MOTILE MODELS OF TELEOST RETINAL CONES

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The general objective of this research program is to use lysed-cell models of teleost retinal cones to investigate the mechanisms and regulation of actomyosin-dependent cell contraction and microtubule-dependent cell elongation. We have developed procedures for obtaining ATP-dependent re-activation of both contraction and elongation in models of teleost cones lysed with 1% Brij-58. By altering cAMP and calcium levels we can produce either reactivated elongation or contraction with sufficiently extensive length changes (20-40u) to permit detailed quantitation. We will analyze the effects of reactivation conditions and various probes by quantifying rates and extents of reactivated movement and by thin section TEM of models. We propose specifically 1) to examine the roles of myosin, myosin light chain kinase and calmodulin in regulating cone contraction and 2) to investigate the possibility that dynein-based inter-microtubule sliding provides motive force for cone elongation. Proposed studies include analyses of the effects of nucleotides, ions, pH, vanadate and EHNA on reactivated elongation and contraction, immunofluorescence localization and ferritin-antibody localization studies with antibodies to platelet myosin, calmodulin, and ciliary and egg dynein, investigations of the effects of exogenous calmodulin, smooth muscle myosin and myosin light chain kinase on reactivated contraction, and morphological analyses of microtubule polarities and intertubule associations in elongating cone myoids.