Biology subjects

Vega, A. R.

Publications and source records attributed to Vega, A. R..

5 recordsLinked to original sources

bioRxiv2022

Network of hotspot interactions cluster tau amyloid folds

Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformation of {beta}-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We test our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements that control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures.

neuroscience↗

bioRxiv2022

Tau seeds translocate across the cell membrane to initiate aggregation

Neurodegenerative tauopathies, including Alzheimers disease and related disorders, are caused by intracellular aggregation of tau protein in ordered assemblies. Experimental evidence suggests that tau assemblies propagate pathology across brain networks. Tau seeds enter cells through endocytosis but must access the cytoplasm to serve as templates for their own replication. The mechanism by which this occurs is unknown. To study tau uptake, we began with a whole-genome CRISPR knockout screen, which indicated a requirement vacuolar H+ ATPase (v-ATPase) components. Treatment with Bafilomycin A1, an inhibitor of the v-ATPase, also reduced tau entry. We next tested direct modifiers of endolysosomal trafficking. Dominant-negative Rab5a expression uniquely decreased tau uptake, as did temporary cold temperature during tau exposure, consistent with a primary role of endocytosis in tau uptake. However, despite reducing tau uptake, these interventions all paradoxically increased intracellular seeding. Consequently, we generated giant plasma membrane vesicles (GPMVs), which cannot undergo endocytosis, and observed that tau fibrils and monomer translocated into the vesicles, in addition to TAT peptide, whereas transferrin and albumin did not. In every case, tau required binding to heparan sulfate proteoglycans (HSPGs) for cell uptake, seeding, or GPMV entry. These findings are most consistent with direct translocation of tau seeds across the lipid bilayer, a novel mechanism of entry into the cytoplasm.

cell biology↗

bioRxiv2022

Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

Tau assembly propagation from the extracellular to intracellular space of a cell may underlie neurodegenerative tauopathies. The first step involves tau binding to heparan sulfate proteoglycans on the cell surface, followed by macropinocytosis. Pathological tau assemblies are thought to exit the vesicular compartment as "seeds" for replication in the cytoplasm. Tau uptake is highly efficient, but only [~]1-10% of cells that take up aggregates exhibit seeding. To investigate the basis for this observation, we used fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells, and "biosensor" cells expressing FL tau or repeat domain fused to mClover (Clo). FL tau-Clo bound tubulin, but seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but imaging revealed a small percentage that slowly and steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure. In fact, most seeded cells had no evidence of lysosome rupture. However, live cell imaging indicated that cells with pre-existing Gal3-positive puncta exhibited seeding at a slightly higher rate than the general population, indicating a potential role for vesicle instability as a predisposing factor. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. Bafilomycin inhibited vesicular clearance, whereas MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds replicate, and are cleared by the proteasome.

cell biology↗

bioRxiv2019

Single-molecule study of the dynamic organization of Vascular Endothelial Growth Factor Receptor 2 on the endothelial cell surface

The nanoscale organization of cell surface receptors plays an important role in signaling. We determined this organization and its relation to receptor activation for VEGF Receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining live-cell single-molecule imaging of endogenous VEGFR-2 with rigorous computational analysis. We found that surface VEGFR-2 can be mobile or immobile/confined, and monomeric or non-monomeric, with a complex interplay between the two. The mobility and interaction heterogeneity of VEGFR-2 in the basal state led to heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we found that VEGF can bind to both monomeric and non-monomeric VEGFR-2, and, when binding to monomeric VEGFR-2, promotes dimer formation but only for immobile/confined receptors. Overall, our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and its complex relationships with receptor activation and signaling.

biophysics↗

bioRxiv2018

A Composition-Dependent Molecular Clutch Between T Cell Signaling Clusters and Actin

Biomolecular condensates play important roles in eukaryotic cells by concentrating molecules into foci without a surrounding membrane. During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of the adaptor protein LAT and its binding partners Grb2, Sos1, SLP-76, Nck and WASP. These condensates move radially at the IS, traversing a radially-oriented and then a concentric actin network. To understand the persistent radial movement, we biochemically reconstituted LAT condensates with mobile actomyosin filaments. We found that basic regions of Nck and N-WASP promote strong association and co-movement of LAT condensates with actin. Condensates lacking these components were instead propelled by steric interactions. In cells, LAT condensates lost Nck while traversing the boundary between the two actin networks, and condensates engineered to constitutively bind actin moved aberrantly. We propose that Nck and WASP form a clutch between LAT condensates and actin, and changes in composition enable condensate movement by distinct actin networks in different regions of the IS.

cell biology↗