Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Tuesday Speaker Abstracts

SYNAPTOTAGMIN-7 ENDOWS A POPULATION OF CHROMAFFIN GRANULES WITH DISTINCT CALCIUM SENSING AND FUSION PROPERTIES Mounir Bendahmane 1 ; Alex J Kreutzberger 2 ; Alina Chapman-Morales 1 ; Noah A Schenk 1 ; Volker Kiessling 2 ; J D Castle 2 ; Lukas K Tamm 2 ; David R Giovannucci 3 ; Arun Anantharam 1 ;

1 University of Michigan, Ann Arbor, Michigan, USA 2 University of Virginia, Charlottesville, Virginia, USA 3 University of Toledo, Toledo, Ohio, USA

Synaptotagmin-7 (Syt7) is one of two major calcium sensors for regulated exocytosis in adrenal chromaffin cells. Its high sensitivity allows tunable secretory responses to a range of stimuli that result in graded increases in intracellular calcium. Despite the undoubted importance of Syt7, questions remain as to whether the protein operates from the granule or plasma membrane and to what degree the functions of chromaffin cell Syts are redundant. Here, these issues were examined using two distinct experimental preparations – primary mouse chromaffin cells lacking endogenous Syt7 and a reconstitution assay employing cell-derived granules expressing either Syt7 or Syt1. First, we find that mouse Syt7 is punctate in appearance, consistent with its sorting to organelles. Antibody-based staining reveals it to be co-localized with plasminogen activator inhibitor 1 (PAI1) – a protein of the granule dense core – but rarely with Syt1. Functionally, chromaffin cells lacking Syt7 (knockout, KO) exhibit properties that readily distinguish them from WT cells. For example, lumenal cargo proteins are released at faster rates from cells only expressing Syt1 than WT cells expressing both Syts. KO cells also exhibit deficits in fusion efficacy, both in response to elevated KCl depolarization and cholinergic stimulation. To further distinguish between the roles of Syt7 and Syt1 in fusion, purified dense core granules expressing only one of the two proteins were triggered to fuse on reconstituted planar supported bilayers bearing t-SNAREs. These studies demonstrate that Syt7 confers substantially greater calcium sensitivity to granule fusion than Syt1 and slows that rate at which cargos are released, just as in primary cells employing overexpressed Syts. By virtue of its sorting and biochemistry, Syt7 serves unique roles in the biology of the chromaffin cell secretory response in ways that distinguish it from Syt1.

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