Biology subjects

Weisgerber, A. W.

Publications and source records attributed to Weisgerber, A. W..

2 recordsLinked to original sources

bioRxiv2023

Syntaxin clusters and cholesterol affect the mobility of Syntaxin1a

Syntaxin1a (Syx1a) is essential for stimulated exocytosis in neuroendocrine cells. The vesicle docking process involves the formation of nanoscale Syx1a domains on the plasma membrane and the Syx1a clusters disintegrate during the fusion process. Syx1a nanodomains are both static yet empty and refill; the process by which these clusters maintain this balance is unclear. In this work, the dynamics of the Syx1a molecules is elucidated relative to the cluster position through a labeling strategy that allows both the bulk position of the Syx clusters to be visualized concurrent with the trajectories of single Syx1a molecules on the surface of PC12 cells. Single Syx1a molecules were tracked in time relative to cluster positions to decipher how Syx1a moves within a cluster and when clusters are not present. Syx1a is mobile on the plasma membrane, more mobile at the center of clusters, and less mobile near the edges of clusters; this depends on the presence of the N-terminal Habc domain and cholesterol, which are essential for proper exocytosis. Simulations of the dynamics observed at clusters support a model where clusters are maintained by a large cage (r = 100 nm) within which Syx1a remains highly mobile within the cluster (r = 50 nm). The depletion of cholesterol dramatically reduces the mobility of Syx1a within clusters and less so over the rest of the plasma membrane. This suggests that fluidity of Syx1a supramolecular clusters is needed for function. STATEMENT OF SIGNIFICANCESyntaxin1a (Syx1a) is essential for exocytosis where the vesicle docking process involves the formation of nanoscale Syx1a domains on the plasma membrane. Syx1a nanodomains are both static yet empty and refill; the process by which these clusters maintain this balance is unclear. In this work, the dynamics of the Syx1a molecules was elucidated relative to the cluster position on the surface of PC12 cells. Single molecules were tracked relative to clusters and Syx1a is mobile on the plasma membrane, more so within a cluster and less mobile near the edges of clusters. This depends on the presence of the N-terminal domain and cholesterol, which are essential for proper exocytosis. This suggests that fluidity of Syx1a clusters is needed for function.

biophysics↗

bioRxiv2022

Exosome secretion kinetics are controlled by temperature

When multivesicular endosomes (MVEs) fuse with the plasma membrane, exosomes are released into the extracellular space where they can affect other cells. Whether exosomes regulate cells nearby or further away depends on whether they remain attached to the secreting cell membrane. The regulation and kinetics of exosome secretion are not well characterized, but probes for directly imaging single MVE fusion events have allowed for visualization of the fusion and release process. In particular, the design of an exosome marker with a pH sensitive dye in the middle of the tetraspanin protein CD63 has facilitated studies of individual MVE fusion events. Using TIRF microscopy, single MVE fusion events were measured in A549 cells held at 23-37{degrees}C and events were identified using an automated detection algorithm. Stable docking precedes fusion almost all of the time and a decrease in temperature was accompanied by decrease in the rate of content loss and a decrease in the frequency of fusion events. The loss of CD63-pHluorin fluorescence was measured at fusion sites and fit with a single or double exponential decay, with approximately 50% of the events requiring two components and a plateau because the loss of fluorescence was typically incomplete. To interpret the kinetics, fusion events were simulated as a point source release of tethered/untethered exosomes coupled with the membrane diffusion of CD63. The experimentally observed decay required three components in the simulation: 1) free exosomes, 2) CD63 membrane diffusion from the endosomal membrane into the plasma membrane at a rate of 0.038 {micro}m2/s, as measured by FRAP, and 3) tethered exosomes. The final component of the decay arises from exosomes being secreted but tethered to the surface with one tether that has a lifetime of 8 seconds at 37{degrees}C and longer at lower temperatures. Simulating with fixed tethers or the absence of tethers fails to replicate the experimental data. This kinetic analysis increases our understanding of exosome secretion and how it is regulated by temperature. Our model suggests that exosome release from the fusion site is incomplete due to post-fusion, membrane attachment.

cell biology↗