Tejeshwar C. Rao

Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules

Adrenal chromaffin cells express two synaptotagmin isoforms, Syt-1 and Syt-7. Isoforms are usually sorted to separate secretory granules, are differentially activated by depolarizing stimuli, and favor discrete modes of exocytosis. It is proposed that stimulus/Ca+-dependent secretion in the chromaffin cell relies on selective Syt isoform activation.

Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules

Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+. Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.

Real-Time Investigation of Plasma Membrane Deformation and Fusion Pore Expansion Using Polarized Total Internal Reflection Fluorescence Microscopy

Polarized Total Internal Reflection Fluorescence Microscopy (pTIRFM) allows for real-time observation of plasma membrane deformations. The technique provides insights into the dynamics of biological processes requiring rapid and localized changes in membrane shape. Such processes include exocytosis, endocytosis, cytokinesis, and cell motility. In this chapter, we describe how to implement a polarization-based TIRF imaging system to monitor exocytosis in adrenal chromaffin cells.