeneration, including the restoration of connectivity, after injury (1). Little is known of the mechanisms underlying these aspects of directed motility in vertebrate neurons, but the participation of a number of molecular motors belonging to the myosin superfamily has been proposed. Thirty years ago, conventional, two-headed myosin II was first shown to be present in brain and neuronally derived material (2, 3). Since then, myosin I (4-6), myosin II (6-11), and myosin V (12, 13) have been observed at the leading edge of neuritic growth cones, often located in close proximity to the plasma membrane. Such observations suggest that these molecular motors may each perform distinct yet integrated tasks that facilitate force generation during the complex motions of growth cone advance.However, defining localization is not the same as defining function. Myosin V has been shown, by the technique of microscale chromophore-assisted laser inactivation, to play a role in neuronal growth cone filopodial extension (13). Yet neither microscale chromophore-assisted laser inactivation (13) nor our preliminary antisense studies have been able to implicate myosin I in the directed motility of neuronal cellseven though myosin I isoforms have been shown to be involved in the mechanism of locomoting amoebae (14,15). Although recent fusion protein expression studies on live amoebae suggest that myosin II also may ...

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.electron microscopy ͉ molluscan muscle ͉ regulation ͉ smooth muscle ͉ image processing