Publications by authors named "Dean Thumkeo"

19 Publications

  • Page 1 of 1

DIAPH1 Variants in Non-East Asian Patients With Sporadic Moyamoya Disease.

JAMA Neurol 2021 Jun 14. Epub 2021 Jun 14.

Yale Center for Genome Analysis, West Haven, Connecticut.

Importance: Moyamoya disease (MMD), a progressive vasculopathy leading to narrowing and ultimate occlusion of the intracranial internal carotid arteries, is a cause of childhood stroke. The cause of MMD is poorly understood, but genetic factors play a role. Several familial forms of MMD have been identified, but the cause of most cases remains elusive, especially among non-East Asian individuals.

Objective: To assess whether ultrarare de novo and rare, damaging transmitted variants with large effect sizes are associated with MMD risk.

Design, Setting, And Participants: A genetic association study was conducted using whole-exome sequencing case-parent MMD trios in a small discovery cohort collected over 3.5 years (2016-2019); data were analyzed in 2020. Medical records from US hospitals spanning a range of 1 month to 1.5 years were reviewed for phenotyping. Exomes from a larger validation cohort were analyzed to identify additional rare, large-effect variants in the top candidate gene. Participants included patients with MMD and, when available, their parents. All participants who met criteria and were presented with the option to join the study agreed to do so; none were excluded. Twenty-four probands (22 trios and 2 singletons) composed the discovery cohort, and 84 probands (29 trios and 55 singletons) composed the validation cohort.

Main Outcomes And Measures: Gene variants were identified and filtered using stringent criteria. Enrichment and case-control tests assessed gene-level variant burden. In silico modeling estimated the probability of variant association with protein structure. Integrative genomics assessed expression patterns of MMD risk genes derived from single-cell RNA sequencing data of human and mouse brain tissue.

Results: Of the 24 patients in the discovery cohort, 14 (58.3%) were men and 18 (75.0%) were of European ancestry. Three of 24 discovery cohort probands contained 2 do novo (1-tailed Poisson P = 1.1 × 10-6) and 1 rare, transmitted damaging variant (12.5% of cases) in DIAPH1 (mammalian diaphanous-1), a key regulator of actin remodeling in vascular cells and platelets. Four additional ultrarare damaging heterozygous DIAPH1 variants (3 unphased) were identified in 3 other patients in an 84-proband validation cohort (73.8% female, 77.4% European). All 6 patients were non-East Asian. Compound heterozygous variants were identified in ena/vasodilator-stimulated phosphoproteinlike protein EVL, a mammalian diaphanous-1 interactor that regulates actin polymerization. DIAPH1 and EVL mutant probands had severe, bilateral MMD associated with transfusion-dependent thrombocytopenia. DIAPH1 and other MMD risk genes are enriched in mural cells of midgestational human brain. The DIAPH1 coexpression network converges in vascular cell actin cytoskeleton regulatory pathways.

Conclusions And Relevance: These findings provide the largest collection to date of non-East Asian individuals with sporadic MMD harboring pathogenic variants in the same gene. The results suggest that DIAPH1 is a novel MMD risk gene and impaired vascular cell actin remodeling in MMD pathogenesis, with diagnostic and therapeutic ramifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1001/jamaneurol.2021.1681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204259PMC
June 2021

Opening the Door to Better Aspirin.

Structure 2021 03;29(3):200-202

Department of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; AMED-FORCE, Japan Agency for Medical Research and Development, Tokyo 100-0004, Japan. Electronic address:

In this issue of Structure, Nojima et al. (2021) report the structure of the PGE-EP4-G complex by cryo-electron microscopy. This work shows unique modes of ligand binding, transduction mechanism, and G protein coupling of EP4, and serves as a starting point for development of more selective drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2021.02.003DOI Listing
March 2021

DAAM2 Variants Cause Nephrotic Syndrome via Actin Dysregulation.

Am J Hum Genet 2020 12 23;107(6):1113-1128. Epub 2020 Nov 23.

Pediatric Genomics Discovery Program, Department of Pediatrics and Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.

The discovery of >60 monogenic causes of nephrotic syndrome (NS) has revealed a central role for the actin regulators RhoA/Rac1/Cdc42 and their effectors, including the formin INF2. By whole-exome sequencing (WES), we here discovered bi-allelic variants in the formin DAAM2 in four unrelated families with steroid-resistant NS. We show that DAAM2 localizes to the cytoplasm in podocytes and in kidney sections. Further, the variants impair DAAM2-dependent actin remodeling processes: wild-type DAAM2 cDNA, but not cDNA representing missense variants found in individuals with NS, rescued reduced podocyte migration rate (PMR) and restored reduced filopodia formation in shRNA-induced DAAM2-knockdown podocytes. Filopodia restoration was also induced by the formin-activating molecule IMM-01. DAAM2 also co-localizes and co-immunoprecipitates with INF2, which is intriguing since variants in both formins cause NS. Using in vitro bulk and TIRF microscopy assays, we find that DAAM2 variants alter actin assembly activities of the formin. In a Xenopus daam2-CRISPR knockout model, we demonstrate actin dysregulation in vivo and glomerular maldevelopment that is rescued by WT-DAAM2 mRNA. We conclude that DAAM2 variants are a likely cause of monogenic human SRNS due to actin dysregulation in podocytes. Further, we provide evidence that DAAM2-associated SRNS may be amenable to treatment using actin regulating compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2020.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820625PMC
December 2020

Disruption of actin dynamics regulated by Rho effector mDia1 attenuates pressure overload-induced cardiac hypertrophic responses and exacerbates dysfunction.

Cardiovasc Res 2021 Mar;117(4):1103-1117

Department of Pharmacology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama, Yufu, Oita 879-5593, Japan.

Aims: Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload and that Rho signalling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy.

Methods And Results : Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10-12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy-related genes, including immediate early genes, in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the filamentous/globular (F/G)-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors and serum response factor were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs.

Conclusion : mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvaa206DOI Listing
March 2021

LPA Induces Keratinocyte Differentiation and Promotes Skin Barrier Function through the LPAR1/LPAR5-RHO-ROCK-SRF Axis.

J Invest Dermatol 2019 05 14;139(5):1010-1022. Epub 2018 Nov 14.

Center for Innovation in Immunoregulation Technology and Therapeutics, Kyoto University Graduate School of Medicine, Kyoto, Japan; Department of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan. Electronic address:

The skin barrier protects the body from water loss, allergens, and pathogens. Profilaggrin is produced by differentiated keratinocytes and is processed into filaggrin monomers. These monomers cross-link keratin filaments and are also decomposed to natural moisturizing factors in the stratum corneum for skin hydration and barrier function. Deficits in FLG expression impair skin barrier function and underlie skin diseases such as dry skin and atopic dermatitis. However, intrinsic factors that regulate FLG expression and their mechanisms of action remain unknown. Here, we show that lysophosphatidic acid induces FLG expression in human keratinocytes via the LPAR1 and LPAR5 receptors and the downstream RHO-ROCK-SRF pathway. Comprehensive gene profiling analysis further showed that lysophosphatidic acid not only induces FLG expression but also facilitates keratinocyte differentiation. Moreover, lysophosphatidic acid treatment significantly up-regulated FLG production in a three-dimensional culture model of human skin and promoted barrier function in mouse skin in vivo. Thus, our work shows a previously unsuspected role for lysophosphatidic acid and its downstream signaling in the maintenance of skin homeostasis, which may serve as a novel therapeutic target for skin barrier dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2018.10.034DOI Listing
May 2019

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility.

PLoS Biol 2018 09 26;16(9):e2004874. Epub 2018 Sep 26.

Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell-germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2004874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175529PMC
September 2018

T cell-intrinsic prostaglandin E-EP2/EP4 signaling is critical in pathogenic T17 cell-driven inflammation.

J Allergy Clin Immunol 2019 02 20;143(2):631-643. Epub 2018 Jun 20.

Core Research for Evolutional Science and Technology (CREST), Medical Innovation Center, Kyoto, Japan; Center for Innovation in Immunoregulation Technology and Therapeutics, Kyoto University Graduate School of Medicine, Kyoto, Japan. Electronic address:

Background: IL-23 is the key cytokine for generation of pathogenic IL-17-producing helper T (T17) cells, which contribute critically to autoimmune diseases. However, how IL-23 generates pathogenic T17 cells remains to be elucidated.

Objectives: We sought to examine the involvement, molecular mechanisms, and clinical implications of prostaglandin (PG) E-EP2/EP4 signaling in induction of IL-23-driven pathogenic T17 cells.

Methods: The role of PGE in induction of pathogenic T17 cells was investigated in mouse T17 cells in culture in vitro and in an IL-23-induced psoriasis mouse model in vivo. Clinical relevance of the findings in mice was examined by using gene expression profiling of IL-23 and PGE-EP2/EP4 signaling in psoriatic skin from patients.

Results: IL-23 induces Ptgs2, encoding COX2 in T17 cells, and produces PGE, which acts back on the PGE receptors EP2 and EP4 in these cells and enhances IL-23-induced expression of an IL-23 receptor subunit gene, Il23r, by activating signal transducer and activator of transcription (STAT) 3, cAMP-responsive element binding protein 1, and nuclear factor κ light chain enhancer of activated B cells (NF-κB) through cyclic AMP-protein kinase A signaling. This PGE signaling also induces expression of various inflammation-related genes, which possibly function in T17 cell-mediated pathology. Combined deletion of EP2 and EP4 selectively in T cells suppressed accumulation of IL-17A and IL-17AIFN-γ pathogenic Th17 cells and abolished skin inflammation in an IL-23-induced psoriasis mouse model. Analysis of human psoriatic skin biopsy specimens shows positive correlation between PGE signaling and the IL-23/T17 pathway.

Conclusions: T cell-intrinsic EP2/EP4 signaling is critical in IL-23-driven generation of pathogenic T17 cells and consequent pathogenesis in the skin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2018.05.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354914PMC
February 2019

Rho signaling research: history, current status and future directions.

FEBS Lett 2018 06 24;592(11):1763-1776. Epub 2018 May 24.

Department of Drug Discovery Medicine, Medical Innovation Center, Kyoto University Graduate School of Medicine, Japan.

One of the main research areas in biology from the mid-1980s through the 1990s was the elucidation of signaling pathways governing cell responses. These studies brought, among other molecules, the small GTPase Rho to the epicenter. Rho signaling research has since expanded to all areas of biology and medicine. Here, we describe how Rho emerged as a key molecule governing cell morphogenesis and movement, how it was linked to actin reorganization, and how the study of Rho signaling has expanded from cultured cells to whole biological systems. We then give an overview of the current research status of Rho signaling in development, brain, cardiovascular system, immunity and cancer, and discuss the future directions of Rho signaling research, with emphasis on one Rho effector, ROCK*. *The Rho GTPase family. Rho family GTPases have now expanded to contain 20 members. Amino acid sequences of 20 Rho GTPases found in human were aligned and the phylogenetic tree was generated by ClustalW2 software (EMBL-EBI) based on NJ algorithm. The subfamilies of the Rho GTPases are highlighted by the circle and labeled on the right side. Rho cited in this review refers to the original members of Rho subfamily, RhoA, RhoB and RhoC, that are C3 substrates, and, unless specified, not to other members of the Rho subfamily such as Rac, Cdc42, and Rnd.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.13087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032899PMC
June 2018

Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss.

EMBO Mol Med 2016 11 2;8(11):1310-1324. Epub 2016 Nov 2.

Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C-terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N-terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient-derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR) at the DAD C-terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID-DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG-tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID-DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201606609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090661PMC
November 2016

Moesin controls clathrin-mediated S1PR1 internalization in T cells.

PLoS One 2013 16;8(12):e82590. Epub 2013 Dec 16.

Department of Fundamental Biosciences, Shiga University of Medical Science, Otsu, Shiga, Japan.

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082590PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865155PMC
October 2014

Physiological roles of Rho and Rho effectors in mammals.

Eur J Cell Biol 2013 Oct-Nov;92(10-11):303-15. Epub 2013 Oct 8.

Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan; Innovation Center for Immunoregulation, Technologies and Drugs (AK Project), Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

Rho GTPase is a master regulator controlling cytoskeleton in multiple contexts such as cell migration, adhesion and cytokinesis. Of several Rho GTPases in mammals, the best characterized is the Rho subfamily including ubiquitously expressed RhoA and its homologs RhoB and RhoC. Upon binding GTP, Rho exerts its functions through downstream Rho effectors, such as ROCK, mDia, Citron, PKN, Rhophilin and Rhotekin. Until recently, our knowledge about functions of Rho and Rho effectors came mostly from in vitro studies utilizing cultured cells, and their physiological roles in vivo were largely unknown. However, gene-targeting studies of Rho and its effectors have now unraveled their tissue- and cell-specific roles and provide deeper insight into the physiological function of Rho signaling in vivo. In this article, we briefly describe previous studies of the function of Rho and its effectors in vitro and then review and discuss recent studies on knockout mice of Rho and its effectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2013.09.002DOI Listing
June 2014

EphA4-dependent axon retraction and midline localization of Ephrin-B3 are disrupted in the spinal cord of mice lacking mDia1 and mDia3 in combination.

Genes Cells 2013 Oct 25;18(10):873-85. Epub 2013 Jul 25.

Department of Pharmacology, Kyoto University Graduate School of Medicine, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

mDia is an actin nucleator and polymerization factor regulated by the small GTPase Rho and consists of three isoforms. Here, we found that mice lacking mDia1 and mDia3, two isoforms expressed in the brain, in combination (mDia-DKO mice) show impaired left-right limb coordination during locomotion and aberrant midline crossing of axons of corticospinal neurons and spinal cord interneurons. Given that mice lacking Ephrin-B3-EphA4 signaling show a similar impairment in locomotion, we examined whether mDia is involved in Ephrin-B3-EphA4 signaling for axon repulsion. In primary cultured neurons, mDia deficiency impairs growth cone collapse and axon retraction induced by chemo-repellants including EphA ligands. In mDia-DKO mice, the Ephrin-B3-expressing midline structure in the spinal cord is disrupted, and axons aberrantly cross the spinal cord midline preferentially through the region devoid of Ephrin-B3. Therefore, mDia plays multiple roles in the proper formation of the neural network in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/gtc.12081DOI Listing
October 2013

Successful delivery following ICSI with macrocephalic sperm head syndrome: a case report.

Reprod Biomed Online 2012 Jun 9;24(6):603-5. Epub 2012 Mar 9.

Department of Obstetrics and Gynecology, Shiga University of Medical Science, Ohtsu, Shiga 520-2192, Japan.

This article reports a case of macrocephalic sperm head syndrome, which is defined as the presence of a very high percentage of spermatozoa with enlarged heads and multiple flagellae, together with detailed morphological analysis. After a couple presented with infertility, sperm analysis showed severe teratozoospermia and almost all of the spermatozoa had macrocephaly with multiple tails. The morphological analysis revealed that most of the sperm heads contained several nuclei and had a similar number of tails as that of nuclei. However, detailed analysis revealed that there were a very few spermatozoa with an almost normal morphology. After genetic counselling, intracytoplasmic sperm injection was performed using a few spermatozoa that had an almost normal morphology, resulting in pregnancy and successful delivery. Even in macrocephalic sperm head syndrome, which may be caused by meiotic division failure, pregnancy is possible if some spermatozoa with almost normal morphology can be utilized, although there may be genetic risks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.rbmo.2012.02.022DOI Listing
June 2012

A role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors.

Nat Neurosci 2012 Jan 15;15(3):373-80, S1-2. Epub 2012 Jan 15.

Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nn.3020DOI Listing
January 2012

Deficiency of mDia, an actin nucleator, disrupts integrity of neuroepithelium and causes periventricular dysplasia.

PLoS One 2011 28;6(9):e25465. Epub 2011 Sep 28.

Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.

During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025465PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182227PMC
January 2012

Impaired vascular remodeling in the yolk sac of embryos deficient in ROCK-I and ROCK-II.

Genes Cells 2011 Oct 5;16(10):1012-21. Epub 2011 Sep 5.

Department of Pharmacology, Kyoto University, Japan.

Rho-associated coiled-coil-forming protein serine/threonine kinase (ROCK) consisting of two isoforms, ROCK-I and ROCK-II, functions downstream of the small GTPase Rho for assembly of actomyosin bundles. To examine the role of ROCK isoforms in vivo, we previously generated and examined mice deficient in each of the two isoforms individually. Here, we further examined the in vivo role of ROCK isoforms by generating mice deficient in both isoforms. Cross-mating of ROCK-I(+/-) ROCK-II(+/-) double heterozygous mice showed that all of the ROCK-I(-/-) ROCK-II(-/-) homozygous mice die in utero before 9.5 days post-coitum (dpc) and ROCK-I(-/-) ROCK-II(+/-) homo-heterozygous or ROCK-I(+/-) ROCK-II(-/-) hetero-homozygous mice die during a period from 9.5 to 12.5 dpc, whereas mice of other genotypes survive until 12.5 dpc with the expected Mendelian ratio. All of the ROCK-I(+/-) ROCK-II(-/-) or ROCK-I(-/-) ROCK-II(+/-) mice showed impaired body turning and defective vascular remodeling in the yolk sac. Impairment of vascular remodeling was also observed in wild-type embryos treated ex vivo with a ROCK inhibitor, Y-27632. These results suggest that ROCK isoforms function redundantly during embryogenesis and play a critical role in vascular development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2443.2011.01546.xDOI Listing
October 2011

ROCK-I and ROCK-II cooperatively regulate closure of eyelid and ventral body wall in mouse embryo.

Genes Cells 2005 Aug;10(8):825-34

Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.

Rho-associated kinase (ROCK) is a serine/threonine kinase working in the Rho signaling to actin cytoskeleton. We previously reported that loss of ROCK-I results in the eyelid open at birth (EOB) and omphalocele phenotype in mice, while loss of ROCK-II results in placental dysfunction leading to intrauterine growth retardation and fetal death. Here, we report that after backcross to the C57BL/6 N genetic background, ROCK-II knockout (KO) neonates are born also with open eyelid and umbilical hernia, a phenotype similar to that of ROCK-I KO mice. ROCK-II KO embryos show impaired extension of the eyelid epithelial sheet with disorganized actin bundles in the leading edge of the sheet. These results suggest that ROCK-I and ROCK-II cooperatively regulates the assembly of actin bundles essential for closure of the eyelid and ventral body wall in mouse embryos. Consistently, ROCK-I(+/-)ROCK-II(+/-) double heterozygous mice also show the EOB and omphalocele phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2443.2005.00882.xDOI Listing
August 2005

ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles.

J Cell Biol 2005 Mar 7;168(6):941-53. Epub 2005 Mar 7.

Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.

Rho-associated kinase (ROCK) I mediates signaling from Rho to the actin cytoskeleton. To investigate the in vivo functions of ROCK-I, we generated ROCK-I-deficient mice. Loss of ROCK-I resulted in failure of eyelid closure and closure of the ventral body wall, which gave rise to the eyes open at birth and omphalocele phenotypes in neonates. Most ROCK-I(-/-) mice died soon after birth as a result of cannibalization of the omphalocele by the mother. Actin cables that encircle the eye in the epithelial cells of the eyelid were disorganized and accumulation of filamentous actin at the umbilical ring was impaired, with loss of phosphorylation of the myosin regulatory light chain (MLC) at both sites, in ROCK-I(-/-) embryos. Stress fiber formation and MLC phosphorylation induced by EGF were also attenuated in primary keratinocytes from ROCK-I(-/-) mice. These results suggest that ROCK-I regulates closure of the eyelids and ventral body wall through organization of actomyosin bundles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200411179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171774PMC
March 2005

Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death.

Mol Cell Biol 2003 Jul;23(14):5043-55

Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.

Rho-associated kinase (ROCK), including the ROCK-I and ROCK-II isoforms, is a protein kinase involved in signaling from Rho to actin cytoskeleton. However, in vivo functions of each ROCK isoform remain largely unknown. We generated mice deficient in ROCK-II by gene targeting. ROCK-II(-/-) embryos were found at the expected Mendelian frequency until 13.5 days postcoitum, but approximately 90% died thereafter in utero. ROCK-II(-/-) mice of both genders that survived were born runts, subsequently developed without gross abnormality, and were fertile. Whole-mount staining for a knocked-in lacZ reporter gene revealed that ROCK-II was highly expressed in the labyrinth layer of the placenta. Disruption of architecture and extensive thrombus formation were found in the labyrinth layer of ROCK-II(-/-) mice. While no obvious alteration in actin filament structures was found in the labyrinth layer of ROCK-II(-/-) placenta and stress fibers were formed in cultured ROCK-II(-/-) trophoblasts, elevated expression of plasminogen activator inhibitor 1 was found in ROCK-II(-/-) placenta. These results suggest that ROCK-II is essential in inhibiting blood coagulation and maintaining blood flow in the endothelium-free labyrinth layer and that loss of ROCK-II leads to thrombus formation, placental dysfunction, intrauterine growth retardation, and fetal death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC162229PMC
http://dx.doi.org/10.1128/MCB.23.14.5043-5055.2003DOI Listing
July 2003