Richard C. Weisenberg - US grants

Isolated microtubule proteins from calf brain will undergo ATP-dependent gelation and contraction. Microtubules in these gels form bundles and structures resembling mitotic asters and spindles. Directed particle movements are observed in contracting gels. The proposed experiments will explore the chemistry and physiology of microtubule gelation-contraction. The proteins necessary for gelation-contraction will be isolated and their physical, chemical and enzymatic properties characterized by standard techniques. Optimum conditions for gelation-contraction will be determined and the role of ATP in microtubule gelation, aster formation and gel contraction will be examined. The nature and role of the motile particulates that form microtubule focal centers will be investigated. The structure of gels and the polarity of microtubules will be explored by electron microscopy. The relationship of microtubule gelation-contraction to nerve cell development will be examined by isolating microtubule proteins from animals at different ages. Actin and neurofilament proteins will be added to microtubule gels to study cytoskeletal interactions in vitro. Motile phenomenon in microtubule gels will be investigated by video enhanced contrast microscopy. The relationship of microtubule gelation-contraction to mitosis will be explored by isolating microtubule proteins from dividing surf clam eggs. Antibodies against gelation-contraction proteins will be prepared and used to localize these proteins in dividing cells and developing neurons. These experiments will help establish the role of microtubules in the morphology and motility of living cells.

Microtubules are a major component of the cytoskeleton of nerve cells and are necessary for the formation and maintenance of neuronal structure. In order to provide material for neuronal growth and to preserve neuronal structure, microtubule proteins, along with other cytoskeletal proteins, are transported at a slow rate down neuronal extensions. The PI has discovered that microtubule preparations can undergo slow, energy-dependent, contractile behavior in vitro and has proposed that this process, microtubule gelation-contraction, is related to the normal process of protein transport in nerve cells. The proposed experiments will help demonstrate the validity of this hypothesis. Experiments will be performed to directly compare the properties of neuronal transport in vivo to microtubule gelation-contraction in vitro. The PI will attempt to identify the enzyme needed for the conversion of chemical energy into kinetic energy in this process. The experiments should provide important information on how nerve cell structure is produced and maintained and could have significance in understanding the processes leading to neuronal degeneration such as occurs in Alzheimer's disease and diabetic neuropathy.

Data is presented suggesting that Slow Component a (SCa) of axonal transport involves the movement along microtubules of an insoluble complex of tubulin, neurofilament, spectrin, and other proteins. These structures have been given the provision acronym "SCAPs." The goal of this research is to gain a fuller understanding of these structures. The protein composition of highly purified SCAPs will be analyzed by routine biochemical and immunochemical approaches. A microtubule-stimulated ATPase is present in SCAP preparations, which appears to be responsible for the in vitro phenomenon of microtubular gelation-contraction. This ATPase activity will be characterized biochemically, with the goal of identifying and understanding the action of the enzyme(s) involved. The properties of this ATPase activity and its relationship to microtubule gelation-contraction in vitro (and possibly to SCAP movement in vivo) will be analyzed. These experiments will provide significant new information relevant to the validity of the hypothesis that SCa involves the translocation of SCAPs along microtubules.

This research is designed to probe the mechanism of slow axonal transport, a phenomenon which is basic for proper functioning of nerve cells. Dr. Weisenberg has discovered a process, called gelation-contraction, which can be observed isolated, in vitro, and which, therefore, is amenable to experimental manipulation. Dr. Weisenberg suggests that gelation-contraction may be a valid model for slow axonal transport and further proposes to test the validity of this hypothesis in a series of experiments designed to see whether the proteins associated with slow axonal transport behave as a tightly bound complex. The results of these experiments should allow him to differentiate between different models for slow axonal transport.