Quantitatively modeling adsorbate diffusion through zeolitic imidazolate frameworks (ZIFs) must account for the inherent flexibility of these materials. The lack of a transferable intramolecular ZIF force field (FF) for use in classical simulations has previously made an accurate simulation of adsorbate diffusion in many ZIFs impossible. We resolve this problem by introducing a density functional theory parameterized force field (FF) for ZIFs named the intraZIF-FF, which includes perturbations to the class I force fields previously used to model ZIFs. This FF outperforms ad hoc force fields at predicting ab initio relative energies and atomic forces taken from fully periodic ab initio molecular dynamics simulations of SALEM-2, ZIF-7, ZIF-8, and ZIF-90. We use the intraZIF-FF to predict the infinite dilution self-diffusion coefficients of 30 adsorbates with molecular diameters ranging from 2.66 to 7.0 Å in these 4 ZIFs. These results greatly increase the number of adsorbates for which accurate information about molecular diffusion in ZIFs is available.

BackgroundMaintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood.MethodsCells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside–induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes.ResultsNebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation.ConclusionsLIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thus establish a significant function of Plexin-B2 in orchestrating cytoskeletal tension and cell-cell/cell-matrix adhesion, therefore solidifying the importance of collective cell mechanics in governing stem cell physiology and tissue morphogenesis.

Despite recent progress in the identification of mediators of podocyte injury, mechanisms underlying podocyte loss remain poorly understood, and cell-specific therapy is lacking. We previously reported that KIBRA, KIdney and BRAin expressed protein, encoded by WWC1, promotes podocyte injury in vitro through activation of the Hippo signaling pathway. KIBRA expression is increased in the glomeruli of patients with focal segmental glomerulosclerosis (FSGS), and KIBRA depletion in vivo is protective against acute podocyte injury. Here, we tested the consequences of transgenic podocyte-specific WWC1 expression in immortalized human podocytes and in mice, and we explored the association between glomerular WWC1 expression and glomerular disease progression. We found that KIBRA overexpression in immortalized human podocytes promoted cytoplasmic localization of YAP (Yes-associated protein), induced actin cytoskeletal reorganization, and altered focal adhesion expression and morphology.Transgenic WWC1 (KIBRA OE) mice were more susceptible to acute and chronic glomerular injury, with evidence of YAP inhibition in vivo. Of clinical relevance, glomerular WWC1 expression negatively correlated with renal survival among patients with primary glomerular diseases. These findings highlight the importance of KIBRA-YAP signaling to the regulation of podocyte structural integrity and identify KIBRA-mediated injury as a potential target for podocyte-specific therapy in glomerular disease.

Background and Aims Diabetic kidney disease (DKD) is a leading cause of end-stage kidney disease (ESKD), however therapies targeting causal pathways have been limited by disease heterogeneity. Integrating electronic health record (EHR) data and genomics may uncover hidden subphenotypes in DKD. In this study, we use deep learning to identify a novel genetic variant of ARHGEF18 associated with significantly higher risk of DKD and ESKD (Figure 1A). We further employed quantitative microscopy techniques and biochemical assays to elucidate the mechanistic role of ARHGEF18 and its variant in podocytes. Method DKD patients from the Mount Sinai BioMe Biobank were used in this study. Unsupervised clustering and accounting for population structure in a deep learning framework identified two clusters: Cluster M (mild) and S (severe). We then performed a genome wide association study (GWAS) of patients within each cluster compared with healthy controls. For mechanistic studies of the novel variant, cytoplasmic, focal adhesion, cytoskeletal morphometrics as well as live-cell motility, Rho GTPase activity, and protein degradation experiments were performed using confocal and total internal reflection fluorescence (TIRF) microscopy as well as cell-free biochemical assays using immortalized human podocytes expressing ARHGEF18 wild-type (WT) and mutant transcripts. Results We employed autoencoders and unsupervised clustering of EHR data on 1,372 DKD patients to establish two clusters with differential prevalence of ESKD. There was a greater prevalence of proteinuria in Cluster S compared to Cluster M (Figure 1B). Further exome sequencing study in these patients identified a novel variant in ARHGEF18, a Rho guanine exchange factor highly enriched in podocytes (Figure 1A). Nephroseq database showed an increased ARHGEF18 expression in chronic kidney disease (CKD) kidney biopsy samples compared to healthy controls (Figure 1C). Overexpression of ARHGEF18 mutant transcripts in human immortalized podocytes led to impairments in cell adhesion, focal adhesion architecture, and cell motility. Live TIRF microscopy experiments showed preferential subcellular localization of GEF18 mutant to the periphery of migrating podocytes whereas GEF18 WT localized at the perinuclear/cytoplasmic region (Figure 2A, B). GEF18 mutant cells also displayed an increased RhoA activation (Figure 2C). Upon inhibition of protein synthesis using cycloheximide (CHX), we observed a significantly slower degradation of GEF18 mutant protein over a 12h period indicating increased protein stability (Figure 2D). GEF18 mutant also showed resistance to ubiquitin mediated degradation leading to pathologically increased protein levels. Conclusions We report a novel gain of function variant of ARHGEF18 that drives podocyte dysfunction through impaired protein localization and degradation. Targeting this pathway could help regulate RhoA activation and cytoskeletal rearrangements preventing podocyte effacement in DKD.