THE ACTIN NETWORK IN THE CILIARY STALK OF PHOTORECEPTORS FUNCTIONS INTHE GENERATION OF NEW OUTER SEGMENT DISCS

Cytochalasin D (CD) interferes with the morphogenesis of outer segment disc membrane in photoreceptors. Disruption of either the actin netwo rk in the ciliary stalk, where membrane evagination is initiated, or t he actin core of the calycal processes, whose position could define th e disc perimeter, could be responsible. We have attempted to determine which of these local F-actin populations is involved in membrane morp hogenesis and what step in the process is actin-dependent. Biocytin ac cumulation in nascent discs, detected by fluorescent avidin and laser scanning confocal microscopy (LSCM), provided a means of labeling abno rmal discs and a measure of disc membrane addition. F-actin content an d distribution were assessed using fluorescent phalloidin and LSCM. Fi rst, we examined the effects of a range of CD dosages (0.1, 1.0, or 10 .0 mu M) on rod photoreceptors in Xenopus laevis eyecup cultures. Ecto pic outgrowth of discs, evaluated by LSCM and transmission electron mi croscopy (TEM), occurred at each concentration. Phalloidin labeling in tensified in the ciliary stalk with increasing CD concentration, indic ating F-actin aggregation. In contrast, it diminished in the calycal p rocesses, indicating dispersal; TEM showed that calycal process collap se ensued. Disruption was evident at a lower concentration in the cili ary stalk (0.1 mu M) than in the calycal processes (1.0 mu M). TEM con firmed that the calycal processes remained intact at 0.1 mu M. Thus, C D's action on the ciliary stalk network is sufficient to disrupt disc morphogenesis. Second, we examined the effect of CD on temperature-ind uced acceleration of the rate of disc formation. In the absence of CD, a 10 degrees C temperature shift increased the disc formation rate ne arly three-fold. CD (5 mu M) caused a 94% inhibition (P < 0.025) of th is response; yet, the rate of membrane addition to ectopically growing discs exhibited the expected three-fold increase. Thus, CD's action i nterferes with the generation of new discs. (C) 1996 Wiley-Liss, Inc.