Biology subjects

Elsayyid, M.

Publications and source records attributed to Elsayyid, M..

4 recordsLinked to original sources

bioRxiv2025

Superoxide dismutase impacts extracellular vesicle biogenesis and uptake

Extracellular vesicles (EVs), which transfer bioactive macromolecules between cells, play an important role in the pathogenesis of multiple neurodegenerative diseases. Focus has centered on how altered EV contents propagate disease and the potential for EVs as diagnostic biomarkers, while the effect of pathogenic factors on EV release is less understood. Here, we defined how the key antioxidant enzyme superoxide dismutase 1 (SOD-1) affects EV shedding from sensory neuron primary cilia, enrichment of ciliary proteins packaged into EVs, and uptake of EVs by surrounding glia in vivo by imaging C. elegans expressing fluorescent protein-tagged EV cargos. We discovered that loss of SOD-1, as well as the SOD-1(G85R) amyotrophic lateral sclerosis (ALS) pathogenic variant, increased EV shedding from the cilium distal tip, and this was associated with greater abundance of EV cargo in this ciliary compartment. In contrast, loss of SOD-1 reduced the glial uptake of a different cargo present in EVs shed from the ciliary base. Together, this suggests that redox balance has a subtype-specific effect on EV biogenesis, influencing neuron communication in vivo.

cell biology↗

bioRxiv2025

Phosphatidylinositol (4,5)-bisphosphate Impacts Ectosome Shedding from C. elegans Ciliated Sensory Neurons

Small secreted extracellular vesicles (EVs) mediate intercellular transport of bioactive macromolecules. How the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which plays a critical role in many cellular processes, impacts EV biogenesis is unclear. The primary cilium, a sensory organelle protruding from most non-dividing cells, transmits signals by shedding EVs called ectosomes. Here, we altered ciliary PI(4,5)P2 by manipulating expression of the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K1) PPK-1 and deletion of the phosphoinositide 5-phosphatase (INPP5E) inpp-1, then determined the impact on release of EVs that carried cargos tagged with fluorescent proteins. We discovered that increasing PI(4,5)P2 differentially affected ectosome shedding from distinct compartments, decreasing biogenesis of an EV subpopulation from the ciliary base, but enhancing budding from the cilium distal tip. Altering PI(4,5)P2 levels also impacted the abundance and distribution of EV cargos in the cilium, but not the sorting of the protein cargos into distinct subsets of ectosomes. Finally, manipulating PI(4,5)P2 did not affect cilium length, suggesting that changing PI(4,5)P2 levels can serve as a mechanism to regulate ectosome biogenesis in response to physiological stimuli without impacting cilium morphology. Summary StatementExtracellular vesicles (EVs), released from most, if not all, cell types, share bioactive cargo that cannot readily cross the plasma membrane with recipient cells. EVs play numerous roles in physiological processes as well as the propagation of pathophysiological conditions, including cancer, neurodegenerative, kidney, and cardiovascular diseases. Here, we sought to determine the impact of the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) on EV biogenesis. We used a genetic approach to manipulate PI(4,5)P2 levels in the primary cilium, a specialized sensory organelle that serves as a platform for signal transmission by shedding EVs. We discovered that high PI(4,5)P2 differentially impacts two distinct EV subpopulations, decreasing shedding of ectosomes derived from the ciliary base, but increasing budding from the cilium distal tip. This work defines a new role for PI(4,5)P2 in the regulation of EV biogenesis and ciliary biology.

cell biology↗

bioRxiv2024

In-cell processing enables rapid and in-depth proteome analysis of low-input Caenorhabditis elegans

Caenorhabditis elegans is a widely used genetic model organism, however, the worm cuticle complicates extraction of intracellular proteins, a prerequisite for typical bottom-up proteomics. Conventional physical disruption procedures are not only time-consuming, but can also cause significant sample loss, making it difficult to perform proteomics with low-input samples. Here, for the first time, we present an on-filter in-cell (OFIC) processing approach, which can digest C. elegans proteins directly in the cells of the organism after methanol fixation. With OFIC processing and single-shot LCMS analysis, we identified over 9,400 proteins from a sample of only 200 worms, the largest C. elegans proteome reported to date that did not require fractionation or enrichment. We systematically evaluated the performance of the OFIC approach by comparing it with conventional lysis-based methods. Our data suggest equivalent and unbiased performance of OFIC processing for C. elegans proteome identification and quantitation. We further evaluated the OFIC approach with even lower input samples, then used this method to determine how the proteome is impacted by loss of superoxide dismutase sod-1, the ortholog of human SOD-1, a gene associated with amyotrophic lateral sclerosis (ALS). Analysis of 8,800 proteins from only 50 worms as the initial input showed that loss of sod-1 affects the abundance of proteins required for stress response, ribosome biogenesis, and metabolism. In conclusion, our streamlined OFIC approach, which can be broadly applied to other systems, minimizes sample loss while offering the simplest workflow reported to date for C. elegans proteomics analysis.

biochemistry↗

bioRxiv2021

Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.

cell biology↗