Biology subjects

Heit, B.

Publications and source records attributed to Heit, B..

12 recordsLinked to original sources

bioRxiv2026

The Actin Cytoskeleton and Caveolae Regulate MERTK Cleavage by ADAM17.

Cell surface receptors are regulated via a variety of mechanisms including proteolytic inactivation by metalloproteases. Cells often co-express both the receptor and its cognate protease simultaneously on the cell surface, often with the proteases in an active state. Despite this, receptor cleavage is generally not constitutive, and rather must be induced. How a receptor and its inactivating protease can both be present in their active form on the cell surface with no cleavage of the target receptor is unclear. Using the efferocytic receptor MERTK and its inactivating protease ADAM17 as a model, along with super-resolution microscopy approaches, we demonstrate that MERTK and ADAM17 are localized to distinct sub-regions of the plasma membrane. These microdomains are distinct subcellular structures, with MERTK contained within actin-based corrals and ADAM17 contained within caveolae. Induction of MERTK cleavage by PMA results in the reorganization of the membrane-proximal actin cytoskeleton that enables MERTK to diffuse into ADAM17-containing microdomains. Only after localizing to ADAM17-containing microdomains is MERTK cleaved. These results demonstrate that the spatial organization of the plasma membrane is dynamically regulated, with reorganization of membrane microdomains playing a role in the regulation of ADAM17 activity.

cell biology↗

bioRxiv2026

Muscle-specific motor unit firing characteristics in elbow flexors and extensors after cervical spinal cord injury

Individuals with cervical spinal cord injury (SCI) often exhibit asymmetric recovery of upper-limb function, with greater weakness in elbow extensors than flexors. To determine whether muscle-specific changes in motor unit (MU) behavior contribute to this disparity, we identified MU firing instants from high-density surface electromyography to characterize MU firing characteristics in the biceps brachii (BIC) and triceps brachii (TRI) of individuals with cervical SCI (n = 20) and non-injured controls (n = 18). We quantified rate-coding behavior and metrics related to persistent inward currents (PICs), including onset-offset hysteresis ({Delta}F), ascending firing rate nonlinearity, and self-sustained firing. At the group level, BIC MUs in SCI participants showed reduced rate coding and altered ascending firing rate nonlinearity relative to controls. In contrast, TRI MUs showed no clear group-level differences. However, subgroup analysis revealed that SCI participants with low-strength during extension (n = 9) exhibited lower {Delta}F and longer self-sustained firing durations in TRI MUs than those with high-strength (n = 6). In BIC, SCI participants with low-strength during flexion (n = 8) showed reduced rate-coding behavior relative to high-strength SCI participants (n = 9), with no differences in PIC-related metrics. Together, these results demonstrate muscle-specific alterations in MU firing after cervical SCI that may relate to strength recovery or preservation and underscore the need for nuanced analyses in heterogeneous SCI populations. Key pointsO_LIRate coding and nonlinear firing behaviors are significantly altered in the biceps brachii, but not triceps brachii, of participants with cervical spinal cord injury. C_LIO_LIStrength based subgroup analyses revealed muscle-specific differences in motor unit behaviors that may be associated with strength preservation or recovery following spinal cord injury. C_LIO_LIFunctional heterogeneity following spinal cord injury may mask group differences in motor unit behaviors and warrants careful interpretation of results of future studies. C_LI

neuroscience↗

bioRxiv2025

GATA2 Induces a Stem Cell-Like Transcriptional Program in Macrophages that Promotes Atherogenesis.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-laden necrotic macrophages within blood vessels walls. GATA2 is a normally hematopoietic transcription factor which in the bone marrow helps maintain the proliferative, non-differentiated phenotype of hematopoietic progenitors. Unexpectedly, GATA2 is upregulated in macrophages within atherosclerotic plaque, where it plays an unknown role in disease progression. Although GATA2 can be expressed from two promoters, we determined that the atherogenic stimuli oxidized low-density lipoprotein and TNF induce GATA2 expression via the internal (IG) GATA2 promoter, with GATA2 transcription initiated by the transcription factors NF-{kappa}B, STAT1, and the aryl hydrocarbon receptor. GATA2 had a divergent effect on promoter activity, with GATA2 upregulating genes associated with stem cell maintenance, proliferation, reactive oxygen species production, and migration, while downregulating genes central to macrophage function including those for cholesterol efflux, pathogen phagocytosis, and for the efferocytosis of apoptotic cells. Consequentially, GATA2-expressing macrophages had a pro-atherogenic phenotype typified by an invasive phenotype, poor cholesterol efflux, and impaired phagocytosis and efferocytosis. These results indicate that GATA2 upregulation induces an immature, stem cell-like phenotype in atheroma macrophages, thereby promoting plaque cellularity while compromising atheroprotective mechanisms such as cholesterol clearance and apoptotic cell removal.

immunology↗

bioRxiv2025

GATA2 Mediates Macrophage Proliferation During Atherosclerosis

Atherosclerosis is fueled by the buildup of lipid-laden macrophages within the vascular intima. These macrophages are derived from monocytes that are recruited from the circulation into the developing lesion, where they proliferate and differentiate into macrophages, with proliferation producing most of the macrophages in the resulting lesions. However, the signals and transcriptional events driving the proliferation of atherosclerotic plaque macrophages remain poorly understood. Analysis of human plaque spanning a range of disease severity identified a subpopulation of macrophages that expressed the hematopoietic transcription factor GATA2. These GATA2-expressing macrophages had a transcriptional profile that was intermediary between monocytes and mature macrophages, and selectively upregulated genes associated with proliferation and apoptosis. The expression of GATA2 was concomitant with plaque macrophage proliferation at all stages of disease, with over 90% of proliferating macrophages expressing GATA2. GATA2 was upregulated in macrophages following exposure to oxLDL, with GATA2 expression being necessary and sufficient for the proliferation of these macrophages. In these cells, GATA2 mediates proliferation by upregulating expression of the proto-oncogene MYB, while simultaneously decreasing sensitivity to apoptosis induced by the unfolded protein response. Together, these data identify GATA2 as a transcription factor upregulated by atherogenic stimuli that functions as the primary mediator of macrophage proliferation in atherosclerotic plaque.

immunology↗

bioRxiv2025

B cell subsets have different capacities for phagocytosis and subsequent presentation of antigen to cognate T cells

B cells have been shown to be phagocytic under some circumstances. However, the phagocytic capacity of different B cell subsets and how this is linked to Antigen (Ag) presentation or other functions has not been characterized. To address this, we developed 2 {micro}m phagocytic Ag conjugated bead targets that target phagocytic pathways including the BCR, scavenger, Fc, and complement receptors to study potential pathways by which B cells phagocytose both cognate and non-cognate Ags. We found that while follicular B2 (Fo B), marginal zone, and B1 B cells are highly phagocytic of BCR-engaging targets through their BCR, only peritoneal cavity B1 cells could uptake non-cognate Ag-coated beads or bacteria. Despite this, B1 cells were not effective at presenting Ag to activate cognate T cells or at killing phagocytosed bacteria. Finally, analysis of scRNA-seq data revealed that these differences in phagocytic capacity could not be explained by differential expression of relevant phagocytic receptors, implying that there is likely some form of regulation in place preventing non-cognate Ag uptake by Fo B cells. Our work will help contribute to a better understanding of non-classical Ag uptake mechanisms employed by B cells and their relevance to inflammation.

immunology↗

bioRxiv2023

MERTK Coordinates Efferocytosis by Regulating Integrin Localization and Activation

Efferocytosis - the phagocytic removal of apoptotic cells - is a central component of tissue homeostasis, and in many tissues is mediated by the efferocytic receptor MERTK expressed by macrophages. Although MERTK is critical for efferocytosis, the mechanism by which it directs the engulfment of apoptotic cells is largely unknown. Using immunoprecipitation, mass spectrometry, and super-resolution microscopy, we have identified a pre-formed receptor complex on the macrophage plasma membrane comprised of [~]180 nm clusters of MERTK, {beta}2 integrins, and multiple signaling molecules including Src-family kinases, PI3-kinases, and the integrin regulatory proteins ILK and FAK. MERTK is unable to mediate efferocytosis in the absence of {beta}2 integrins or their opsonins, while {beta}2 integrins require activation via MERTK signaling to induce the engulfment of apoptotic cells. Using FRET microscopy, we determined that MERTK directly induces the conformational change of {beta}2 integrins from the low to high-affinity form via a PI3-kinase-dependent signaling pathway. MERTK and {beta}2 integrins then form a highly structured synapse in which MERTK is retained by ligand-induced clustering in the synapse centre, while {beta}2 integrins and actin form a Src family kinase-, ILK- and FAK-dependent expanding ring which defines the leading edge of the synapse that ultimately engulfs the apoptotic cell. The identification of the MERTK membrane-proximal signaling pathway and the role of {beta}2 integrins in this pathway provides new insights into the function of this critical homeostatic receptor and provides new insights into how MERTK mutations and signaling defects may contribute to inflammatory and autoimmune diseases.

immunology↗

bioRxiv2023

SARS-CoV-2 NSP5 Antagonizes the MHC II Antigen Presentation Pathway by Hijacking Histone Deacetylase 2

SARS-CoV-2 interferes with antigen presentation by downregulating MHC II on antigen presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. This downregulation of CIITA is independent of NSP5s proteolytic activity, but rather, NSP5 delivers HDAC2 to IRF3 at an IRF binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response. ImportanceSARS-CoV-2 alters the expression of many immunoregulatory proteins to limit and delay the host antiviral response, thereby producing a more severe and longer-lasting infection. Preventing and limiting the activation of helper T cells by reducing MHC II expression on antigen presenting cells is one of these strategies, but while this mechanism was identified early in the pandemic, the mechanism allowing SARS-CoV-2 to limit MHC II expression has remained unclear. Herein, we demonstrate that this occurs via a tripartite interaction between viral NSP5 and host HDAC2 and IRF3, where a complex of NSP5 and HDAC2 is recruited to IRF3 bound to the promoter of CIITA--the master regulator of MHC II expression--with the delivery of HDAC2 then mediating the deacetylation of the CIITA promoter and the suppression of MHC II expression.

immunology↗

bioRxiv2020

Customizable Live-Cell Imaging Chambers for Fluorescence and Super-Resolution Microscopy.

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires provision of appropriate experimental conditions in a format that meets the microscopys optical requirements. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized live-cell imaging chambers that are compatible with a range of imaging modalities including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and which supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting PDMS microfluidic devices. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with modern microscopy techniques.

cell biology↗

bioRxiv2019

GATA2 Expression by Intima-Infiltrating Macrophages Drives Early Atheroma Formation

Aberrant macrophage polarization is a major contributor to the onset and progression of atherosclerosis. Despite this, macrophage polarization during in early stages of human atherosclerotic disease is poorly understood. Using transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions, we have identified a unique gene expression profile dissimilar to that observed in later stages of disease that is characterized by upregulation of the hematopoietic transcription factor GATA2. GATA2 overexpression in vitro recapitulated defects observed in patient macrophages, including deficiencies in the uptake and processing of apoptotic cells, and in the catalysis of atherogenic protein modifications, with GATA2 knockdown abrogating these defects. Our data describe a previously unreported macrophage differentiation state present in early atheroma formation and identifies GATA2 as a driver of macrophage functional defects during the early stages of atherosclerosis in humans.

immunology↗

bioRxiv2018

PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway

The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein - 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

cell biology↗

bioRxiv2018

Soluble CD93 is an Apoptotic Cell Opsonin Recognized by the αxβ2 Integrin

AO_SCPLOWBSTRACTC_SCPLOWEfferocytosis - the phagocytic removal of apoptotic cells - is essential for the maintenance of homeostasis and prevention of the inflammatory and autoimmune diseases which can follow the lysis of uncleared apoptotic cells. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis and angiogenesis, and upon mutation, results in the onset of efferocytosis-associated diseases such as atherosclerosis and rheumatoid arthritis. CD93 is produced as a cell surface protein which is shed as soluble CD93, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane-bound form. Herein, we demonstrate that the membrane bound form of CD93 has no phagocytic, efferocytic, or tethering activity, whereas soluble CD93 potently opsonizes apoptotic cells but not a broad range of Gram-Negative, Gram-Positive or fungal microorganisms. Using mass spectrometry, we identified the x{beta}2 integrin as the receptor required for soluble CD93-mediated efferocytosis, and via deletion mutagenesis determined that soluble CD93 binds to apoptotic cells via its C-Type Lectin-Like domain, and to x{beta}2 by its EGF-like repeats. This bridging of apoptotic cells to the x{beta}2 integrin markedly enhanced efferocytosis by macrophages, and could be abrogated by knockdown of x{beta}2 integrin. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identify a previously unreported opsonin-receptor system utilized by the immune system for the efferocytic clearance of apoptotic cells.

immunology↗

bioRxiv2017

Membrane Diffusion Occurs by a Continuous-Time Random Walk Sustained by Vesicular Trafficking.

Diffusion in cellular membranes is regulated by processes which occur over a range of spatial and temporal scales. These processes include membrane fluidity, inter-protein and inter-lipid interactions, interactions with membrane microdomains, interactions with the underlying cytoskeleton, and cellular processes which result in net membrane movement. The complex, non-Brownian diffusion that results from these processes has been difficult to characterize, and moreover, the impact of factors such as membrane recycling on membrane diffusion remains largely unexplored. We have used a careful statistical analysis of single-particle tracking data of the single-pass plasma membrane protein CD93 to show that the diffusion of this protein is well-described by a continuous-time random walk in parallel with an aging process mediated by membrane corrals. The overall result is an evolution in the diffusion of CD93: proteins initially diffuse freely on the cell surface, but over time, become increasingly trapped within diffusion-limiting membrane corrals. Stable populations of freely diffusing and corralled CD93 are maintained by an endocytic/exocytic process in which corralled CD93 is selectively endocytosed, while freely diffusing CD93 is replenished by exocytosis of newly synthesized and recycled CD93. This trafficking not only maintained CD93 diffusivity, but also maintained the heterogeneous distribution of CD93 in the plasma membrane. These results provide insight into the nature of the biological and biophysical processes that can lead to significantly non-Brownian diffusion of membrane proteins, and demonstrate that ongoing membrane recycling is critical to maintaining steady-state diffusion and distribution of proteins in the plasma membrane.

biophysics↗