ATP and calmodulin dependent actomyosin aggregates induced by cytochalasin D in goldfish retinal ganglion cell axons in vitro

Growing retinal ganglion cell (RGC) axons of the goldfish have mobile varicosities, which play a role in rapid bulk redistribution of axoplasm (Koenig, Kinsman, Repasky, and Sultz, 1985; Edmonds and Koenig, 1987). Varicosities contain a tubulo-vesicular SER embedded in an actin-containing cytomatrix (Koenig et al., 1985). Cytochalasin D (CD) induces the formation of focal cytoskeletal aggregates throughout preterminal axons and especially in varicosities. The aggregates are visible when labelled with fluoroscein isothiocyanate (FITC)-conjugated phalloidin. Double-labelling experiments show that Texas red-myosin or rhodamine isothiocyanate (RITC)-calmodulin immunofluorescence co-localizes with FITC-phalloidin-labelled aggregates. Formation of aggregates is blocked by calmidazolium, a calmodulin antagonist. Axon models permeabilized with digitonin retain the capacity to form focal aggregates in response to CD, when ATP or adenosine-5'-O(3-thiotriphosphate) (ATP-gamma S) is present in the permeabilization buffer, but not when 5'-adenylylimidodiphosphate (AMP-PNP) is present. The latter result indicates that formation of focal aggregates depends on ATP. The findings suggest that the formation of focal aggregates in immature axons is a manifestation of actomyosin interactions after free actin-filament ends are generated by CD treatment.