Biology subjects

Schmidtke, D. W.

Publications and source records attributed to Schmidtke, D. W..

4 recordsLinked to original sources

bioRxiv2024

Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions is unclear. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two nucleation promoting factors (NPFs) for the Arp2/3 complex, WAVE and N-WASP, exhibit different localization patterns in the protrusions. While WAVE is closely associated with the plasma membrane at the leading edge of the protrusive structures, N-WASP is enriched with WIP along the actin bundles in the shafts of the protrusions. During protrusion initiation and growth, the Arp2/3 complex nucleates branched actin filaments to generate low density actin clouds, in which the large GTPase dynamin organizes the new branched actin filaments into bundles, followed by actin-bundle stabilization by WIP, the latter functioning as a novel actin-bundling protein. Disrupting any of these components results in defective protrusions and failed myoblast fusion in cultured cells and mouse embryos. Taken together, our study has revealed the intricate spatiotemporal coordination between two NPFs and two actin-bundling proteins in building invasive protrusions at the mammalian fusogenic synapse, and has general implications in understanding invasive protrusion formation in cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2024

Treatment with both TGF-β1 and PDGF-BB disrupts the stiffness-dependent myofibroblast differentiation of corneal keratocytes

During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-{beta}1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-{beta}1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-{beta}1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-{beta}1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-{beta}1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-{beta}1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of -SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of -SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of -SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-{beta}1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes. Statement of SignificanceIn vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-{beta}1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated -SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-{beta}1 and PDGF-BB.

bioengineering↗

bioRxiv2024

Investigating transcriptional differences in mechanotransductive and ECM related genes in cultured primary corneal keratocytes, fibroblasts and myofibroblasts

PurposeAfter stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. MethodsPrimary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-{beta}1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. ResultsGenes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-{beta}1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. ConclusionsOverall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

bioengineering↗

bioRxiv2023

Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions remains a longstanding question in cell biology. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two Arp2/3 nucleation promoting factors (NPFs), WAVE and N-WASP, exhibit distinct and complementary localization patterns in the protrusions. While WAVE is at the leading edge, N-WASP is recruited by its interacting protein, WIP, to the shaft of the protrusion. During protrusion growth, new branched actin filaments are polymerized at the periphery of the shaft and crosslinked to preexisting actin bundles by the "pioneer" actin-bundling protein dynamin. The thickened actin bundles are further stabilized by WIP, which functions as a WH2 domain-mediated actin-bundling protein. Disrupting any of these components results in defective protrusions and myoblast fusion in cultured cells and/or in mouse embryos. Thus, our study has revealed the intricate spatiotemporal coordination between two NPFs and two actin-bundling proteins in creating invasive protrusions and has general implications in understanding protrusion formation in many cellular processes beyond cell-cell fusion.

cell biology↗