Bills, B. L. | Search bioRxiv

bioRxiv2023

Phospholipase D1 Produces Phosphatidic Acid at Sites of Secretory Vesicle Docking and Fusion

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently-tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

cell biology↗

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↗