Biology subjects

Pandey, T.

Publications and source records attributed to Pandey, T..

8 recordsLinked to original sources

bioRxiv2026

Evolution of Vesicle Shape Parameters Based on A Deep-Learning Based Image Analysis Framework Over Extended Time Periods

A precise analysis of shape instabilities in cell-sized membrane compartments requires thorough quantification of the shape dynamics of individual components over extended periods. Herein, we have used phase-contrast microscopy to image giant unilamellar membrane vesicles (GUVs), which are widely used as model systems for cellular structures. GUVs were prepared in the fluid lamellar phase of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine with and without cholesterol dissolved within a potato starch solution. The morphological changes and budding dynamics of individual GUVs with different chemical compositions were quantified using a deep-learning based image analysis framework over extended time periods, by using phase-contrast micrographs as input data. This automated framework integrates contour-based detection of the mother vesicle with precise segmentation of daughter compartments, thereby enabling explicit identification and tracking of mother and daughter vesicles at high temporal resolution. These findings further facilitate the measurement of the temporal evolution of vesicle morphometrics including area, and local membrane curvature during budding, that can be used as an input to develop computational models aimed at elucidating curvature-driven membrane remodeling in complex soft materials.

biophysics↗

bioRxiv2024

Shapes of Plasma Membrane Vesicles and PC-Cholesterol Vesicles Reveal Their Effective Spontaneous Curvature

Giant membrane vesicles (GUVs) and giant plasma membrane vesicles (GPMVs) are useful models for studying cellular membrane properties. Our research analyzed the reduced volume of vesicles made from phospholipid and 10% cholesterol to investigate transbilayer sugar asymmetries. We found that GPMVs have an average reduced volume of (0.88 {+/-} 0.06) with buffer asymmetry of 323 mM, lower than the (0.92 {+/-} 0.08) observed for DOPC: cholesterol vesicles with sucrose/glucose asymmetry of 390 mM. GUVs with different sugars inside and outside were more deflated, demonstrating a greater volume reduction than those with the same sugar inside and out. We applied the area-difference elasticity (ADE) model to map GPMVs and used the spontaneous curvature (SC) model to analyze DOPC: cholesterol GUVs, extracting spontaneous curvature based on their reduced volume. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/622000v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@178828forg.highwire.dtl.DTLVardef@c38f8eorg.highwire.dtl.DTLVardef@105c783org.highwire.dtl.DTLVardef@6da36d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Shape analysis of biomimetic and plasma membrane vesicles

Giant membrane vesicles (GUVs) and Giant plasma membrane vesicles (GPMVs) are models for examining membrane properties. We attempted to conduct a comparative study of reduced volume of vesicles with different lipid compositions, solution symmetry, solution asymmetry, and membrane charge. The vesicular morphology is studied using three-dimensional visualization techniques. The vesicles may not be exactly similar to spheres, but they may have some fluctuations resulting in changes in their shape. To understand these shape deformations, we visualize them as confocal image stacks. Our experimental observations indicate that the charge of the membrane affects the deflation of the GUVs in the presence of trans-bilayer sugar asymmetries. The lipid bilayers of our GUVs contain a uniform distribution of the lipids in the two leaflets, which implies that the two leaflets have the same lipid composition and there is no asymmetries in this composition. However, they obtain trans-bilayer asymmetries through asymmetric adsorption or desorption layers of different solution compositions adjacent to each leaflet. The deformation of the GPMVs shapes extracted from cells with trans-bilayer buffer asymmetries and the composition asymmetries is also estimated and compared with biomimetic membranes.

biophysics↗

bioRxiv2024

ER-GUARD: an evolutionarily conserved antioxidant defense system at ER membranes

Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2-based and functional reporter screens in C. elegans to discover a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting C. elegans ERGU-1 causes excessive H2O2 and transcriptional gene up-regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behavioral responses to H2O2. Both C. elegans and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. Human and Drosophila homologs of ERGU-1 can rescue C. elegans mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. In addition, purified ERGU-1 and human homolog TMEM161B exhibit redox-modulated oligomeric states. Together, our results reveal an ER-membrane-specific protein machinery for peroxide detoxification and suggest a previously unknown and conserved mechanisms for antioxidant defense in animal cells.

cell biology↗

bioRxiv2023

Acquired stress resilience through bacteria-to-nematode horizontal gene transfer

Natural selection drives acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisition of functions in immunity, metabolism, and reproduction via interdomain HGT (iHGT) from bacteria. We report that the nematode gene rml-3, which was acquired by iHGT from bacteria, enables exoskeleton resilience and protection against environmental toxins in C. elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most highly similar to bacterial enzymes that biosynthesize L-rhamnose to build cell wall polysaccharides. C. elegans rml-3 is regulated in developing seam cells by heat stress and stress-resistant dauer stage. Importantly, rml-3 deficiency impairs cuticle integrity, barrier functions and organismal stress resilience, phenotypes that are rescued by exogenous L-rhamnose. We propose that iHGT of an ancient bacterial rml-3 homolog enables L-rhamnose biosynthesis in nematodes that facilitates cuticle integrity and organismal resilience in adaptation to environmental stresses during evolution. These findings highlight the remarkable contribution of iHGT on metazoan evolution that is conferred by the domestication of bacterial genes.

genetics↗

bioRxiv2023

Insulin-mTOR hyperfunction drives C. elegans aging opposed by the megaprotein LPD-3

Insulin-mTOR signaling drives anabolic growth during organismal development, while its late-life dysregulation may detrimentally contribute to aging and limit lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin INS-7 is drastically over-produced in early life and shortens lifespan in lpd-3 mutants, a C. elegans model of human Alkuraya-Ku[c]inskas syndrome. LPD-3 forms a bridge-like tunnel megaprotein to facilitate phospholipid trafficking to plasma membranes. Lipidomic profiling reveals increased abundance of hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1 (Homolog of Yeast Longevity). Reducing HYL-1 activity decreases INS-7 levels and rescues the lifespan of lpd-3 mutants through insulin receptor/DAF-2 and mTOR/LET-363. LPD-3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age in wild type animals. We propose that LPD-3 acts as a megaprotein brake for aging and its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

physiology↗

bioRxiv2022

A megaprotein-based molecular bridge critical for lipid trafficking and cold resilience

Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through homeostatic mechanisms conserved in nearly all forms of life. As most eukaryotic enzymes for lipid synthesis and desaturation localize on endoplasmic reticulum (ER) membranes, it remains unknown how ER-resident lipids rapidly distribute to plasma membranes (PM). Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that plays critical roles in lipid trafficking and cold resilience. We identified lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7, and found that the 452 kDa megaprotein LPD-3 bridges ER and PM, consisting of a structurally predicted hydrophobic tunnel for lipid trafficking. Loss of LPD-3 caused abnormal cellular distribution of phospholipids, diminished FAT-7 abundance, and organismic vulnerability to cold. These phenotypic defects of lpd-3 mutants were rescued by Lecithin comprising unsaturated phospholipids. Importantly, we found that deficient lpd-3 homologues in Zebrafish and mammalian cells led to defects similar to those observed in C. elegans. As mutations in KIAA1109/BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, we propose that the LPD-3 family proteins may serve as evolutionarily conserved "highway bridges" critical for ER-associated non-vesicular trafficking of lipids and resilience to cold stress in eukaryotic cells.

cell biology↗

bioRxiv2022

Co-opted Genes of Algal Origin Protect C. elegans against Cyanogenic Toxins

Amygdalin is a cyanogenic glycoside widely used by many plants in herbivore defense. Poisonous to most animals, amygdalin-derived cyanide is detoxified by potent enzymes commonly found in bacteria and plants but not most animals. Here we show that the nematode C. elegans can detoxify amygdalin by a genetic pathway comprising cysl-1, egl-9, hif-1 and cysl-2. Essential for amygdalin resistance, cysl-1 encodes a protein similar to cysteine synthetic enzymes in bacteria and plants, but functionally co-opted in C. elegans. We identify exclusively HIF-activating egl-9 mutations in a cysl-1 suppressor screen and show that cysl-1 confers amygdalin resistance by regulating HIF-1-dependent cysl-2 transcription to protect against amygdalin toxicity. Phylogenetic analysis suggests cysl-1 and cysl-2 were likely acquired from green algae through horizontal gene transfer (HGT) and functionally co-opted in protection against amygdalin. Our studies reveal that HGT-mediated evolutionary changes can facilitate host survival and adaptation to adverse environment stresses and biogenic toxins.

genetics↗