简介

We study the molecular mechanisms underlying polarized targeting of proteins in neurons. We have discovered an actin- and myosin-based mechanism by which transmembrane proteins such as ion channels and receptors are targeted to either the surface of the axon or to the somatodendritic compartment. We showed that interaction with Myosin Va is both necessary and sufficient for the localization of dendritic proteins and Myosin VI plays a similar role in localization of proteins to the surface of the axon. We also showed recently that vesicles carrying dendritic or axonal proteins enter axons and dendrites with equal frequencies immediately following release from the Golgi apparatus. In the axon initial segment, however, vesicles carrying dendritic proteins almost all halt and some reverse in an actin- and Myosin Va-dependent manner, while vesicles carrying axonal proteins proceed to the distal axon. These results suggest that an actin-dependent vesicle filter is present in the AIS that mediates the selective trafficking of vesicles depending on their contents. (2) We have developed recombinant probes known as intrabodies that can be used to label endogenous proteins in living neurons, or to mediate the degradation of endogenous proteins inducibly, specifically and very quickly. To make intrabodies we use mRNA display, an in vitro selection procedure developed by our collaborator Richard Roberts. We have used intrabodies to report the localization of endogenous proteins in neurons in dissociated culture, as well as in slices and in vivo following in utero electroporation. We have made intrabodies against PSD95, Gephyrin, CAM Kinase II and Kv4.2. When expressed in neurons intrabodies accurately report the localization and trafficking of target proteins without affecting their localization, expression level or function. Modified intrabodies known as ablating intrabodies can mediate the degradation of their endogenous target proteins inducibly, specifically, and very quickly. Ablating intrabodies mediate the direct ablation of their target proteins and, thus, work much faster than traditional, nucleic acid-based methods of protein degradation, such as RNAi. Furthermore, intrabodies are capable of specifically degrading proteins in particular conformations or with specific post-translational modifications. Thus, they could be used to specifically degrade pathological proteins involved in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s, without degrading wild-type forms of these proteins.