Lymphocytes play a critical role in the survival of vertebrates. To accomplish this task, they constantly sample the environment and respond to it, events which rely on vesicular trafficking at the plasma membrane. Despite the importance of this process, there are major gaps in our understanding of the molecular events that regulate constitutive trafficking at the plasma membrane. We propose to study this process using B lymphocytes, especially high-rate antibody secreting (plasma) cells, as model systems. The overall goals of these experiments are to characterize the secretory compartment in plasma cells and define the proteins and structures that determine the specificity of constitutive vesicular trafficking from the Golgi to the plasma membrane. To accomplish this, we will carry out the following specific aims: 1) Isolate and characterize IgM-containing post-Golgi transport vesicles from plasma cells, and investigate the developmental expression of vesicle-associated SNARE proteins; 2) Characterize the IgM-containing secretory vesicle compartment in plasma cells, and determine if it co-localizes with other structures especially the cytoskeleton; 3) Localize the docking sites for the IgM-containing vesicles at the plasma membrane, and determine the role of t-SNAREs and the cytoskeleton in specifying the localization of these sites; 4) Determine the specificity of cargo loading in post-Golgi secretory vesicles by asking if membrane and secretory proteins, including immunoglobulins, are transported to the plasma membrane in the same or different secretory vesicles; 5) Produce mice carrying lymphocyte-specific targeted mutations in genes encoding v- and t-SNARE proteins expressed in plasma cells, in order to define the role of these proteins in antibody secretion as well as in other aspects of T and B cell biology. The results of these experiments will not only provide new information concerning the regulation of high-rate antibody secretion, but should also provide the first molecular insights into the specificity of constitutive vesicular trafficking in lymphocytes, and contribute to a general understanding of the late steps in constitutive exocytosis. These experiments may also provide novel insights into the molecular basis for immunodeficiencies, and provide the tools necessary to study the intracellular transport of viruses and viral particles, and other infectious agents.
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