Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Arun Anantharam

Abstract

The sympathetic nervous system is activated by a variety of threats to organismal homeostasis. The adrenomedullary chromaffin cell is the core effector of sympathetic activity in the peripheral nervous system. By design, the chromaffin cell secretory response is mutable so that release can be rapidly tuned to drive context-dependent changes in physiological function. However, the mechanisms by which this tuning is achieved with such high temporal fidelity and context specificity remain unclear. This represents a major gap in our understanding of the sympatho-adrenal system since it is known to modify the function of nearly every organ system in the body.

In chromaffin cells, the trigger for stimulus-evoked exocytosis is a rise in intracellular Ca2+. The level of intracellular Ca2+ accumulation varies with the stimulus intensity and secretagogue. Ca2+ regulates release by acting on the Ca2+-binding synaptotagmin (Syt) protein family, driving their penetration into membranes that harbor anionic lipids, and possibly bending those membranes. Chromaffin cells only express two of the 17 known Syt isoforms: Syt-1 and Syt-7. Previous studies had identified that Syt-1 and Syt-7 possess distinct affinities for Ca2+ in the presence of phospholipid membranes and, once bound release membranes differentially. An underlying assumption in previous studies of chromaffin cell secretion was that granules are homogeneous, with the same biochemical constituents and the potential for similar rates of content discharge. This idea is challenged by the work in this dissertation. We discovered that granules harbor functionally different isoforms of Syt, and therefore reasoned that these isoforms (Syt-1 and Syt-7) may confer different Ca2+ sensitivities to the granules in situ. This would thereby enable them to respond differentially to depolarizing stimuli. In order to study this, we first focused our attention on developing an imaging tool (pTIRFM) that could combine nanoscale measurements of membrane curvature changes with single-molecule imaging. This tool would allow us to study the interaction of synaptotagmin with the plasma membrane in unprecedented kinetic and topological detail, and would be necessary to resolve how specific structural differences in Syt isoforms relate to their distinct functional properties.

Using pTIRFM we first observed that topological changes associated with Syt-1 granules are more short-lived than those of Syt-7 granules. This finding led credence to the idea that fused Syt-7 granules maintain a narrow fusion pore and more often undergo the “kiss-and-run” mode of exocytosis. Conversely, Syt-1 granules collapse onto the plasma membrane following fusion with rapid dilation of the fusion pore (“full-collapse” mode of exocytosis). Also, lumenal and membrane proteins are released more slowly from Syt-7 than from Syt-1 granules. The above findings led us to propose the hypothesis that chromaffin granules are not in fact homogenous but instead may exploit the molecular and functional heterogeneity conferred by Syt to regulate release in an activity-dependent manner.

To explore this, we decided to focus our attention on identifying novel differences between the spatial and functional properties of granules expressing the two Syt isoforms. These isoforms are not only sorted to separate granule populations, but Syt-1 granules are also distributed further away from the plasma membrane in comparison to Syt-7 granules. Lower stimulation conditions are more effective in activating Syt-7 granules, and on average they begin fusing with the membrane earlier following depolarization. On the contrary, more Ca2+ is needed for Syt-1 activation, and these granules require less time to transition into a fusion-competent state. Thus, the behavior of Syt isoforms is strongly Ca2+ and stimulus-dependent. Therefore these experiments demonstrate that heterogeneity of Syt granules is utilized by the chromaffin cell to modulate the release of cargo in a physiologically relevant context.

The performed studies have led to a paradigm shift in our current understanding of the basic molecular organization of secretory systems. They have provided insight into how heterogeneity of individual secretory granules may affect the properties of regulated exocytosis in other cell types. This is a major conceptual advance, as intrinsic functional differences among granules have not been considered as a factor in the regulation of fusion modalities.

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