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Biology subjects

Combs, C. A.

Publications and source records attributed to Combs, C. A..

10 recordsLinked to original sources

Remodeling of cholesterol metabolism in melanoma progression regulates nuclear membrane mechanics.

Metastatic cancer cells migrate through physically restrictive environments where nuclear deformation ruptures the nuclear envelope (NE) resulting in genomic instability. Although structural proteins of the NE are established regulators of nuclear mechanics, how nuclear membrane (NM) lipid composition contributes to its biophysical response to mechanical stress remains poorly understood. Here, we show that melanoma progression is associated with broad transcriptional remodeling of cholesterol homeostasis that is established early in disease progression, with altered expression of cholesterol biosynthesis genes associated with reduced patient survival. Building on our previous identification of the inner NM sterol reductase lamin B receptor (LBR) as a regulator of NE fragility, we find that LBR-dependent cholesterol biosynthesis promotes cholesterol enrichment within the NM and alters its spatial organization during cellular confinement. Quantitative fluorescence and lifetime imaging of biosensors of cholesterol distribution, lipid order, and tension reveals that cholesterol remodeling alters NM lipid organization and results in high NM tension in melanoma cells that is selectively reduced by cholesterol depletion. Cellular confinement generates nuclear blebs where cholesterol becomes preferentially enriched at highly curved, rupture-prone regions of the NM. Together, our results establish cholesterol-dependent NM remodeling as a regulator of nuclear mechanics and link metabolic changes acquired during cancer progression to the biophysical response of the NM to mechanical stress.

cell biology↗

DVT-primed neutrophils reshape the brain microenvironment to promote brain metastasis

Brain metastasis (BrM) is a devastating complication of triple-negative breast cancer (TNBC), yet how host macroenvironmental conditions influence brain metastatic susceptibility remains poorly understood. Here, we investigated the impact of deep vein thrombosis (DVT), a common complication in patients with cancer, on TNBC brain metastasis. Analysis of a large clinical cohort identified DVT as an independent risk factor for BrM in patients with metastatic breast cancer. Using two syngeneic TNBC models, we demonstrated that DVT selectively enhanced brain metastasis without significantly affecting primary tumor growth or lung metastasis. Mechanistically, DVT increased neutrophil accumulation in the brain metastatic microenvironment, and neutrophil depletion completely abrogated the metastasis-promoting effect of DVT. Single-cell RNA sequencing of CD45+ cells in peripheral blood revealed that DVT reprogrammed circulating neutrophils toward migratory and inflammatory transcriptomic profiles characterized by neutrophil extracellular trap (NET) formation. DVT increased CXCR2 expression on circulating neutrophils and enhanced neutrophil recruitment in brain. Moreover, DVT significantly increased circulating NETs. Collectively, our findings identify DVT as a systemic driver of TNBC brain metastasis and reveal that DVT primes circulating neutrophils toward enhanced vascular recruitment and NET formation, providing a mechanistic link between cancer-associated thrombosis and brain metastatic susceptibility.

molecular biology↗

APOE4 and INPP5D converge on membrane mechanics to regulate endocytosis in human astrocytes

Disrupted endocytosis is an early feature of Alzheimers disease (AD), but how genetic risk factors functionally impact this pathway remains unclear. Using isogenic human iPSC-derived astrocytes, we show that the AD risk variant, APOE4, impairs clathrin-mediated membrane curvature, clathrin-mediated endocytosis, and alters plasma membrane lipid saturation and tension. Compared to APOE3 astrocytes, APOE4 cells display an accumulation of flat clathrin structures, reduced maturation of clathrin-coated pits, decreased early endosomes, and reduced endocytic uptake. We then identify the AD risk gene INPP5D as a modifier that restores early endocytosis in APOE4 astrocytes by promoting clathrin curvature and maturation through a mechanism distinct from membrane tension regulation. Beyond effects on trafficking, INPP5D overexpression also reduces lipid droplet accumulation and attenuates inflammatory signaling, linking membrane dynamics to disease-associated astrocytic phenotypes. Together, these findings establish altered membrane mechanics as a proximal consequence of the APOE4 variant and identify endocytosis as a common node linking AD risk genes.

cell biology↗

BLOC1S1 regulates autolysosomal and exosomal dynamics during CD4⁺ T cell differentiation

Although the endolysosome system is central to intracellular recycling, signal transduction, and intercellular communication via exocytosis, its role in immunoregulation remains incompletely defined. We recently identified that CD4+ T cell-specific depletion of BLOC1S1, a component of multiprotein complexes regulating endolysosomal biology, predisposes toward type 2 (Th2) immunity. We therefore hypothesized that the study of BLOC1S1-deficient CD4+ T cells would expand our understanding of endolysosomal dynamics in Th2 function. Here, we demonstrate that CD4+ T cell BLOC1S1 deficiency resulted in aberrant lysosomal distribution, accumulation of endosomal vesicles, and increased exocytosis, which collectively correlated with enhanced Th2 immune responses. The phenotype was associated with upregulation of key components of the exocytosis machinery, including RAB11 and VAMP7. Functional inhibition of these vesicle trafficking proteins following siRNA knockdown of RAB11 and VAMP7 significantly attenuated Th2 cytokine secretion in BLOC1S1-deficient CD4+ T cells, highlighting their essential role in exosome-mediated cytokine export. Furthermore, exosomes derived from BLOC1S1-deficient CD4+ T cells promoted Th2 polarization in recipient cells, indicating a mechanism of intracellular amplification. Together, these findings identify BLOC1S1 as a critical regulator of lysosomal dynamics and exocytic vesicle fusion, thereby linking intracellular trafficking mechanisms to Th2 immune regulation.

immunology↗

OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

Peripheral pain sensation is regulated by interactions between sensory nerves and various tissue cells. In obese patients with painful small fiber neuropathy, skin sensory nerves are often hypersensitive. While obesity is known to cause circulation-related vascular abnormalities, how these changes affect sensory dysfunction is not fully understood. In this study, we found that in a diet-induced obesity mouse model, skin capillaries become fenestrated, allowing insulin to diffuse into the avascular epidermis. This exposure triggers the production and secretion of nerve growth factor (NGF) from epidermal keratinocytes via insulin signaling with the forkhead box O1 (FOXO1) transcription factor. Elevated NGF leads to heightened sensory hypersensitivity by enhancing transient receptor potential vanilloid subtype 1 (TRPV1) in sensory nerves. Controlling capillary permeability reduces abnormal NGF expression and attenuates pain hypersensitivity. These findings nominate peripheral nerve-associated capillary permeability as a novel therapeutic target in obesity-associated sensory dysfunction.

neuroscience↗

β-III tubulin identifies anti-fibrotic state of pericytes in pulmonary fibrosis

Pericytes have been implicated in pulmonary fibrosis, yet their activated cellular states and functional roles remain largely unclear. Here, we identified {beta}-III tubulin (Tuj1) as a distinctive marker for fibrosis-associated lung pericytes. Most pericytes in fibrotic regions are Tuj1-positive and interact uniquely with multiple endothelial cells, localizing near collagen-producing fibroblasts and pro-fibrotic SPP1+/arginase+ macrophages. Tuj1 expression is predominantly induced in pericytes during the fibrotic phase, and Tuj1 gene (Tubb3) knockout in mice exacerbates lung fibrosis, accompanied by an increase in the neighboring pro-fibrotic fibroblasts and macrophages, suggesting an anti-fibrotic role for Tuj1-expressing pericytes. Mechanistically, the anti-fibrotic chemokine CXCL10 is upregulated in Tuj1-expressing pericytes, whereas this upregulation is not observed in Tubb3 knockout. Moreover, CXCL10 inhibits the pro-fibrotic differentiation of macrophages induced by lung fibroblasts in culture, implying that CXCL10 may mediate the anti-fibrotic effects of Tuj1-expressing pericytes. These findings underscore the role of lung pericytes in negatively regulating fibrotic process and their potential as therapeutic targets for pulmonary fibrosis patients. SummaryIn response to fibrotic stimuli, lung pericytes upregulate {beta}-III tubulin (Tuj1) expression, adopting an anti-fibrotic phenotype. This phenotype acts as a negative immune regulator of pro-fibrotic macrophage differentiation by releasing CXCL10, thereby counteracting the progression of pulmonary fibrosis.

pathology↗

BLOC1S1 control of vacuolar organelle fidelity modulates TH2 cell immunity and allergy susceptibility.

The levels of biogenesis of lysosome organelles complex 1 subunit 1 (BLOC1S1) control mitochondrial and endolysosome organelle homeostasis and function. Reduced fidelity of these vacuolar organelles is increasingly being recognized as important in instigating cell-autonomous immune cell activation. We reasoned that exploring the role of BLOC1S1 in CD4+ T cells, may further advance our understanding of regulatory events linked to mitochondrial and/or endolysosomal function in adaptive immunity. Transcript levels of the canonical transcription factors driving CD4+T cell polarization in response to activation showed that, the TH2 regulator GATA3 and phosphorylated STAT6 were preferentially induced in BLOC1S1 depleted primary CD4+ T (TKO) cells. In parallel, in response to both T cell receptor activation and in response to TH2 polarization the levels of IL-4, IL-5 and IL-13 were markedly induced in the absence of BLOC1S1. At the organelle level, mitochondrial DNA leakage evoked cGAS-STING and NF-kB pathway activation with subsequent TH2 polarization. The induction of autophagy with rapamycin reduced cytosolic mtDNA and reverses these TH2 signatures. Furthermore, genetic knockdown of STING and STING and NF-{kappa}B inhibition ameliorated this immune regulatory cascade in TKO cells. Finally, at a functional level, TKO mice displayed increased susceptible to allergic conditions including atopic dermatitis and allergic asthma. In conclusion, BLOC1S1 depletion mediated disruption of mitochondrial integrity to initiate a predominant TH2 responsive phenotype via STING-NF-{kappa}B driven signaling of the canonical TH2 regulatory program.

immunology↗

Impact of Capillary and Sarcolemmal Proximity on Mitochondrial Structure and Energetic Function in Skeletal Muscle

Mitochondria within skeletal muscle cells are considered to be located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or in large pools beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies suggest that proximity to capillaries embedded in sarcolemmal grooves, rather than proximity to the sarcolemma itself, may drive the accumulation of mitochondria near the cell periphery (paravascular mitochondria, PVM). To evaluate the impact of capillary versus sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either in large pools lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM), or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation while mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, while PVM and SSM were similarly larger than IFM, PVM were more compact and had greater mitochondrial connectivity compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate proximity to capillaries has a greater impact on mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone.

physiology↗

Transgelin: A New Gene Involved in LDL Endocytosis Identified by a Genome-wide CRISPR-Cas9 Screen

To identify new genes involved in the cellular uptake of low-density lipoprotein (LDL), we applied a novel whole genome CRISPR/Cas9 knockout-screen on HepG2 cell lines. We identified TAGLN (transgelin), an actin-binding protein, as a new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were associated with plasma lipids (triglycerides, total cholesterol, HDL, and LDL cholesterol) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. Decreased cellular LDL uptake observed in TAGLN-knockout cells due to decreased LDL receptor internalization, led to alterations in cellular cholesterol content and compensatory changes in cholesterol biosynthesis. Transgelin was also shown to be involved in the actin-dependent phase of clathrin-mediated endocytosis of other cargo besides LDL. The identification of novel genes involved in LDL uptake may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.

cell biology↗

Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling [~]2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of [~]1.4-fold laterally and [~]3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

bioengineering↗