Biology subjects

Hoppe, A. D.

Publications and source records attributed to Hoppe, A. D..

9 recordsLinked to original sources

bioRxiv2026

High-resolution CRISPR/Cas9 screens identify PAK2 as a suppressor of macrophage proliferation

Macrophage abundance is regulated by pathways controlling survival and proliferation, yet the genetic determinants of macrophage fitness remain poorly understood. Here, we performed replicate genome-wide CRISPR/Cas9 screens in primary murine bone marrow-derived macrophages (BMDMs) to identify positive and negative regulators of macrophage fitness. Validity of the screens is supported by finding previously characterized core essential genes such as those encoding translation machinery, while disruption of established tumor suppressors increased BMDM representation in the culture. As expected, Csf1r encoding the macrophage growth factor emerged as a positive regulator of macrophage fitness. Unexpectedly, Pak2, encoding p21-activated kinase 2 (PAK2), emerged as a negative regulator of macrophage fitness. Targeted Pak2 disruption increased BMDM accumulation and was associated with elevated cyclin-D1 expression, identifying PAK2 as a suppressor of macrophage proliferation. PAK2 also localized to CSF1-induced actin-rich membrane ruffles, yet its depletion did not impair ruffle formation. Instead, PAK2-depleted macrophages exhibited persistent F-actin-rich ruffles, larger macropinosomes, and increased fluid-phase uptake. These changes were accompanied by altered LIMK/cofilin signaling and persistent cofilin localization at macropinocytic structures. Phosphoproteomic analysis further identified reduced phosphorylation of proteins associated with cytoskeletal regulation, phosphoinositide signaling, and endosomal trafficking. Together, these findings identify PAK2 as a context-dependent regulator that restrains macrophage proliferation and membrane-remodeling activity and demonstrate that PAK2 has markedly different fitness functions in primary macrophages than previously identified in several transformed cell types.

cell biology↗

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↗

bioRxiv2024

Mannose receptor (MRC1) mediates uptake of dextran in macrophages via receptor-mediated endocytosis

Macrophages maintain surveillance of their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows macrophages to recognize and internalize specific ligands whereas macropinocytosis non-selectively internalizes extracellular fluids and solutes. Here, CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone-marrow derived macrophages (BMDM). The endocytic mannose receptor c-type 1 (Mrc1, also known as CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on uptake of Lucifer yellow, a fluid-phase marker. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating the solutes are internalized by different mechanisms. We further deduced that BMDMs take up dextran via MRC1-mediated endocytosis by showing that competition with mannan, a ligand of MRC1, as well as treatment with Dyngo-4a, a dynamin inhibitor, reduced dextran uptake. Finally, we observed that IL4-treated BMDM internalize more dextran than untreated BMDM by upregulating MRC1 expression. These results demonstrate that dextran is not an effective marker for the bulk uptake of fluids and solutes by macropinocytosis since it is internalized by both macropinocytosis and receptor-mediated endocytosis in cells expressing MRC1. This report identifies numerous genes that regulate dextran internalization in primary murine macrophages and predicts cellular pathways and processes regulating MRC1. This work lays the groundwork for identifying specific genes and regulatory networks that regulate MRC1 expression and MRC1-mediated endocytosis in macrophages. Significance StatementO_LIMacrophages constantly survey and clear tissues by specifically and non-specifically internalizing debris and solutes. However, the molecular mechanisms and modes of regulation of these endocytic and macropinocytic processes are not well understood. Here, CRISPR/Cas9 whole genome screens were used to identify genes regulating uptake of dextran, a sugar polymer that is frequently used as a marker macropinocytosis, and compared with Lucifer yellow, a fluorescent dye with no known receptors. C_LIO_LIThe authors identified the mannose receptor as well as other proteins regulating expression of the mannose receptor as top hits in the screen. Targeted disruption of Mrc1, the gene that encodes mannose receptor, greatly diminished dextran uptake but had no effect on cellular uptake of Lucifer yellow. Furthermore, exposure to the cytokine IL4 upregulated mannose receptor expression on the cell surface and increased uptake of dextran with little effect on Lucifer yellow uptake. Studies seeking to understand regulation of macropinocytosis in macrophages will be confounded by the use of dextran as a fluid-phase marker. C_LIO_LIMRC1 is a marker of alternatively activated/anti-inflammatory macrophages and is a potential target for delivery of therapeutics to macrophages. This work provides the basis for mechanistic underpinning of how MRC1 contributes to the receptor-mediated uptake of carbohydrates and glycoproteins from the tissue milieu and distinguishes genes regulating receptor-mediated endocytosis from those regulating the bona fide fluid-phase uptake of fluids and solutes by macropinocytosis. C_LI

cell biology↗

bioRxiv2024

Self-growth suppression in Bradyrhizobium diazoefficiens is caused by a diffusible antagonist

Microbes in soil navigate interactions by recognizing kin, forming social groups, exhibiting antagonistic behavior, and engaging in competitive kin rivalry. Here, we investigated a novel phenomenon of self-growth suppression (sibling rivalry) observed in Bradyrhizobium diazoefficiens USDA 110. Swimming colonies of USDA 110 developed a distinct demarcation line and inter-colony zone when inoculated adjacent to each other. In addition to self, USDA 110 suppressed growth of other Bradyrhizobium strains and several other soil bacteria. We demonstrated that the phenomenon of sibling rivalry is due to growth suppression but not cell death. The cells in the inter-colony zone were culturable but have reduced respiratory activity, ATP levels and motility. The observed growth suppression was due to the presence of a diffusible effector compound. This effector was labile, preventing extraction, and identification, but it is unlikely a protein or a strong acid or base. This counterintuitive phenomenon of self-growth suppression suggests a strategic adaptation for conserving energy and resources in competitive soil environments. Bradyrhizobiums utilization of antagonism including self-growth suppression likely provides a competitive advantage for long-term success in soil ecosystems.

microbiology↗

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

Systematic Prediction of Regulatory Motifs from Human ChIP-Sequencing Data Based on a Deep Learning Framework

Identification of transcription factor binding sites (TFBSs) and cis-regulatory motifs (motifs for short) from genomics datasets, provides a powerful view of the rules governing the interactions between TFs and DNA. Existing motif prediction methods however, are limited by high false positive rates in TFBSs identification, contributions from non-sequence-specific binding, and complex and indirect binding mechanisms. High throughput next-generation sequencing data provides unprecedented opportunities to overcome these difficulties, as it provides multiple whole-genome scale measurements of TF binding information. Uncovering this information brings new computational and modeling challenges in high-dimensional data mining and heterogeneous data integration. To improve TFBS identification and novel motifs prediction accuracy in the human genome, we developed an advanced computational technique based on deep learning (DL) and high-performance computing, named DESSO. DESSO utilizes deep neural network and binomial distribution to optimize the motif prediction. Our results showed that DESSO outperformed existing tools in predicting distinct motifs from the 690 in vivo ENCODE ChIP-Sequencing (ChIP-Seq) datasets for 161 human TFs in 91 cell lines. We also found that protein-protein interactions (PPIs) are prevalent among human TFs, and a total of 61 potential tethering binding were identified among the 100 TFs in the K562 cell line. To further expand DESSOs deep-learning capabilities, we included DNA shape features and found that (i) shape information has a strong predictive power for TF-DNA binding specificity; and (ii) it aided in identification of the shape motifs recognized by human TFs which in turn contributed to the interpretation of TF-DNA binding in the absence of sequence recognition. DESSO and the analyses it enabled will continue to improve our understanding of how gene expression is controlled by TFs and the complexities of DNA binding. The source code and the predicted motifs and TFBSs from the 690 ENCODE TF ChIP-Seq datasets are freely available at the DESSO web server: http://bmbl.sdstate.edu/DESSO.

bioinformatics↗

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↗