Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

48-POS Board 48 MULTIPLE ISOFORMS OF SYNAPTOTAGMIN REGULATE PEPTIDE SECRETION

FROM NOCICEPTIVE NEURONS Judit Meszaros 1 ; Elizabeth P Seward 1 ; 1 University of Sheffield, Biomedical Science, Sheffield, United Kingdom

In response to inflammation and nerve injury, pain signaling is transduced from the periphery to the CNS through nociceptors. While much is known about the changes in ion channel function which underlie pain signaling, less is known about what changes occur at the level of the exocytotic machinery to support enhanced signaling, hyperalgesia and peptide secretion. Here we present research aimed at characterizing molecules regulating peptide secretion from nociceptors, focusing on the well-known vesicular regulator of exocytosis, synaptotagmin (SYT). We found that SYT4, -11, -2 and -7 are expressed in nociceptors isolated from adult dorsal root ganglia (DRG). Co-localization studies with CGRP indicated that multiple pools of peptidergic vesicles exist within nociceptors, characterized through their differential expression of distinct Syt isoforms. CGRP secretion, as measured by ELISA from DRGs prepared from heterozygous or litter-mate knockout (KO) SYT4 or Syt7 mice showed no significant difference, implying either that these two isoforms of SYT play a modulatory rather than essential role in controlling peptide secretion from nociceptors or that compensatory changes have taken place in the gene knockout animals. To investigate the involvement of SYT4 and SYT7 in peptide release in greater detail, we switched to expressing SYT4-pHluorin or SYT7-pHluorin in combination with NPY-mCherry in adult nociceptors and imaging fusion using TIRFM. We compared results obtained with the SYT-pHlourins with synapto-pHlourin (VAMP2-pHluorin). Analysis of single events showed that fusion of vesicles expressing SYT4 or SYT7 are relatively slow and long- lived and have the characteristics associated with ‘kiss-and-run’ exocytosis. Expressing SYT7- pHluorin in SYT4 KO nociceptors significantly altered the timing and kinetics of single fusion events. In the absence of SYT4, exocytosis of SYT7-bearing vesicles is slowed and reduced, consistent with the view that SYT4 is increasing the pool of releasable peptidergic vesicles in nociceptors.

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