• Satir, Birgit H. (PI)

Project: Research project

Project Details


This proposal concerns detailed mechanisms of signal transduction
that lead to exocytosis during regulative secretion induced by
specific secretagogues in eukaryotic cells. There are least three
cellular compartments involved in the process of signal
transduction in exocytosis: (1) the plasma membrane which
contains secretagogue receptors and other transmembrane
proteins (2) the cytoplasm particularly in the region between the
cell and secretory vesicle membrane, where molecules may
influence interactions of the membranes and (3) the secretory
vesicle itself. The properties of the vesicle content and its
membrane are also crucial to the interactions that finally
accompany product release. The details of signal transduction
during exocytosis will be studied in Paramecium tetraurelia.
Gilligan and Satir demonstrated that when normal exocytosis was
induced in wild type cells a dephosphorylation could be detected
in a number of Paramecium phosphoproteins, most prominently in
a Mr 63kDa species. Preincubation of cells in high Mg2+ (no
added Ca2+) inhibited both exocytosis and dephosphorylation in
response to secretagogue. Further, a mutant nd9, when grown at
18 degree C (permissive temperature), had the normal rosettes at
the secretory site, secreted normally and dephosphorylated the
63kDa polypeptide in response to secretagogue, but when grown at
27 degree C (non-permissive temperature) where rosettes were
not assembled, did not secrete nor dephosphorylate the 63kDa
polypeptide in response to the secretagogue. We have succeeded
in purifying the 63kDa protein and have made a polyclonal
antibody against it. We intend to investigate its intracellular
localization, exploring the possibility that the protein becomes an
integral part of the cytoskeleton in the rosette region upon
dephosphorylation. Using Western blot analysis with the purified
antibody on other cells and mammalian tissues, we will examine
whether the 63kDa protein may be of general significance in other
secretory processes. We shall attempt to define, characterize and
localize the cellular phosphatase(s) and kinase(s) that act on the
63kDa protein. In addition, the influx of extracellular Ca2+ after
appropriate stimulation appears to be mediated by specific
exocytic Ca2+ channels, separate and distinct from the well
known voltage dependent ciliary Ca2+ channels. We intend to
characterize and localize these channels more completely.
Effective start/end date12/1/8312/31/93


  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)


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