Application of quantitative stereology to conventional transmission electron microscopy (TEM) and immunoelectron microscopy (IEM) serves as a powerful tool to visualize intermediates involved in vesicular traffic through the secretory pathway. Our studies will now focus on the morphological characterization of the role of the COPII transport machinery in cargo selection from the ER, and the role of COPI in the function of pre-Golgi intermediates. We will morphologically define structural intermediates which appear in vitro in response to the assembly of COPII coat components into a molecular scaffold which direct vesicle budding from the ER and we will examine the morphological basis for the assembly of tubular elements on pre-Golgi intermediates. The following specific aims will be pursued:
Specific Aim 1 : Test the hypothesis that carbo and the Sar1 GTPase coordinate the assembly of COPII coat components into a coat lattice which drives vesicle formation by examine sequential structural intermediates formed during coat recruitment.
Specific Aim 2 : Examine the hypothesis that the assembly of pre-Golgi tubular elements involves homotypic fusion of COPII derived vesicles.
Specific Aim 3 : Analyze the structural basis for the maturation of pre-Golgi intermediates by testing the hypothesis that Golgi processing enzymes are acquired through COPI mediated retrograde transport of these enzymes from distal Golgi compartments.
Specific Aim 4 : Apply real-time imaging to visualize the spatial and temporal dynamics of cargo movement from the ER to the Golgi complex. These studies will focus directly on the thematic aspects of the Program Project in that we envision that the small GTPases mediating ER to Golgi transport serve as transducers in a signal transduction pathway involving cargo molecules to promote the specific assembly of COP-coated vesicles mediating anterograde and retrograde transport.
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