Biology subjects

van Loon, A. P.

Publications and source records attributed to van Loon, A. P..

5 recordsLinked to original sources

bioRxiv2022

Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. Their exquisite sensitivity to chemical gradients and ability to rapidly migrate make them especially suited to protect against infection. However, their terminal differentiation status and short lifetime (on the order of days) have hindered their study. Furthermore, while modern CRISPR-based gene perturbation strategies now allow comprehensive, genome-scale screens in human cells, their application to complex and dynamic processes like cell migration remain limited. Using HL-60 cells, a leukemia cell line that can be differentiated into neutrophil-like cells, we have developed multiple cell migration screen strategies that provide comprehensive, genome-wide discovery of molecular factors that are critical for directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. Combining these assays with additional, pooled, genome-wide CRISPR interference dropout screens of cell proliferation and neutrophil differentiation, we have identified a comprehensive set of genes that are important across the processes of cellular growth, differentiation, and migration. This combined dataset highlights a particular reliance upon mTORC1 signaling that alters neutrophil lifetime, migration phenotype, and sensitivity to chemotactic cues. Across our cell migration screens, we identified several hundred genes important for migration including those with specific roles only in particular migratory contexts. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

cell biology↗

bioRxiv2022

Cell surface receptors TREM2, CD14 and integrin αMβ2 drive sinking engulfment in phosphatidylserine-mediated phagocytosis

Macrophages phagocytose and thereby eliminate a wide array of extracellular threats, ranging from antibody-coated bacteria to apoptotic cells. Precision modulation of phagocytosis has emerged as a therapeutic strategy across a range of diseases, but is limited by our incomplete understanding of how macrophages recognize, engulf, and respond to different phagocytic targets. Here, we undertook a systematic investigation of the morphological, biophysical and regulatory differences between two major types of phagocytosis: an immunostimulatory form of phagocytosis triggered by antibody-coated targets and an immunosuppressive form triggered by phosphatidylserine (PS)-coated targets. We confirmed classic observations that antibody-mediated phagocytosis involves the extension of thin actin-rich protrusions around the target, but find that PS-mediated phagocytosis involves an unexpected combination of filopodial probing, piecemeal phagocytosis and a distinct sinking mechanism of uptake. Using a genome-wide screening approach, we identified genes specifically required for each form of phagocytosis, including actin regulators, cell surface receptors and intracellular signaling molecules. Three cell surface receptors - TREM2, CD14 and integrin M{beta}2 - were revealed as essential for PS-mediated uptake. Strikingly, each receptor exhibited a distinct pattern of localization at the plasma membrane and contributed uniquely to the organization of the PS-dependent phagocytic cup. Overall, this work reveals divergent genetic requirements for the morphologically and mechanically distinct forms of PS-mediated and antibody-mediated phagocytosis, thereby informing therapeutic strategies for substrate-specific phagocytosis modulation.

cell biology↗

bioRxiv2020

Cortical myosin minifilaments orchestrate the arrangement of microridge protrusions on epithelial cell surfaces

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally-elongated protrusions arranged in maze-like patterns on mucosal epithelial cells that rearrange dynamically by fission and fusion. To characterize how microridges mature and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridges continued to lengthen and microridge alignment became increasingly well ordered. Imaging F-actin and Non-Muscle Myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging revealed that individual cortical NMII minifilaments are tethered to protrusions, often connecting adjacent microridges. NMII minifilaments connecting the ends of microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. Our findings demonstrate that as cells mature, microridges continue to remodel and form an increasingly orderly arrangement through a process orchestrated by cortical NMII contraction.

cell biology↗

bioRxiv2019

Cortical contraction drives the 3D patterning of epithelial cell surfaces

Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regulating their formation and arrangement are largely unknown. To study how protrusions form, we focused on the morphogenesis of microridges, elongated actin-based structures projecting from the apical surfaces of zebrafish skin cells that are arranged in labyrinthine patterns. Microridges form by accreting simple finger-like precursors. Live imaging demonstrated that microridge morphogenesis is linked to apical constriction. A non-muscle myosin II (NMII) reporter revealed pulsatile contractions of the actomyosin cortex; inhibiting NMII demonstrated that contractions are required for apical constriction and microridge formation. A biomechanical model suggested that contraction reduces surface tension to permit the fusion of precursors into microridges. Indeed, reducing surface tension with hyperosmolar media promoted microridge formation. In anisotropically stretched cells, microridges formed by precursor fusion along the stretch axis, which computational modeling explained as a consequence of stretch-induced cortical flow. Collectively, our results demonstrate how contraction within the 2D plane of the cortex patterns 3D cell surfaces.\n\nSUMMARYMicroridges, elongated 3D protrusions arranged in maze-like patterns on zebrafish skin cells, form by the accretion of simple precursor projections. Modeling and in vivo experiments showed that cortical contractions promote the coalescence of precursors into microridges by reducing membrane tension.

cell biology↗

bioRxiv2017

The Drosophila Formin Fhod Nucleates Actin Filaments

Formins are a conserved group of proteins that nucleate and processively elongate actin filaments. Among them, the formin homology domain-containing protein (FHOD) family of formins contributes to contractility of striated muscle and cell motility in several contexts. However, the mechanisms by which they carry out these functions remain poorly understood. Unlike other formins, mammalian FHOD1 and FHOD3 do not accelerate actin assembly in vitro, and have instead been suggested to act as barbed end cappers or bundlers. Here, we show that purified Drosophila Fhod, in contrast with the mammalian homologues, potently accelerates actin assembly by nucleation. We found that Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows elongation in the presence of profilin. Fhod protects barbed ends from capping protein, but dissociates from barbed ends relatively quickly. Finally, we used cosedimentation assays to determine that Fhod binds the sides of actin filaments and bundles filaments. This work establishes that Fhod shares the capacity of other formins to nucleate and bundle actin filaments, but is notably less effective at processively elongating barbed ends.

cell biology↗