Biology subjects

Marchando, P.

Publications and source records attributed to Marchando, P..

4 recordsLinked to original sources

bioRxiv2024

Polarized exocytosis and anionic phospholipid species implicated in the initiation of clathrin-mediated endocytosis

Understanding of the mechanisms that initiate clathrin-mediated endocytosis (CME) is incomplete. Recent studies in budding yeast identified the endocytic adaptor proteins Yap1801/Yap1802 (budding yeast AP180) as key CME factors that promote CME initiation in daughter cells during polarized growth, but how Yap1801/2 are recruited preferentially to the plasma membrane of daughter cells is not clear. The only known cargos for Yap1801/2 in yeast are the synaptobrevins Snc1 and Snc2, which serve as v-SNARES for exocytic vesicles reaching the plasma membrane and are crucial for polarized cell growth. In this study, we examine the spatiotemporal dynamics of functional, fluorescent protein-tagged Snc2 expressed from its endogenous locus and provide evidence that, along with anionic phospholipids, Snc2 specifically recruits Yap1802 to growing daughter cells. This protein-protein interaction creates a direct link between polarized secretion and CME and has further implications in CME initiation.

cell biology↗

bioRxiv2024

The WAVE complex forms linear arrays at negative membrane curvature to instruct lamellipodia formation.

Cells generate a wide range of actin-based membrane protrusions for various cell behaviors. These protrusions are organized by different actin nucleation promoting factors. For example, N-WASP controls finger-like filopodia, whereas the WAVE complex controls sheet-like lamellipodia. These different membrane morphologies likely reflect different patterns of nucleator self-organization. N-WASP phase separation has been successfully studied through biochemical reconstitutions, but how the WAVE complex self-organizes to instruct lamellipodia is unknown. Because WAVE complex self-organization has proven refractory to cell-free studies, we leverage in vivo biochemical approaches to investigate WAVE complex organization within its native cellular context. With single molecule tracking and molecular counting, we show that the WAVE complex forms highly regular multilayered linear arrays at the plasma membrane that are reminiscent of a microtubule-like organization. Similar to the organization of microtubule protofilaments in a curved array, membrane curvature is both necessary and sufficient for formation of these WAVE complex linear arrays, though actin polymerization is not. This dependency on negative membrane curvature could explain both the templating of lamellipodia and their emergent behaviors, including barrier avoidance. Our data uncover the key biophysical properties of mesoscale WAVE complex patterning and highlight an integral relationship between NPF self-organization and cell morphogenesis.

cell biology↗

bioRxiv2024

Aggregation of an FG nucleoporin under crowded conditions

Macromolecular crowding can affect the aggregation behavior of intrinsically disordered proteins in unexpected ways. We studied the aggregation of a peptide derived from the disordered FG nucleoporins which line the nuclear pore complex. We measured its aggregation kinetics in the presence of both inert and non-specifically interacting crowding agents. Using fluorescence emission and NMR spectroscopy, we probed differences in the local chemical microenvironment of the peptides residues. Our results indicate differences in aggregation kinetics and residue microenvironment depending on the identity of the crowder, including differences between crowding with PEG and PVP, two polymers which are often used interchangeably as inert crowding agents.

biophysics↗

bioRxiv2019

Cargo licenses maturation of stalled endocytic sites through a regulatory checkpoint

During clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches clustered near exocytic sites are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abortive events, our data instead identify maturation through a checkpoint in the pathway as the cargo-sensitive step. SummaryPedersen, Hassinger, et al. investigate steps of the clathrin-mediated endocytosis pathway that are subject to regulation. They report position-dependent differences in endocytic site maturation rates in polarized cells and suggest that cargo controls endocytic internalization through tuning site maturation rather than site initiation.

cell biology↗