Dean Thumkeo

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

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.

Targeted Disruption of the Mouse Rho-Associated Kinase 2 Gene Results in Intrauterine Growth Retardation and Fetal Death

ABSTRACT 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.

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

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.

Physiological roles of Rho and Rho effectors in mammals

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.

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

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.

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

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.

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

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.

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

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 (RRKR1204–1207) 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.

Moesin Controls Clathrin-Mediated S1PR1 Internalization in T Cells

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.

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

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.