scholarly journals Replacing nonmuscle myosin 2A with myosin 2C1 permits gastrulation but not placenta vascular development in mice

2018 ◽  
Vol 29 (19) ◽  
pp. 2326-2335
Author(s):  
Yingfan Zhang ◽  
Chengyu Liu ◽  
Robert S. Adelstein ◽  
Xuefei Ma
Keyword(s):  
Homologous Recombination ◽  
Focal Adhesions ◽  
Nonmuscle Myosin ◽  
Developmentally Delayed ◽  
Mouse Development ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Heavy Chains ◽  
Vascular Formation

Three paralogues of nonmuscle myosin 2 (NM 2A, 2B, and 2C) are expressed in mammals, and the heavy chains are the products of three different genes (Myh9, Myh10, and Myh14, respectively). NM 2A and 2B are essential for mouse development, while 2C is not. Studies on NM 2C are limited and the in vivo function of this paralogue is not clear. Using homologous recombination, cDNA encoding nonmuscle myosin heavy chain 2C1 fused with GFP was introduced into the first coding exon of Myh9, replacing NM 2A expression with NM 2C1 expression in mice. In contrast to A–/A– embryos, which die by embryonic day (E) 6.5, AC1*gfp/AC1*gfp embryos survive through E8.5, demonstrating that NM 2C1 can support mouse development beyond gastrulation. At E9.5 and E10.5, however, AC1*gfp/AC1*gfp embryos are developmentally delayed, with abnormalities in placental vascular formation. The defect in vascular formation is confirmed in allantois explants from AC1*gfp/AC1*gfp embryos. Thus, NM 2C1 cannot support normal placental vascular formation. In addition, AC1*gfp/AC1*gfp mouse embryonic fibroblasts (MEFs) migrate rapidly but with impaired persistence and develop smaller, less mature focal adhesions than A+/A+ MEFs. This is attributed to enhanced NM 2C1 actomyosin stability and different NM 2C1 subcellular localization than in NM 2A.

scholarly journals Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

2016 ◽  
Vol 473 (17) ◽  
pp. 2671-2685 ◽  
Author(s):  
Genta Ito ◽  
Kristina Katsemonova ◽  
Francesca Tonelli ◽  
Pawel Lis ◽  
Marco A.S. Baptista ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Mouse Lung ◽  
Rab Gtpase ◽  
Leucine Rich Repeat ◽  
Serine Residue ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
The Impact ◽  
Effector Binding

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

scholarly journals Establishment and Identification of a CiPSC Lineage Reprogrammed from FSP-tdTomato Mouse Embryonic Fibroblasts (MEFs)

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Ruiping Chen ◽  
Wenxiu Xie ◽  
Baomei Cai ◽  
Yue Qin ◽  
Chuman Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecule ◽  
Fluorescent Protein ◽  
Day Treatment ◽  
Tracking System ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Underlying Mechanisms

Safety issues associated with transcription factors or viruses may be avoided with the use of chemically induced pluripotent stem cells (CiPSCs), thus promoting their clinical application. Previously, we had successfully developed and standardized an induction method using small-molecule compound, with simple operation, uniform induction conditions, and clear constituents. In order to verify that the CiPSCs were indeed reprogrammed from mouse embryonic fibroblasts (MEFs), and further explore the underlying mechanisms, FSP-tdTomato mice were used to construct a fluorescent protein-tracking system of MEFs, for revealing the process of CiPSC reprogramming. CiPSCs were identified by morphological analysis, mRNA, and protein expression of pluripotency genes, as well as teratoma formation experiments. Results showed that after 40-day treatment of tdTomato-MEFs with small-molecule compounds, the cells were presented with prominent nucleoli, high core-to-cytoplasmic ratio, round shape, group and mass arrangement, and high expression of pluripotency gene. These cells could differentiate into three germ layer tissues in vivo. As indicated by the above results, tdTomato-MEFs could be reprogrammed into CiPSCs, a lineage that possesses pluripotency similar to mouse embryonic stem cells (mESCs), with the use of small-molecule compounds. The establishment of CiPSC lineage, tracked by fluorescent protein, would benefit further studies exploring its underlying mechanisms. With continuous expression of fluorescent proteins during cellular differentiation, this cell lineage could be used for tracking CiPSC transplantation and differentiation into functional cells.

scholarly journals SCCRO (DCUN1D1) Promotes Nuclear Translocation and Assembly of the Neddylation E3 Complex

2011 ◽  
Vol 286 (12) ◽  
pp. 10297-10304 ◽  
Author(s):  
Guochang Huang ◽  
Andrew J. Kaufman ◽  
Y. Ramanathan ◽  
Bhuvanesh Singh
Keyword(s):  
Nuclear Localization ◽  
Essential Role ◽  
Nuclear Translocation ◽  
Nuclear Localization Sequence ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Transgenic Expression ◽  
Localization Sequence ◽  
Efficient Transfer

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO−/− mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO−/− mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.

scholarly journals The Histidine Triad Protein Hint Is Not Required for Murine Development or Cdk7 Function

2003 ◽  
Vol 23 (11) ◽  
pp. 3929-3935 ◽  
Author(s):  
Nina Korsisaari ◽  
Derrick J. Rossi ◽  
Keijo Luukko ◽  
Kay Huebner ◽  
Mark Henkemeyer ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Protein Gene ◽  
Wild Type ◽  
Mouse Development ◽  
Embryonic Fibroblasts ◽  
Normal Life Span ◽  
Abnormal Pathology ◽  
Deficient Mice ◽  
Apparently Healthy

ABSTRACT The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28. To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint −/−) mice. Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward. Despite this widespread expression, disruption of Hint did not impair murine development. Moreover, Hint-deficient mice had a normal life span and were apparently healthy. Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint −/− mice. Functional redundancy within the HIT family was addressed by crossing Hint −/− mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint +/+ and Hint −/− tissues. Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint −/− mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.

scholarly journals PPARγ-Independent Increase in Glucose Uptake and Adiponectin Abundance in Fat Cells

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3648-3660 ◽  
Author(s):  
Olga Dubuisson ◽  
Emily J. Dhurandhar ◽  
Rashmi Krishnapuram ◽  
Heather Kirk-Ballard ◽  
Alok K. Gupta ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Glycemic Control ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Human Adipose Tissue ◽  
Peroxisome Proliferator ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Glucose Transporter 1

Although thiazolidinediones (TZD) effectively improve hyperglycemia and increase adiponectin, a proinsulin-sensitizing adipokine, they also increase adipogenesis via peroxisome proliferator-activated receptor (PPAR)γ induction, which may be undesirable. Recent safety concerns about some TZD have prompted the search for next generation agents that can enhance glycemic control and adiponectin independent of PPARγ or adipogenesis. Reminiscent of TZD action, a human adenovirus, adenovirus 36 (Ad36), up-regulates PPARγ, induces adipogenesis, and improves systemic glycemic control in vivo. We determined whether this effect of Ad36 requires PPARγ and/or adipogenesis. Glucose uptake and relevant cell signaling were determined in mock-infected or human adenoviruses Ad36 or Ad2-infected cell types under the following conditions: 1) undifferentiated human-adipose-tissue-derived stem cells (hASC), 2) hASC differentiated as adipocytes, 3) hASC in presence or absence of a PPARγ inhibitor, 4) NIH/3T3 that have impaired PPARγ expression, and 5) PPARγ-knockout mouse embryonic fibroblasts. Mouse embryonic fibroblasts with intact PPARγ served as a positive control. Additionally, to determine natural Ad36 infection, human sera were screened for Ad36 antibodies. In undifferentiated or differentiated hASC, or despite the inhibition, down-regulation, or the absence of PPARγ, Ad36 significantly enhanced glucose uptake and PPARγ, adiponectin, glucose transporter 4, and glucose transporter 1 protein abundance, compared with mock or Ad2-infected cells. This indicated that Ad36 up-regulates glucose uptake and adiponectin secretion independent of adipogenesis or without recruiting PPARγ. In humans, natural Ad36 infection predicted greater adiponectin levels, suggesting a human relevance of these effects. In conclusion, Ad36 provides a novel template to metabolically remodel human adipose tissue to enhance glycemic control without the concomitant increase in adiposity or PPARγ induction associated with TZD actions.

scholarly journals Appl1andAppl2are Expendable for Mouse Development But Are Essential for HGF-Induced Akt Activation and Migration in Mouse Embryonic Fibroblasts

2015 ◽  
Vol 231 (5) ◽  
pp. 1142-1150 ◽  
Author(s):  
Yinfei Tan ◽  
Xiaoban Xin ◽  
Francis J. Coffey ◽  
David L. Wiest ◽  
Lily Q. Dong ◽  
...  
Keyword(s):  
Mouse Development ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Akt Activation ◽  
And Migration

scholarly journals MAPK-induced miR-29 targets MAFG and suppresses melanoma development

2020 ◽  
Author(s):  
Olga Vera ◽  
Ilah Bok ◽  
Neel Jasani ◽  
Koji Nakamura ◽  
Xiaonan Xu ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mapk Signaling ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Oncogenic Potential ◽  
Human Melanocytes ◽  
Signaling Axis ◽  
Bona Fide ◽  
Mouse Modeling

ABSTRACTThe tumor suppressive miR-29 family of microRNAs is encoded by two clusters, miR-29b1∼a and miR-29b2∼c, and is regulated by several oncogenic and tumor suppressive stimuli. Here we investigated whether oncogenic MAPK hyperactivation regulates miR-29 abundance and how this signaling axis impacts melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1∼a and pri-miR-29b2∼c, respectively. Expression analyses revealed that while pri-miR-29a∼bl remains elevated, pri-miR-29b2∼c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we showed that inactivation of miR-29 in vivo accelerates melanoma development and decreases overall survival. We identified the transcription factor MAFG as a bona fide miR-29 target that has oncogenic potential in melanocytes and is required for growth of melanoma cells. Our findings suggest that MAPK-induced miR-29 contributes to a tumor suppressive barrier by targeting MAFG, which is overcome by attenuation of miR-29b2∼c expression.

scholarly journals Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Markus Terrey ◽  
Scott I Adamson ◽  
Jeffrey H Chuang ◽  
Susan L Ackerman
Keyword(s):  
Quality Control ◽  
Signaling Pathways ◽  
Mutant Mouse ◽  
Cellular Responses ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Nonsense Mediated Decay ◽  
Development Analysis ◽  
Toxic Peptides

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.

scholarly journals Translocation of CrkL to Focal Adhesions Mediates Integrin-Induced Migration Downstream of Src Family Kinases

2003 ◽  
Vol 23 (8) ◽  
pp. 2883-2892 ◽  
Author(s):  
Leiming Li ◽  
Deborah L. Guris ◽  
Masaya Okura ◽  
Akira Imamoto
Keyword(s):  
G Proteins ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Adapter Protein ◽  
Related Protein ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Src Signaling ◽  
Downstream Mediator

ABSTRACT The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130 Cas (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.

scholarly journals Expression of Caveolin-1 Induces Premature Cellular Senescence in Primary Cultures of Murine Fibroblasts

2002 ◽  
Vol 13 (7) ◽  
pp. 2502-2517 ◽  
Author(s):  
Daniela Volonte ◽  
Kun Zhang ◽  
Michael P. Lisanti ◽  
Ferruccio Galbiati
Keyword(s):  
Hydrogen Peroxide ◽  
Cellular Senescence ◽  
Premature Senescence ◽  
3T3 Cells ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Caveolin 1 ◽  
Senescent Phenotype ◽  
Nih 3T3

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a “transformation suppressor” protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G0/G1 phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in β-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.

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