Localization of myosin II A and B isoforms in cultured neurons

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Abstract
Tension generated by growth cones regulates both the rate and the direction of neurite growth. The most likely effectors of tension generation are actin and myosins. We are investigating the role of conventional myosin in growth cone advance. In this paper we report the localization of the two most prominent isoforms of brain myosin II in growth cones, neurites and cell bodies of rat superior cervical ganglion neurons. Affinity purified polyclonal antibodies were prepared against unique peptide sequences from human and rat A and B isoforms of myosin heavy chain. Although each of these antibodies brightly stained nonneuronal cells, antibodies to myosin heavy chain B stained neurons with greater intensity than antibodies to myosin heavy chain A. In growth cones, myosin heavy chain B was most concentrated in the margin bordering the thickened, organelle-rich central region and the thin, actin-rich peripheral region. The staining colocalized with actin bundles proximal and distal to the marginal zone, though the staining was more prominent proximally. The trailing edge of growth cones and the distal portion of the neurite often had a rimmed appearance, but more proximal regions of neurites had cytoplasmic labelling. Localizing MHC-B in growth cones previously monitored during advance (using differential interference contrast microscopy) revealed a positive correlation with edges at which retraction had just occurred and a negative correlation with lamellipodia that had recently undergone protrusion. Cell bodies were brightly labelled for myosin heavy chain B. Myosin heavy chain A staining was dimmer and its colocalization with filamentous actin bundles in growth cones was less striking than that of myosin heavy chain B. Growth cones stained for both myosin heavy chain A and B revealed that the two antigens overlapped frequently, but not exclusively, and that myosin heavy chain A lacked the elevation in the marginal zone that was characteristic of myosin heavy chain B. The pattern of staining we observed is consistent with a prominent role for myosin heavy chain B in either generating tension between widely separated areas of the growth cone, or bundling of actin filaments, which would enable other motors to effect this tension. These data support the notion that conventional myosin is important in growth cone advance and turning.