Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix. (A) The native and synthetic mucin biopolymers that were genetically encoded and used throughout this work. (B) Quantification of membrane tube density in epithelial cells. Mucin polymers induce dramatic tubularization compared to wild-type (Control) cells and compared to a similarly sized biopolymer composed of EGF-like repeats from Notch1 and the Muc1 transmembrane anchor with GFP reporter (EGF-repeats GFP-DCT) cells. Number of cells analyzed is shown on the x axis for each condition. Box notches here and elsewhere indicate 95% confidence intervals. The number of tandem repeats (TRs) are indicated in Muc1 constructs. (C) Scanning electron microscopy (SEM) images of cells expressing the indicated biopolymer. (D) Labelled glycans and membrane morphologies resolved with single-molecule localization microscopy in Muc1-42TR DCT-expressing cells before and after mucin backbone digestion with the StcE mucinase. Images are shown as 2D color-coded histograms of localizations with 32 nm bin width. (E) Representative confocal images of GUVs with and without anchorage of recombinant Podocalyxin. (F) (Left) Cartoons of Muc1 GFP-DCT polymers of varying length. (Right) Flow cytometry data showing similar cell-surface expression levels of the mucins using a GFP-binding nanobody, n = 3, >40,000 cells per population. (G) Representative SEM images of cells expressing mucins with a varying number of TRs. (H) Quantification of membrane tube density for cells expressing the indicated mucins, significance compared to 42TR. ***p < 0.001; ns, not significant (post-hoc Student's two-tailed t test). See also Figure S1.
The tumor microbiome is increasingly implicated in cancer progression and resistance to chemotherapy. In pancreatic ductal adenocarcinoma (PDAC), high intratumoral loads of Fusobacterium nucleatum correlate with shorter survival in patients. Here, we investigated the potential mechanisms underlying this association. We found that F. nucleatum infection induced both normal pancreatic epithelial cells and PDAC cells to secrete increased amounts of the cytokines GM-CSF, CXCL1, IL-8, and MIP-3α. These cytokines increased proliferation, migration, and invasive cell motility in both infected and noninfected PDAC cells but not in noncancerous pancreatic epithelial cells, suggesting autocrine and paracrine signaling to PDAC cells. This phenomenon occurred in response to Fusobacterium infection regardless of the strain and in the absence of immune and other stromal cells. Blocking GM-CSF signaling markedly limited proliferative gains after infection. Thus, F. nucleatum infection in the pancreas elicits cytokine secretion from both normal and cancerous cells that promotes phenotypes in PDAC cells associated with tumor progression. The findings support the importance of exploring host-microbe interactions in pancreatic cancer to guide future therapeutic interventions.
HIV positive patients on highly active antiretroviral therapy (HAART) have shown elevated incidence of a number of non-AIDS defining co-morbidities, including cardiovascular disease. Given that HAART regimens contain a combination of at least three drugs, that disease management often requires adjustment of these regimens, and HIV, independent of HAART, also plays a role in development of co-morbidities, determining the role of specific HAART drugs and HIV infection itself from clinical data remains challenging. To characterize specific mediators and underlying mechanisms of disease, in vitro and in vivo animal models are required, in parallel with clinical data. Given its low cost azidothymidine (AZT) contributes to the backbone of a large proportion of HAART treated patients in the developing world where much of the global burden of HIV resides. The goal of this study was to test the hypothesis that AZT can lead to proatherogenic changes including the subclinical markers of arterial stiffening and intima-media thickening in mice. AZT (100 mg/kg) or vehicle was administered to wild-type FVB/N mice via oral gavage for 35 days. Cylindrical biaxial biomechanical tests on the common carotid arteries and suprarenal aortas exhibited arterial stiffening in AZT mice compared to controls. Multiphoton microscopy and histology demonstrated that AZT led to increased intima-media thickness. These data correlated with decreased elastin content and increased protease activity as measured by cathepsin zymography; no differences were observed in collagen content or organization, in vivo axial stretch, or opening angle. Thus, this study suggests the drug AZT has significant effects on the development of subclinical markers of atherosclerosis.
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