Molecular mechanism of GPCR spatial organization at the plasma membrane.

G-protein-coupled receptors (GPCRs) mediate many critical physiological processes. Their spatial organization in plasma membrane (PM) domains is believed to encode signaling specificity and efficiency. However, the existence of domains and, crucially, the mechanism of formation of such putative domains remain elusive. Here, live-cell imaging (corrected for topography-induced imaging artifacts) conclusively established the existence of PM domains for GPCRs. Paradoxically, energetic coupling to extremely shallow PM curvature (<1 µm-1) emerged as the dominant, necessary and sufficient molecular mechanism of GPCR spatiotemporal organization. Experiments with different GPCRs, H-Ras, Piezo1 and epidermal growth factor receptor, suggest that the mechanism is general, yet protein specific, and can be regulated by ligands. These findings delineate a new spatiomechanical molecular mechanism that can transduce to domain-based signaling any mechanical or chemical stimulus that affects the morphology of the PM and suggest innovative therapeutic strategies targeting cellular shape.

OpenCell: Endogenous tagging for the cartography of human cellular organization.

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic 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. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

WASP integrates substrate topology and cell polarity to guide neutrophil migration.

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.

The WAVE complex associates with sites of saddle membrane curvature.

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

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 timescales. 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. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and 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.

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging.

Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as "fluorescence correlation spectroscopy super-resolution optical fluctuation imaging" or "fcsSOFI". Simulations demonstrate that structural features and diffusion properties can be accurately obtained at sub-diffraction-limited resolution. We apply our technique to image agarose hydrogels and aqueous lyotropic liquid crystal gels. The heterogeneous pore resolution is improved by up to a factor of 2, and diffusion coefficients are accurately obtained through our method compared to diffraction-limited fluorescence imaging and single-particle tracking. Moreover, fcsSOFI allows for rapid and high-throughput characterization of porous materials. fcsSOFI could be applied to soft porous environments such hydrogels, polymers, and membranes in addition to hard materials such as zeolites and mesoporous silica.