Biology subjects

Knowles, M. K.

Publications and source records attributed to Knowles, M. K..

4 recordsLinked to original sources

bioRxiv2026

Phospholipase D1 and Phosphatidic Acid are required for MVE Fusion and Exosome Secretion

Extracellular vesicles (EVs) mediate critical intercellular communication, yet the molecular mechanisms that govern multivesicular endosome (MVE) fusion with the plasma membrane and exosome release remain poorly understood. Phospholipase D1 (PLD1) produces phosphatidic acid (PA), a lipid involved in membrane remodeling, but when and how PLD1 and PA act during exosome secretion has not been defined. Here, we used immunofluorescence and total internal reflection fluorescence microscopy (TIRFM) to track individual CD63+ MVEs together with fluorescent PLD1 or a PA reporter (GFP-PASS) in A549 cells. PLD1 localized to CD63+ MVEs during visiting, docking, and fusion. Inhibition or knockdown of PLD1 significantly reduced MVE fusion frequency and decreased the number of secreted small EVs, while causing only a modest reduction in vesicle availability at the plasma membrane. PLD1 inhibition also increased the number of membrane-proximal lysosomes, suggesting that MVEs are diverted toward degradation when fusion is impaired. Meanwhile, PA dynamics were stage-specific: PA remained low on visiting vesicles, gradually accumulated during docking, and exhibited a sharp spike followed by loss during fusion. PA has been reported to stabilize negative curvature and potentially play a role during fusion. To test whether PA influences fusion pore behavior, we quantified CD63 decay duration (t1/2) for individual fusion events. K-means clustering revealed that vesicles with longer decay durations had higher PA levels, whereas short-decay events showed minimal PA. The PA intensity correlated positively with decay duration, while cytosolic GFP did not, indicating a specific relationship between PA and exosome release rates. Furthermore, pharmacological activation of PLD1 increased the proportion of long-decay events. Together, these findings demonstrate that PLD1-generated PA regulates MVE fate at two levels: it promotes the docking-to-fusion transition and prolongs exosome release. This identifies a lipid-based mechanism that controls both the efficiency and kinetics of exosome secretion. SIGNIFICANCE STATEMENTExosome release is essential for cell-cell communication in processes such as development, immunity, and cancer. However, the mechanism that determines whether a multivesicular endosome fuses with the plasma membrane to release exosome cargo remains unclear. We identified a lipid-based mechanism that regulates both fusion and the rate of content release. We show that phospholipase D1 (PLD1) and its product phosphatidic acid (PA) act at late stages of exosome secretion: PLD1 promotes the docking-to-fusion transition and PA levels quantitatively correlate with content release rate, suggesting that PA stabilizes the pore. These findings demonstrate that lipids can control secretion frequency and kinetics, providing a new framework for understanding and modulating exosome output.

biophysics↗

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↗

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↗