Biology subjects

Pokrovskaya, I. D.

Publications and source records attributed to Pokrovskaya, I. D..

4 recordsLinked to original sources

bioRxiv2024

Single-Platelet Mapping of Jugular, Puncture-Wound Thrombi Reveals the Spatial Evolution of Platelet Activation

BackgroundThe contributions of platelet activation to thrombus formation during hemostatic bleeding cessation likely involve multiple activation states. However, the spatial and temporal distribution of platelets in these states has not been defined in a clot. ObjectivesTo use single-platelet mapping of activation states within jugular vein puncture thrombi to determine how the spatial distribution of platelet state evolves during hemostasis. MethodsMontaged, wide-area electron micrographs (EM) were taken at various time points, post-puncture, and annotated for platelet activation state. These classifications were mapped onto the images to identify regions of platelet activation and calculate neighbor associations. The importance of -granule secretion was tested using VAMP8-/- mice. Resultsmapping of platelet activation states at 1 min post-puncture showed extensive spatial intermixing of most platelet activation classes. No high-activation-state, platelet-rich core was observed in 5-min post-puncture thrombi, rather such platelets tended to be localized on the interior surfaces of thrombus vaults open to the circulation. Only at a later stage, 20 min post-puncture, was distinct clustering of high activation, degranulated, cytosol-rich platelets observed. These clusters localized to the central portion of the intravascular platelet-rich crown, and they were now inaccessible to the circulation. Counterintuitively, deletion of the primary platelet v-SNARE, VAMP8, increased the frequency and spatial clustering of highly activated, degranulated platelets in association with intra-thrombus vault surfaces at 5 min post puncture. ConclusionsWe conclude recent multi-activation state models can provide a realistic thrombus formation framework if linked together to encompass the dynamics of puncture wound formation.

systems biology↗

bioRxiv2023

Essential role of the Conserved Oligomeric Golgi complex in Toxoplasma gondii

Survival of the apicomplexan parasite Toxoplasma gondii depends on the proper functioning of many glycosylated proteins. Glycosylation is performed in the major membranous organelles ER and Golgi apparatus that constitute a significant portion of the intracellular secretory system. The secretory pathway is bidirectional: cargo is delivered to target organelles in the anterograde direction, while the retrograde flow maintains the membrane balance and proper localization of glycosylation machinery. Despite the vital role of the Golgi in parasite infectivity, little is known about its biogenesis in apicomplexan parasites. In this study we examined T. gondii Conserved Oligomeric Golgi (COG) complex and determined that, contrary to predictions, T. gondii expresses the entire eight-subunit complex and each complex subunit is essential for tachyzoite growth. Deprivation of the COG complex induces a pronounced effect on Golgi and ER membranes, which suggests the T. gondii COG complex has wider role in intracellular membrane trafficking. We demonstrated that besides its conservative role in protein glycosylation and retrograde intra-Golgi trafficking, the COG complex also interacted with anterograde and novel transport machinery. Furthermore, we identified coccidian-specific components of the Golgi transport system: TgUlp1 and TgGlp1. Protein structure and phylogenetic analyses revealed that TgUlp1 is an adaptation of the conservative Golgi tethering factor Uso1/p115, and together with Golgi-localized TgGlp1, TgUlp1 showed dominant interactions with the trafficking machinery that predicted to operate the endosome-to-Golgi recycling. Together, our study showed that T. gondii has expanded function of the conservative Golgi tethering COG complex and evolved additional regulators of the transport likely to serve parasite-specific secretory organelles.

cell biology↗

bioRxiv2022

STX5's flexibility in SNARE paring supports Golgi functions

The intracellular transport system is an evolutionally conserved, essential, and highly regulated network of organelles and transport vesicles that traffic protein and lipid cargoes within the cell. The events of vesicle formation, budding and fusion are orchestrated by the trafficking machinery - an elaborate set of proteins including small GTPases, vesicular coats, tethers, and SNAREs. The Golgi - the central organelle in this transport network, receives, modifies and sorts secretory and endocytic cargo. Glycosylation is one of the major modifications that occur within the Golgi, which houses enzymes and other components of glycosylation machinery. According to the current Golgi maturation model, Golgi resident proteins are constantly recycled from the late (trans) Golgi compartments to the early compartment (cis) by the evolutionary conserved vesicular trafficking machinery. The key modulator of vesicular trafficking and glycosylation at the Golgi is the Conserved Oligomeric Golgi (COG) complex - its interaction vesicular trafficking machinery particularly Golgi SNAREs (STX5, GS28 (GOSR1), GS15 (BET1L) and YKT6) that drive fusion of incoming vesicles. Since the COG complex functions upstream of SNARE-mediated vesicle fusion, we hypothesize that depletion of Golgi v-SNAREs would mirror defects observed in COG deficient cells. To test this, we created single and double knockouts (KO) of GS28 and GS15 in HEK293T cells and analyzed resulting mutants using a comprehensive set of biochemical, mass-spectrometry (MS) and microscopy approaches. Deletion of GS28 significantly affected GS15, but not the other two partners, STX5 and YKT6. Surprisingly, our analysis revealed that COG dysfunction is more deleterious for Golgi function than disrupting the canonical Golgi SNARE complex. Quantitative MS analysis of STX5-interacting SNAREs revealed unexpected flexibility of Golgi SNARE pairing in mammalian cells. We uncovered two novel non-canonical Golgi SNARE complexes - STX5/VTI1B/GS15/YKT6 and STX5/SNAP29/VAMP7 which were upregulated in GS28 KO cells. Analysis of cells co-depleted for GS28/SNAP29 or GS28/VTI1B SNAREs revealed escalated defects in Golgi glycosylation, indicating that upregulation of these complexes functionally substitutes deleted GS28. Our data points to the remarkable plasticity in the intra-Golgi membrane fusion machinery which is controlled by the COG complex.

cell biology↗

bioRxiv2022

Acute COG inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra-Golgi recycling vesicles

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats- COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.

cell biology↗