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The ubiquitously expressed adapter proteins Nck1/2 interact with a multitude of effector molecules to regulate diverse cellular functions including cytoskeletal dynamics. Here we show that Nck1, but not Nck2, is a substrate of c-Cbl-mediated ubiquitination. We uncover lysine 178 in Nck1 as the evolutionarily conserved ubiquitin acceptor site. We previously reported that synaptopodin, a proline-rich actin-binding protein, induces stress fibres by blocking the Smurf1-mediated ubiquitination of RhoA. We now find that synaptopodin competes with c-Cbl for binding to Nck1, which prevents the ubiquitination of Nck1 by c-Cbl. Gene silencing of c-Cbl restores Nck1 protein abundance and stress fibres in synaptopodin knockdown cells. Similarly, expression of c-Cbl-resistant Nck1(K178R) or Nck2 containing the SH3 domain 2 of Nck1 restores stress fibres in synaptopodin-depleted podocytes through activation of RhoA signalling. These findings reveal proteasomal regulation as a key factor in the distinct and non-redundant effects of Nck on RhoA-mediated actin dynamics.

Tropomyosins are widespread actin‐binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin‐binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1‐mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half‐life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin‐deficient human MDA‐MB 231 breast cancer cells and TPMα‐depleted fibroblasts. Gene silencing of TPMα but not TPMβ causes loss of stress fibres by promoting Smurf1‐mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA‐MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin‐binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type‐specific fashion.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

The Kidney Precision Medicine Project (KPMP) plans to construct a spatially specified tissue atlas of the human kidney at a cellular resolution with near comprehensive molecular details. The atlas will have maps of healthy, acute kidney injury and chronic kidney disease tissues. To construct such maps, we integrate different data sets that profile mRNAs, proteins and metabolites collected by five KPMP Tissue Interrogation Sites. Here, we describe a set of hierarchical analytical methods to process, combine, and harmonize single-cell, single-nucleus and subsegmental laser microdissection (LMD) transcriptomics with LMD and near single-cell proteomics, 3-D nondestructive and immunofluorescence-based Codex imaging and spatial metabolomics datasets. We use nephrectomy, healthy living donor and surveillance transplant biopsy tissues to create a harmonized reference tissue map. Our results demonstrate that different assays produce reliable and coherent identification of cell types and tissue subsegments. They further show that the molecular profiles and pathways are partially overlapping yet complementary for cell type-specific and subsegmental physiological processes. Focusing on the proximal tubules, we find that our integrated systems biologybased analyses identify different subtypes of tubular cells with potential for different levels of lipid oxidation and energy generation. Integration of our omics data with pathways from the literature, enables us to construct predictive computational models to develop a smart kidney atlas. These integrated models can describe physiological capabilities of the tissues based on the underlying cell types and pathways in health and disease.

Molecular assessments at the single cell level can accelerate biological research by providing detailed assessments of cellular organization and tissue heterogeneity in both disease and health. The human kidney has complex multi-cellular states with varying functionality, much of which can now be completely harnessed with recent technological advances in tissue proteomics at a near single-cell level. We discuss the foundational steps in the first application of this mass spectrometry (MS) based proteomics method for analysis of subsections of the normal human kidney, as part of the Kidney Precision Medicine Project (KPMP). Using ∼30-40 laser captured micro-dissected kidney cells, we identified more than 2,500 human proteins, with specificity to the proximal tubular (PT; n = 25 proteins) and glomerular (Glom; n = 67 proteins) regions of the kidney and their unique metabolic functions. This pilot study provides the roadmap for application of our near-single-cell proteomics workflow for analysis of other renal micro-compartments, on a larger scale, to unravel perturbations of renal sub-cellular function in the normal kidney as well as different etiologies of acute and chronic kidney disease.

Glucocorticoid induced leucine zipper protein (GILZ) is an aldosterone-regulated protein that controls sodium transport in cultured kidney epithelial cells. Mice lacking GILZ have been reported previously to have electrolyte abnormalities. However, the mechanistic basis has not been explored. Here we provide evidence supporting a role for GILZ in modulating the balance of renal sodium and potassium excretion by regulating the sodium-chloride cotransporter (NCC) activity in the distal nephron. GILZ−/− knockout mice have a higher plasma potassium concentration and lower fractional excretion of potassium than wild type mice. Furthermore, knockout mice are more sensitive to NCC inhibition by thiazides than are the wild type mice, and their phosphorylated NCC expression is higher. Despite increased NCC activity, knockout mice do not have higher blood pressure than wild type mice. However, during sodium deprivation, knockout mice come into sodium balance more quickly, than do the wild type, without a significant increase in plasma renin activity. Upon prolonged sodium restriction, knockout mice develop frank hyperkalemia. Finally, in HEK293T cells, exogenous GILZ inhibits NCC activity at least in part by inhibiting SPAK phosphorylation. Thus, GILZ promotes potassium secretion by inhibiting NCC and enhancing distal sodium delivery to the epithelial sodium channel. Additionally, Gilz knockout mice have features resembling familial hyperkalemic hypertension, a human disorder that manifests with hyperkalemia associated variably with hypertension.

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