An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis

During mammalian embryogenesis, extensive cellular remodeling is needed for tissue morphogenesis. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely involved, but their daunting complexity has hindered progress in dissecting their functions. We overcome this hurdle by employing high throughput in utero RNAi-mediated screening to identify key Rho regulators of skin morphogenesis. Our screen unveiled hitherto unrecognized roles for Rho-mediated cytoskeletal remodeling events that impact hair follicle specification, differentiation, downgrowth and planar cell polarity. Coupling our top hit with gain/loss-of-function genetics, interactome proteomics and tissue imaging, we show that RHOU, an atypical Rho, governs the cytoskeletal-junction dynamics that establish columnar shape and planar cell polarity in epidermal progenitors. Conversely, RHOU downregulation is required to remodel to a conical cellular shape that enables hair bud invagination and downgrowth. Our findings underscore the power of coupling screens with proteomics to unravel the physiological significance of complex gene families.

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Regulation of Rho GTPases in the Vasculature by Cullin3-Based E3 Ligase Complexes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.680901 ◽

2021 ◽

Vol 9 ◽

Author(s):

Fabienne Podieh ◽

Peter L. Hordijk

Keyword(s):

Rho Gtpases ◽

Vascular Function ◽

Rho Gtpase ◽

Current Knowledge ◽

E3 Ligase ◽

Small Gtpases ◽

E3 Ligases ◽

Cellular Processes ◽

Ubiquitin E3 Ligase ◽

Cytoskeletal Dynamics

Cullin3-based ubiquitin E3 ligases induce ubiquitination of substrates leading to their proteasomal or lysosomal degradation. BTB proteins serve as adaptors by binding to Cullin3 and recruiting substrate proteins, which enables specific recognition of a broad spectrum of targets. Hence, Cullin3 and its adaptors are involved in myriad cellular processes and organ functions. Cullin3-based ubiquitin E3 ligase complexes target small GTPases of the Rho subfamily, which are key regulators of cytoskeletal dynamics and cell adhesion. In this mini review, we discuss recent insights in Cullin3-mediated regulation of Rho GTPases and their impact on cellular function and disease. Intriguingly, upstream regulators of Rho GTPases are targeted by Cullin3 complexes as well. Thus, Rho GTPase signaling is regulated by Cullin3 on multiple levels. In addition, we address current knowledge of Cullin3 in regulating vascular function, focusing on its prominent role in endothelial barrier function, angiogenesis and the regulation of blood pressure.

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Dysregulation of Rho GTPases in Human Cancers

Cancers ◽

10.3390/cancers12051179 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1179 ◽

Cited By ~ 5

Author(s):

Haiyoung Jung ◽

Suk Ran Yoon ◽

Jeewon Lim ◽

Hee Jun Cho ◽

Hee Gu Lee

Keyword(s):

Cancer Progression ◽

Rho Gtpases ◽

Cell Cycle Progression ◽

Rho Gtpase ◽

Current Knowledge ◽

New Drugs ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Human Cancers ◽

Cytoskeletal Dynamics

Rho GTPases play central roles in numerous cellular processes, including cell motility, cell polarity, and cell cycle progression, by regulating actin cytoskeletal dynamics and cell adhesion. Dysregulation of Rho GTPase signaling is observed in a broad range of human cancers, and is associated with cancer development and malignant phenotypes, including metastasis and chemoresistance. Rho GTPase activity is precisely controlled by guanine nucleotide exchange factors, GTPase-activating proteins, and guanine nucleotide dissociation inhibitors. Recent evidence demonstrates that it is also regulated by post-translational modifications, such as phosphorylation, ubiquitination, and sumoylation. Here, we review the current knowledge on the role of Rho GTPases, and the precise mechanisms controlling their activity in the regulation of cancer progression. In addition, we discuss targeting strategies for the development of new drugs to improve cancer therapy.

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Loss of Rho-GDIα sensitizes podocytes to lipopolysaccharide-mediated injury

AJP Renal Physiology ◽

10.1152/ajprenal.00225.2014 ◽

2015 ◽

Vol 308 (11) ◽

pp. F1207-F1216 ◽

Cited By ~ 5

Author(s):

Richard Robins ◽

Cindy Baldwin ◽

Lamine Aoudjit ◽

Indra R. Gupta ◽

Tomoko Takano

Keyword(s):

Nephrotic Syndrome ◽

Rho Gtpases ◽

Rho Gtpase ◽

Cathepsin L ◽

Loss Of Function ◽

Filtration Barrier ◽

Guanine Nucleotide Dissociation Inhibitor ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Lps Treatment

Nephrotic syndrome is a disease of glomerular permselectivity that can arise as a consequence of heritable or acquired changes to the integrity of the glomerular filtration barrier. We recently reported two siblings with heritable nephrotic syndrome caused by a loss of function mutation in the gene ARHGDIA, which encodes for Rho guanine nucleotide dissociation inhibitor-α (GDIα). GDIs are known to negatively regulate Rho-GTPase signaling. We hypothesized that loss of GDIα sensitizes podocytes to external injury via hyperactivation of Rho-GTPases and p38 MAPK. We examined the response of cultured podocytes with and without knockdown of GDIα to LPS injury by assessing the levels of phospho-p38 as well as the degree of synaptopodin loss. GDIα knockdown podocytes showed more pronounced and sustained p38 phosphorylation in response to LPS compared with control podocytes, and this was blunted significantly by the Rac1 inhibitor. In LPS-treated control podocytes, synaptopodin degradation occurred, and this was dependent on p38, the proteasome, and cathepsin L. In GDIα knockdown podocytes, the same events were triggered, but the levels of synaptopodin after LPS treatment were significantly lower than in control podocytes. These experiments reveal a common pathway by which heritable and environmental risk factors converge to injure podocytes, from Rac1 hyperactivation to p38 phosphorylation and synaptopodin degradation via the ubiquitin-proteasome pathway and cathepsin L.

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Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix

Open Biology ◽

10.1098/rsob.190148 ◽

2019 ◽

Vol 9 (8) ◽

pp. 190148 ◽

Cited By ~ 2

Author(s):

Maria Thuveson ◽

Konstantin Gaengel ◽

Giovanna M. Collu ◽

Mei-ling Chin ◽

Jaskirat Singh ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Polarity ◽

Planar Cell Polarity ◽

Rho Kinase ◽

Cell Movement ◽

Interaction Network ◽

Loss Of Function ◽

Ommatidial Rotation ◽

Cytoskeletal Elements ◽

Function Activity

Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. Here, we demonstrate that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. We further demonstrate that integrin localization changes dynamically during the rotation process. Our data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90° rotation movement.

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The Exo70 Subunit of the Exocyst Is an Effector for Both Cdc42 and Rho3 Function in Polarized Exocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e09-06-0501 ◽

2010 ◽

Vol 21 (3) ◽

pp. 430-442 ◽

Cited By ~ 75

Author(s):

Hao Wu ◽

Courtney Turner ◽

Jimmy Gardner ◽

Brenda Temple ◽

Patrick Brennwald

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Biochemical Properties ◽

Loss Of Function ◽

Gain Of Function ◽

Present Evidence ◽

Spatial Regulation ◽

Exocyst Complex ◽

Polarized Exocytosis

The Rho3 and Cdc42 members of the Rho GTPase family are important regulators of exocytosis in yeast. However, the precise mechanism by which they regulate this process is controversial. Here, we present evidence that the Exo70 component of the exocyst complex is a direct effector of both Rho3 and Cdc42. We identify gain-of-function mutants in EXO70 that potently suppress mutants in RHO3 and CDC42 defective for exocytic function. We show that Exo70 has the biochemical properties expected of a direct effector for both Rho3 and Cdc42. Surprisingly, we find that C-terminal prenylation of these GTPases both promotes the interaction and influences the sites of binding within Exo70. Finally, we demonstrate that the phenotypes associated with novel loss-of-function mutants in EXO70, are entirely consistent with Exo70 as an effector for both Rho3 and Cdc42 function in secretion. These data suggest that interaction with the Exo70 component of the exocyst is a key event in spatial regulation of exocytosis by Rho GTPases.

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Turnover Regulation of the Rho GTPase Cdc42 by Heat Shock Protein Chaperones and the MAPK Pathway Scaffold Bem4

10.1101/2021.07.13.452164 ◽

2021 ◽

Author(s):

Paul J Cullen ◽

Beatriz Gonzalez

Keyword(s):

Heat Shock ◽

Cell Polarity ◽

Rho Gtpases ◽

Rho Gtpase ◽

Mapk Pathway ◽

Cell Types ◽

Cell Polarization ◽

Extrinsic Cues ◽

Dependent Cell ◽

Map Kinase Pathway

All cells maintain an axis of polarity that directs the orientation of growth. Cell polarity can be reorganized during development and in response to extrinsic cues to produce new cell types. Rho GTPases are central regulators of cell polarity and signal-dependent cell differentiation. We show here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned over by members of the Heat Shock family of Proteins (HSPs). The Hsp40p chaperone, Ydj1p, was required for turnover of Cdc42p by the NEDD4 E3 ubiquitin ligase, Rsp5p, in the proteosome. Cdc42p turnover was regulated by HSPs at high temperatures, and in aging cells where the protein formed aggregates, implicating HSPs in Rho GTPase quality control. We also show that Cdc42pQ61L, which mimics the active (GTP-bound) conformation of the protein, was turned over at elevated levels by Ydj1p and Rsp5p. A turnover-defective version of Cdc42pQ61L led to multibudding phenotypes, implicating Cdc42 turnover in singularity in cell polarization. Cdc42p turnover also impacted MAP kinase pathway specificity. A pathway-specific scaffold, Bem4p, stabilized Cdc42p levels, which biased Cdc42p function in one MAPK pathway over another. Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the regulation of cell polarity and signal-dependent morphogenesis.

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Wnt ligands are not required for planar cell polarity in the Drosophila wing or notum

10.1101/2020.06.05.137182 ◽

2020 ◽

Author(s):

Ben Ewen-Campen ◽

Typhaine Comyn ◽

Eric Vogt ◽

Norbert Perrimon

Keyword(s):

Cell Polarity ◽

Planar Cell Polarity ◽

Wnt Signaling Pathway ◽

Loss Of Function ◽

Knock Out ◽

The Core ◽

Drosophila Wing ◽

Wnt Ligands ◽

Pcp Signaling ◽

Dual Functions

AbstractThe frizzled (fz) and disheveled (dsh) genes are highly conserved members of the core planar cell polarity (PCP) pathway and of the Wnt signaling pathway. Given these dual functions, a number of studies have examined whether Wnt ligands may provide a global, tissue-scale orientation cue for PCP establishment during development, and these studies have reached differing conclusions. In this study, we re-examine this issue in the Drosophila melanogaster wing and notum using split-Gal4 co-expression analysis, systematic pairwise and triple somatic CRISPR-based knock-outs and double RNAi experiments. Pairwise loss-of-function experiments targeting wg together with other Wnt genes does not produce PCP defects, neither via somatic CRISPR nor RNAi. In addition, somatic knock-out of evi (aka wntless), which is required for the secretion of all Wnt ligands expressed in these tissues, did not produce detectable PCP phenotypes. Altogether, we were unable to find support for the hypothesis that Wnt ligands contribute to PCP signaling in the Drosophila wing or notum.

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Roles of Rho GTPases in leucocyte and leukaemia cell transendothelial migration

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0013 ◽

2013 ◽

Vol 368 (1629) ◽

pp. 20130013 ◽

Cited By ~ 14

Author(s):

Elvira Infante ◽

Anne J. Ridley

Keyword(s):

Cell Migration ◽

Blood Vessels ◽

Rho Gtpases ◽

Rho Gtpase ◽

Immune Surveillance ◽

Transendothelial Migration ◽

Cell Entry ◽

Leukaemia Cell ◽

Multiple Points ◽

Cytoskeletal Dynamics

Leucocytes migrate into and out of blood vessels at multiple points during their development and maturation, and during immune surveillance. In response to tissue damage and infection, they are rapidly recruited through the endothelium lining blood vessels into the tissues. Leukaemia cells also move in and out of the bloodstream during leukaemia progression. Rho GTPases are intracellular signalling proteins that regulate cytoskeletal dynamics and are key coordinators of cell migration. Here, we describe how different members of the Rho GTPase family act in leucocytes and leukaemia cells to regulate steps of transendothelial migration. We discuss how inhibitors of Rho signalling could be used to reduce leucocyte or leukaemia cell entry into tissues.

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Rho GTPase function in flies: insights from a developmental and organismal perspective

Biochemistry and Cell Biology ◽

10.1139/o04-118 ◽

2004 ◽

Vol 82 (6) ◽

pp. 643-657 ◽

Cited By ~ 11

Author(s):

James E Johndrow ◽

Craig R Magie ◽

Susan M Parkhurst

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Fruit Fly ◽

Genetic System ◽

Small Gtpases ◽

Dominant Negative ◽

Loss Of Function ◽

Cellular Behavior ◽

Essential Components ◽

Work Done

Morphogenesis is a key event in the development of a multicellular organism and is reliant on coordinated transcriptional and signal transduction events. To establish the segmented body plan that underlies much of metazoan development, individual cells and groups of cells must respond to exogenous signals with complex movements and shape changes. One class of proteins that plays a pivotal role in the interpretation of extracellular cues into cellular behavior is the Rho family of small GTPases. These molecular switches are essential components of a growing number of signaling pathways, many of which regulate actin cytoskeletal remodeling. Much of our understanding of Rho biology has come from work done in cell culture. More recently, the fruit fly Drosophila melanogaster has emerged as an excellent genetic system for the study of these proteins in a developmental and organismal context. Studies in flies have greatly enhanced our understanding of pathways involving Rho GTPases and their roles in development.Key words: Rho GTPases, Drosophila, development, effectors, loss-of-function mutation, dominant-negative, constitutively active.

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Neural tube closure requires the endocytic receptor Lrp2 and its functional interaction with intracellular scaffolds

10.1101/2020.07.15.205252 ◽

2020 ◽

Author(s):

Izabela Kowalczyk ◽

Chanjae Lee ◽

Elisabeth Schuster ◽

Josefine Hoeren ◽

Valentina Trivigno ◽

...

Keyword(s):

Cell Polarity ◽

Neural Tube ◽

Planar Cell Polarity ◽

Adaptor Proteins ◽

Neural Tube Closure ◽

Model Organisms ◽

Loss Of Function ◽

Functional Interaction ◽

Apical Constriction ◽

Tube Closure

AbstractRecent studies have revealed that pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we combined analysis of neural tube closure in mouse and in the African Clawed Frog Xenopus laevis to elucidate the etiology of Lrp2-related NTDs. Lrp2 loss-of-function (LOF) impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction as well as PCP component trafficking in a temporospatial manner.Summary statementAnalysis of neurulation in mouse and Xenopus reveals novel roles for Lrp2-mediated endocytosis in orchestrating apical constriction and planar cell polarity essential for neural tube closure.

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