Biology subjects

Brunetti, R. M.

Publications and source records attributed to Brunetti, R. M..

4 recordsLinked to original sources

bioRxiv2021

WASP integrates substrate topology and cell polarity to guide neutrophil migration

To control their shape and movement, cells leverage nucleation promoting factors (NPFs) to regulate when and where they polymerize actin. Here we investigate the role of the immune-specific NPF WASP during neutrophil migration. Endogenously-tagged WASP localizes to substrate-induced plasma membrane deformations. Super-resolution imaging of live cells reveals that WASP preferentially enriches to the necks of these substrate-induced membrane invaginations, a distribution that could support substrate pinching. Unlike other curvature-sensitive proteins, WASP only enriches to membrane deformations at the cell front, where it controls Arp2/3 complex recruitment and actin polymerization. Despite relatively normal migration on flat substrates, WASP depletion causes defects in topology sensing and directed migration on textured substrates. WASP therefore both responds to and reinforces cell polarity during migration. Surprisingly, front-biased WASP puncta continue to form in the absence of Cdc42. We propose that WASP integrates substrate topology with cell polarity for 3D guidance by selectively polymerizing actin around substrate-induced membrane deformations at the leading edge. A misregulation of WASP-mediated contact guidance could provide insight into the immune disorder Wiskott-Aldrich syndrome.

cell biology↗

bioRxiv2021

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

cell biology↗

bioRxiv2019

WAVE complex self-organization templates lamellipodial formation

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. For example, why does the WAVE complex build lamellipodia, the broad sheet-like protrusions that power cell migration, whereas the homologous actin regulator N-WASP forms spiky finger-like actin networks? N-WASP is known to oligomerize into focal condensates that generate an actin finger. In contrast, the WAVE complex exhibits the linear distribution needed to generate an actin sheet. This linear organization of the WAVE complex could either arise from interactions with the actin cytoskeleton or could represent an ability of the complex to self-organize into a linear template. Using super-resolution microscopy, we find that the WAVE complex forms higher-order linear oligomers that curve into 270 nanometer-wide ring structures in the absence of actin polymer. These rings localize to the necks of membrane invaginations, which display saddle point geometries with positive curvature in one axis and negative curvature in the orthogonal axis. To investigate the molecular mechanism of saddle curvature enrichment, we show that the WAVE complex and IRSp53, a membrane curvature-sensitive protein, collaborate to recognize saddle curvature that IRSp53 cannot sense alone. This saddle preference for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes. Our work highlights how partnering protein interactions enable complex shape sensing and how feedback between cell shape and actin regulators yields self-organized cell morphogenesis.

cell biology↗

bioRxiv2017

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.\n\nBuilds onMavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchuir SO, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife. 2016.\n\nImpact StatementWe organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.

bioinformatics↗