Microtubule motor proteins are responsible for numerous transport functions in cells. This proposal focuses on a novel mechanism for the motor protein cytoplasmic dynein in mammalian autophagy. Autophagy is a critical cellular function responsible for recycling old or damaged proteins and organelles, and for clearing toxic protein aggregates. Autophagy is also implicated in neurodevelopmental and neurodegenerative diseases. Cytoplasmic dynein is a major motor protein responsible for a broad range of basic cellular roles, including retrograde axonal transport, cell division, and nuclear and cell migration. We have now found that the cytoplasmic dynein regulator, RILP (Rab-interacting Lysosomal Protein) acts as a novel master regulator of neuronal and nonneuronal autophagy. We find that RILP recruits dynein to autophagosomes at a succesion of stages throughout this process via a sequence of distinct recruitment mechanisms involving interactions with the autophagosomal proteins LC3 and ATG5, as well as the late endosomal/lysosomal protein Rab7. We find RILP mediates not only autophagosome transport, but has a surprising role in autophagosome biogenesis as well. Of further interest we find RILP expression to be controlled by the mTOR kinase, which plays a central role in the cellular response to nutrient deprivation, injury, and toxic protein aggregation. We find further that RILP is necessary for processing of p62(/SQSTM1), direct evidence for a physiological role in clearance of protein aggregates. RILP appears, therefore, to represent a missing link in understanding how mTOR regulates the cellular machinery in response to diverse forms of insult or stress. This proposal is to work out the detailed mechanisms for RILP regulation and function, especially in neurons.
Aim 1 will test the role of mTOR in controlling RILP expression, and of PKA in controlling RILP/dynein-mediated autophagosome transport.
Aim 2 Will define the roles of RILP in autophagosome biogenesis and maturation.
Aim 3 will define the role of a novel RILP-dynein-dynactin-LIS1 supercomplex we have isolated in regulating autophagosome transport. The proposed studies should provide important insight into a basic new autophagy pathway, with fundamental implications for understanding the etiology and control of neurodegenerative and neurodevelopmental diseases.
Autophagosomes are cellular organelles responsible for clearance of subcellular protein aggregates and defective organelles, and protect against the effects of starvation and other forms of cell stress. Autophagosomes also play critical roles in neurodegeneration and neurodevelopment. We have discovered that the protein RILP serves as a master controller of autophagosome formation, distribution, and clearance in neurons. We will determine the underlying mechanisms for these behaviors, which have important implications for understanding the causes and control of neurodegenerative, neurological, and other forms of disease.
