Biology subjects

Scott, B. L.

Publications and source records attributed to Scott, B. L..

6 recordsLinked to original sources

bioRxiv2026

PI(3)P Signaling by VPS34 Complex II Orchestrates Macropinocytosis

Macrophage macropinocytosis contributes to wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Here, CRISPR/Cas9 whole-genome screens in murine bone-marrow derived macrophages (BMDM) identified genes regulating unstimulated, PMA-, and CSF1-stimulated uptake of the fluorescent pinocytosis solute tracer, Lucifer yellow. UVRAG and other members of VPS34 complex II (VPS34-II), which catalyze PI(3)P formation from phosphatidylinositol, were identified as positive regulators of macropinocytosis. Targeted gene disruption of Uvrag and Pik3r3 revealed that VPS34-II is required for efficient macropinocytosis with Uvrag-disrupted BMDM having fewer but larger macropinosomes. In contrast, depletion of ATG14, a unique component of VPS34 complex I, increased solute uptake and the number of macropinosomes. Live-cell imagining of macrophages expressing 2xFYVE-fluorescent protein fusions showed PI(3)P present on the plasma membrane, nascent macropinosomes, and endosomes. The presence of PI(3)P on the plasma membrane prior to macropinocytic cup closure, indicates a novel role on the plasma membrane. Quantitative imaging of fixed cells shows a decrease in concentration of PI(3)P in Uvrag-disrupted BMDMs while increase in concentration of PI(3)P in Atg14-disrupted BMDM compared to WT BMDMs. Treatment with the VPS34 inhibitor SAR-405 acutely decreased macropinocytosis but maintained AKT phosphorylation suggesting class I PI3K activity and PI(3,4,5)P3 production are independent of class III PI3K activity. These results suggest that PI(3)P is a key phosphoinositide governing macropinocytosis at the plasma membrane and that it is primarily formed via direct phosphorylation of PI rather than via the sequential dephosphorylation of PIP3.

cell biology↗

bioRxiv2021

IgG surface mobility promotes antibody-dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages.

By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fc{gamma} receptors (Fc{gamma}R) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to Fc{gamma}R signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger redistribution of Rituximab-Fc{gamma}R during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of Fc{gamma}R-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and Fc{gamma}R-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens. Significance StatementADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange Fc{gamma}R-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.

immunology↗

bioRxiv2021

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

As the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

cell biology↗

bioRxiv2020

The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice lightsheet microscopy

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

cell biology↗

bioRxiv2018

Different Munc18 Proteins Mediate Baseline and Stimulated Airway Mucin Secretion

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens, but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all three Munc18 isoforms. Using conditional airway epithelial deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane, and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.

cell biology↗

bioRxiv2017

Membrane bending begins at any stage of clathrin-coat assembly and defines endocytic dynamics

Summary ParagraphClathrin-mediated endocytosis internalizes membrane from the cell surface by reshaping flat regions of membrane into spherical vesicles(1, 2). The relationship between membrane bending and clathrin coatomer assembly has been inferred from electron microscopy and structural biology, without directly visualization of membrane bending dynamics (3-6). This has resulted in two distinct and opposing models for how clathrin bends membrane (7-10). Here, polarized Total Internal Reflection Fluorescence microscopy was improved and combined with electron microscopy, atomic force microscopy, and super-resolution imaging to measure membrane bending during endogenous clathrin and dynamin assembly in living cells. Surprisingly, and not predicted by either model, the timing of membrane bending was variable relative to clathrin assembly. Approximately half of the time, membrane bending occurs at the start of clathrin assembly, in the other half, the onset of membrane bending lags clathrin arrival, and occasionally completely assembled flat clathrin transitions into a pit. Importantly, once the membrane bends, the process proceeds to scission with similar timing. We conclude that the pathway of coatomer formation is versatile and can bend the membrane during or after the assembly of the clathrin lattice. These results highlight the heterogeneity in this fundamental biological process, and provide a more complete nanoscale view of membrane bending dynamics during endocytosis.

cell biology↗