9506279 Langford This project will investigate the specific functions of organelle-associated myosins in nerve cells. The number of new myosin motors identified by molecular and genetic techniques has grown explosively in the past few years but the functional roles of these new motors have not been determined. This project tests the hypothesis that microtubule-dependent motors drive movement of organelles over long distances while actin-dependent motors drive movement to local sites along the axon. The broad objectives of the research project are to determine the actin-dependent motors involved in fast axonal transport, the interrelationship between the actin-dependent and microtubule-dependent forms of organelle motility and the mechanism by which the motors involved in organelle motility are regulated. The squid giant axon and optic lobes (brain) will be the primary biological materials used in these studies. Squid axoplasm is one of a small number of in vitro systems where myosin-dependent organelle motility can be studied directly. Therefore, it represents a unique system in which to study (a) organelle associated myosins, (b) the regulation of these motors and (c) the relationship between membrane trafficking on microtubules and actin filaments. Extruded axoplasm is particularly useful for motility studies but the amount of material is too small for biochemical purification of myosin motors. Squid brain, however, represents an alternate source of material for purification of myosins, therefore, motors identified in axoplasm will be subsequently isolated from squid optic lobes. The primary goal of the research project is to use an identified organelle, the tubulovesicular organelles (TVOs) in the squid giant axon, to study the properties and regulation of organelle-associated myosins. Purification of TVOs will be achieved by generating axoplasmic ghosts; the highly extracted remnant of axoplasm. Motility of purified TVOs will be reconstituted on actin f ilaments and the parameters of movement determined by quantitative motion analysis. The function of these compartments as Ca stores will be determined with Ca-indicator dyes. In addition, antibodies to known smooth ER proteins will be used to establish the identity and purity of the isolated compartments. Another specific goal is to identify factors that influence binding of motors to the tubulovesicular organelles. The location of the actin-dependent motors on the surfaces of TVOs will be determined using immunofluorescence microscopy. This technique will make it possible to establish whether the motors are uniformly distributed or localized to the vesicular domains of TVOs. Properties of the motor, such as whether the motor is a peripheral protein and whether soluble factors are involved in its attachment to the membrane will be determined. Myosins will be purified from squid brain by conventional biochemical methods or from axoplasm by immunoprecipitation. Purified motors will be biochemically and functionally characterized using in vitro motility assays. An antibody to squid brain myosin V has been generated for use in localization and functional studies. Antibodies to other organelle-associated unconventional myosins will be generated and used to inhibit motility in extruded axoplasm thereby verifying the role of these motors in the movement of organelles. %%% The work described in this proposal will provide new knowledge on molecular motors and will aid our understanding of fast axonal transport as well as our understanding of many other fundamental cellular processes including secretion, cell division, cell motility and the organization and transport of organelles. The new knowledge from these studies has applications in biotechnology and research on molecular nanostructures. ***
