224 results on '"Keith E. Mostov"'
Search Results
2. Vascular and Liver Homeostasis in Juvenile Mice Require Endothelial Cyclic AMP-Dependent Protein Kinase A
- Author
-
Pavel I. Nedvetsky, Ivo Cornelissen, Thomas Mathivet, Claire Bouleti, Phalla Ou, Pieter Baatsen, Xiaocheng Zhao, Frans Schuit, Fabio Stanchi, Keith E. Mostov, and Holger Gerhardt
- Subjects
angiogenesis ,edema ,liver sinusoidal endothelium ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1–P3) of later (P28–P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.
- Published
- 2022
- Full Text
- View/download PDF
3. The phospholipid PI(3,4)P2 is an apical identity determinant
- Author
-
Álvaro Román-Fernández, Julie Roignot, Emma Sandilands, Marisa Nacke, Mohammed A. Mansour, Lynn McGarry, Emma Shanks, Keith E. Mostov, and David M. Bryant
- Subjects
Science - Abstract
During de novo establishment of apical-basal polarity, a basolateral membrane must be converted into an apical delivery zone. Here, the authors use MDCK 3D cysts to uncover that the phospholipid PI(3,4)P2 is an apical membrane determinant.
- Published
- 2018
- Full Text
- View/download PDF
4. A Qualitative Change in the Transcriptome Occurs after the First Cell Cycle and Coincides with Lumen Establishment during MDCKII Cystogenesis
- Author
-
Tianfang Wang, Sang-Ho Kwon, Xiao Peng, Severine Urdy, Zefu Lu, Robert J. Schmitz, Stephen Dalton, Keith E. Mostov, and Shaying Zhao
- Subjects
Developmental Biology ,Embryology ,Transcriptomics ,Science - Abstract
Summary: Madin-Darby canine kidney II (MDCKII) cells are widely used to study epithelial morphogenesis. To better understand this process, we performed time course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome that occurs after the first cell cycle and coincides with lumen establishment. This change appears to be linked to translocation of β-catenin, supported by analyses with AVL9- and DENND5A-knockdown clones, and regulation by HNF1B, supported by ATAC-seq study. These findings indicate a qualitative change model for transcriptome remodeling during epithelial morphogenesis, leading to cell proliferation decrease and cell polarity establishment. Furthermore, our study reveals that active mitochondria are retained and chromatin accessibility decreases in 3D cysts but not in 2D polarized cells. This indicates that 3D culture is a better model than 2D culture for studying epithelial morphogenesis.
- Published
- 2020
- Full Text
- View/download PDF
5. Intussusceptive Angiogenesis in Human Metastatic Malignant Melanoma
- Author
-
Jan Borén, Levent M. Akyürek, Per Fogelstrand, Matias Ekstrand, Joakim Karlsson, Max Levin, Malin Levin, Keith E. Mostov, Lars Ny, Martin O. Bergo, Jonas Nilsson, Sara Bjursten, Zhiyuan Zhao, Kristell Le Gal, Andrew J. Ewald, and Ankur Pandita
- Subjects
Male ,Skin Neoplasms ,Angiogenesis ,MMP9 ,Matrix metalloproteinase ,Medical and Health Sciences ,Pathology and Forensic Medicine ,Mice ,chemistry.chemical_compound ,80 and over ,Genetics ,Pathology ,medicine ,Animals ,Humans ,2.1 Biological and endogenous factors ,PTEN ,Tensin ,Aetiology ,Intussusceptive angiogenesis ,Melanoma ,Neovascularization ,Aged ,Cancer ,Aged, 80 and over ,Pathologic ,Neovascularization, Pathologic ,biology ,Regular Article ,Middle Aged ,medicine.disease ,Vascular endothelial growth factor ,Matrix Metalloproteinase 9 ,chemistry ,Cancer research ,biology.protein ,Heterografts ,Female ,Biotechnology - Abstract
Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, invitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation invitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.
- Published
- 2021
6. A robust in silico analogue of MDCK cystogenesis mimics growth in multiple culture conditions.
- Author
-
Jesse Engelberg, Anirban Datta, Keith E. Mostov, and C. Anthony Hunt
- Published
- 2011
7. Systems Modeling of Alveolar Morphogenesis In Vitro.
- Author
-
Sean H. J. Kim, Sunwoo Park, Wei Yu, Keith E. Mostov, Michael A. Matthay, and C. Anthony Hunt
- Published
- 2007
8. Simple Rules Determine Distinct Patterns of Branching Morphogenesis
- Author
-
Wallace F. Marshall, Leonardo Morsut, Wendell A. Lim, Keith E. Mostov, Paul Brakeman, Wei Yu, and Ross J. Metzger
- Subjects
Kidney Disease ,Histology ,Body Patterning ,Morphogenesis ,Biology ,Kidney ,Models, Biological ,Article ,Epithelium ,Pathology and Forensic Medicine ,Branching (linguistics) ,03 medical and health sciences ,0302 clinical medicine ,Models ,Branching morphogenesis ,Animals ,Humans ,Developmental ,Lung ,030304 developmental biology ,0303 health sciences ,Extramural ,Mammalian ,Gene Expression Regulation, Developmental ,Cell Biology ,Anatomy ,Embryo, Mammalian ,Biological ,Gene Expression Regulation ,Embryo ,Ureteric bud ,Organ surface ,Biochemistry and Cell Biology ,030217 neurology & neurosurgery ,Metanephric kidney - Abstract
Many metazoan organs are comprised of branching trees of epithelial tubes; how patterning occurs in these trees is a fundamental problem of development. Commonly, branch tips fill the volume of the organ approximately uniformly, e.g. in mammalian lung, airway branch tips are dispersed roughly uniformly throughout the volume of the lung. Contrastingly, in the developing metanephric kidney, the tips of the ureteric bud tree are located close to the outer surface of the kidney, rather than filling the kidney. Here we describe a simple alteration in the branching rules that accounts for the difference between the kidney pattern that leads to tips near the organ surface, versus previously known patterns that lead to the branch tips being dispersed throughout the organ. We further use a simple toy model to deduce from first principles how this rule change accounts for the differences in the two types of trees.
- Published
- 2019
9. The phospholipid PI(3,4)P2 is an apical identity determinant
- Author
-
Keith E. Mostov, Álvaro Román-Fernández, Mohammed A. Mansour, Lynn McGarry, David M. Bryant, Julie Roignot, Emma Shanks, Marisa Nacke, and Emma Sandilands
- Subjects
0301 basic medicine ,Endosome ,1.1 Normal biological development and functioning ,Science ,Endocytic cycle ,General Physics and Astronomy ,Endosomes ,Phosphatidylinositols ,Article ,General Biochemistry, Genetics and Molecular Biology ,Madin Darby Canine Kidney Cells ,Extracellular matrix ,03 medical and health sciences ,chemistry.chemical_compound ,Dogs ,Underpinning research ,MD Multidisciplinary ,Cell polarity ,Pi ,Animals ,Phosphatidylinositol ,lcsh:Science ,Multidisciplinary ,Vesicle ,5-Trisphosphate 5-Phosphatases ,Phosphatidylinositol-3 ,General Chemistry ,Intracellular Membranes ,Apical membrane ,Cell biology ,030104 developmental biology ,chemistry ,Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases ,lcsh:Q - Abstract
Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P2 localization and function is distinct from the basolateral determinant PI(3,4,5)P3. PI(3,4)P2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P2 is a determinant of apical membrane identity., During de novo establishment of apical-basal polarity, a basolateral membrane must be converted into an apical delivery zone. Here, the authors use MDCK 3D cysts to uncover that the phospholipid PI(3,4)P2 is an apical membrane determinant.
- Published
- 2018
10. MDCK Cystogenesis Driven by Cell Stabilization within Computational Analogues.
- Author
-
Jesse Engelberg, Anirban Datta, Keith E. Mostov, and C. Anthony Hunt
- Published
- 2011
- Full Text
- View/download PDF
11. Apical targeting of the formin Diaphanous in Drosophila tubular epithelia
- Author
-
Tal Rousso, Annette M Shewan, Keith E Mostov, Eyal D Schejter, and Ben-Zion Shilo
- Subjects
Diaphanous ,formin ,tubular epithelia ,apical localization ,PI(4,5)P2 ,Rho1 ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane.
- Published
- 2013
- Full Text
- View/download PDF
12. Ciliary signaling-patterned smooth muscle drives tubular elongation
- Author
-
Jeremy F. Reiter, Chang Xie, Keith E. Mostov, Alexis Leigh Krup, Ying Yang, Pekka Paivinen, and Tomi P. Mäkelä
- Subjects
0303 health sciences ,Hippo signaling pathway ,Chemistry ,Cilium ,Mesenchyme ,Smooth muscle layer ,Strain (injury) ,medicine.disease ,Small intestine ,Epithelium ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,medicine ,Elongation ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
SummaryDuring development, many tubular organs undergo extensive longitudinal growth to reach their defined length, essential for their function, but how they lengthen is poorly understood. Here, we found that primary cilia are critical for the elongation of the small intestine and esophagus during murine embryonic development. More specifically, HH ligands produced by the epithelium signaled via cilia in the surrounding mesenchyme to pattern the smooth muscle. Like attenuated ciliary HH signaling, partial ablation of the smooth muscle reduced elongation, revealing an essential role for smooth muscle in longitudinal growth. Disruption of cilia, HH signaling or the smooth muscle reduced residual stress within the gut wall, indicating that smooth muscle contributes to the mechanical properties of the developing gut. Reducing residual stress decreased nuclear YAP, an effector of the mechanotransductive Hippo pathway. Removing YAP in the mesenchyme did not affect smooth muscle formation, but attenuated proliferation and elongation, demonstrating that YAP interprets smooth muscle-generated force to promote proliferation. Together, our results reveal that ciliary signaling directs the formation of the smooth muscle layer which, in turn, generates mechanical forces that activate YAP-mediated proliferation. As this interplay of biochemical and mechanical signals drives elongation of both the esophagus and small intestine, we propose that this mechanism may underlie tubular organ elongation generally.HighlightsPrimary cilia are essential for the elongation of the small intestine and esophagus during embryonic developmentCiliary signaling patterns the smooth muscle in the developing intestine and esophagusThe smooth muscle contributes to tissue mechanicsSmooth muscle-generated strain activates YAP to drive longitudinal growth of the tubular organs
- Published
- 2020
13. Guidelines and definitions for research on epithelial–mesenchymal transition
- Author
-
Kyra Campbell, Ruby Yun-Ju Huang, Binhua P. Zhou, Yoshiko Takahashi, Yibin Kang, Cédric Blanpain, Antonio García de Herreros, Ben Z. Stanger, Amparo Cano, Gerhard Christofori, Jordi Casanova, Jean Paul Thiery, Jing Yang, Chaya Kalcheim, Geert Berx, Raghu Kalluri, Sendurai A. Mani, Marc P. Stemmler, Yeesim Khew-Goodall, Marianne E. Bronner, Thomas Brabletz, Heide L. Ford, Masatoshi Takeichi, Jonas Fuxe, Rik Derynck, Anna-Katerina Hadjantonakis, Shoukat Dedhar, Parker B. Antin, M. Angela Nieto, Donald F. Newgreen, Herbert Levine, Raymond B. Runyan, Guojun Sheng, Robert A. Weinberg, Gregory J. Goodall, Keith E. Mostov, Erik W. Thompson, Roberto Mayor, Eric Theveneau, Pierre Savagner, Alain Puisieux, Jinsong Liu, Joan Massagué, Elizabeth D. Williams, Gregory D. Longmore, Jianhua Xing, David R. McClay, Yang, Jing, Antin, Parker, Berx, Geert, Blanpain, Cédric, Goodall, Gregory J, Khew-Goodall, Yeesim, Sheng, Guojun, EMT International Association (TEMTIA), UAM. Departamento de Bioquímica, and Instituto de Investigaciones Biomédicas 'Alberto Sols' (IIBM)
- Subjects
Web of science ,cell migration ,Medicina ,Lung metastasis ,Diversity in experimental ,Regulació cel·lular -- Guies ,Definitions ,Guidelines ,Cellular organization ,Terminology ,EMBRYONIC HEART ,03 medical and health sciences ,0302 clinical medicine ,E-CADHERIN EXPRESSION ,Developmental biology ,Medicine and Health Sciences ,Epithelial–mesenchymal transition ,PLASTICITY ,epithelial–mesenchymal transition (EMT) ,Molecular Biology ,TRANSCRIPTION FACTOR SNAIL ,030304 developmental biology ,Research data ,FIBROBLAST-GROWTH-FACTOR ,Epithelial–mesenchymal transition (EMT) ,Cancer ,Cognitive science ,0303 health sciences ,Cèl·lules epitelials ,Nomenclature ,EMT ,Consensus Statement ,Biology and Life Sciences ,Biologie moléculaire ,Cell movement ,Cell Biology ,COLLAGEN GELS ,Epithelial-mesenchymal transition ,3. Good health ,Conceptual framework ,NEURAL CREST CELLS ,030220 oncology & carcinogenesis ,REPRESSES E-CADHERIN ,Consensus statement ,embryonic structures ,LUNG METASTASIS ,Biologie cellulaire ,Psychology - Abstract
On behalf of the EMT International Association (TEMTIA)., Epithelial–mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by ‘the EMT International Association’ (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.
- Published
- 2020
14. A Qualitative Change in the Transcriptome During MDCKII 3D Epithelial Morphogenesis is Linked to the First Cell Cycle and Intracellular Trafficking
- Author
-
Robert J. Schmitz, Xiao Peng, Zefu Lu, Tianfang Wang, Sang Ho Kwon, Keith E. Mostov, Shaying Zhao, Stephen Dalton, and Severine Urdy
- Subjects
Transcriptome ,Cell growth ,Cell polarity ,Biology ,Cell cycle ,Mitosis ,Transcription factor ,Intracellular ,Chromatin ,Cell biology - Abstract
Madin-Darby canine kidney II (MDCKII) cells are used widely to study epithelial morphogenesis. To better understand this process, we performed time-course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome after the 1st cell cycle. This change appears to be linked to translocation of β-catenin, supported by analyses with AVL9- or DENND5A-knockdown clones, and HNF1B mitotic bookmarking, supported by ATAC-seq study. Specifically, β-catenin is depleted from the nucleus and enriched at the cell-cell junctions following the 1st cell cycle, downregulating the MYC network and decreasing cell proliferation. Meanwhile, HNF1B is retained in the nucleus, upregulating its targets and contributing to the cell polarity establishment. Our study supports a qualitative change model for transcriptome remodeling during epithelial morphogenesis and that this qualitative change results from transcription factor redistribution during the first cell cycle.
- Published
- 2020
15. Simulating Properties of In Vitro Epithelial Cell Morphogenesis.
- Author
-
Mark R. Grant, Keith E. Mostov, Thea D. Tlsty, and C. Anthony Hunt
- Published
- 2006
- Full Text
- View/download PDF
16. Author Correction: Guidelines and definitions for research on epithelial–mesenchymal transition
- Author
-
Jing Yang, Geert Berx, Elizabeth D. Williams, Sendurai A. Mani, Gregory J. Goodall, David R. McClay, Binhua P. Zhou, Parker B. Antin, Raghu Kalluri, Pierre Savagner, Erik W. Thompson, Roberto Mayor, Amparo Cano, Jinsong Liu, Robert A. Weinberg, Masatoshi Takeichi, Thomas Brabletz, Chaya Kalcheim, Donald F. Newgreen, Shoukat Dedhar, Kyra Campbell, Herbert Levine, Raymond B. Runyan, Jordi Casanova, Rik Derynck, Anna-Katerina Hadjantonakis, M. Angela Nieto, Marc P. Stemmler, Yeesim Khew-Goodall, Jean Paul Thiery, Heide L. Ford, Marianne E. Bronner, Yibin Kang, Antonio García de Herreros, Ben Z. Stanger, Cédric Blanpain, Keith E. Mostov, Jonas Fuxe, Eric Theveneau, Yoshiko Takahashi, Alain Puisieux, Ruby Yun-Ju Huang, Gerhard Christofori, Guojun Sheng, Joan Massagué, Gregory D. Longmore, and Jianhua Xing
- Subjects
Biomedical Research ,Consensus ,Epithelial-Mesenchymal Transition ,business.industry ,Published Erratum ,Cell Plasticity ,MEDLINE ,Cell Biology ,Cell Movement ,Neoplasms ,Terminology as Topic ,Cancer research ,Animals ,Humans ,Medicine ,Epithelial–mesenchymal transition ,Author Correction ,business ,Molecular Biology ,Developmental Biology - Abstract
Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.
- Published
- 2021
17. Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex
- Author
-
Sekyung Oh, Keith E. Mostov, Joshua H. Lipschutz, Sang Ho Kwon, and Marisa Nacke
- Subjects
0301 basic medicine ,Golgi Apparatus ,CD2AP ,Exosomes ,Medical and Health Sciences ,Biochemistry ,Receptors, G-Protein-Coupled ,Receptors ,Golgi ,LMAN2 ,Internalization ,media_common ,membrane trafficking ,Adaptor Proteins ,Signal transducing adaptor protein ,Biological Sciences ,Cell biology ,symbols ,Additions and Corrections ,extracellular vesicles ,Intracellular ,urinary proteins ,Biochemistry & Molecular Biology ,Active ,Endosome ,media_common.quotation_subject ,Biological Transport, Active ,exosomes ,Biology ,multivesicular ,Exosome ,G-Protein-Coupled ,03 medical and health sciences ,symbols.namesake ,Dogs ,Animals ,Humans ,G protein-coupled receptor ,endosome ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Endoplasmic reticulum ,Signal Transducing ,Membrane Transport Proteins ,Biological Transport ,Cell Biology ,Golgi apparatus ,Microvesicles ,Vesicular Transport ,Adaptor Proteins, Vesicular Transport ,Cytoskeletal Proteins ,Mannose-Binding Lectins ,HEK293 Cells ,030104 developmental biology ,Chemical Sciences ,Generic health relevance - Abstract
Exosomes, 40-150 nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. While exosomes are known to originate from maturation of endosomes into multivesicular endosomes (MVEs; also known as multivesicular bodies, MVBs) with subsequent fusion of the MVEs with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network (TGN)-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum (ER). Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B, in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively.
- Published
- 2016
18. A computational approach to resolve cell level contributions to early glandular epithelial cancer progression.
- Author
-
Sean H. J. Kim, Jayanta Debnath, Keith E. Mostov, Sunwoo Park, and C. Anthony Hunt
- Published
- 2009
- Full Text
- View/download PDF
19. A Qualitative Change in the Transcriptome Occurs after the First Cell Cycle and Coincides with Lumen Establishment during MDCKII Cystogenesis
- Author
-
Robert J. Schmitz, Stephen Dalton, Tianfang Wang, Sang Ho Kwon, Severine Urdy, Keith E. Mostov, Zefu Lu, Shaying Zhao, and Xiao Peng
- Subjects
0301 basic medicine ,Embryology ,Kidney Disease ,1.1 Normal biological development and functioning ,Chromosomal translocation ,02 engineering and technology ,Mitochondrion ,Biology ,Article ,Transcriptome ,03 medical and health sciences ,Underpinning research ,Cell polarity ,Genetics ,lcsh:Science ,Transcriptomics ,Multidisciplinary ,Cell growth ,Cell cycle ,021001 nanoscience & nanotechnology ,Cell biology ,Chromatin ,030104 developmental biology ,lcsh:Q ,0210 nano-technology ,Developmental biology ,Developmental Biology - Abstract
Summary Madin-Darby canine kidney II (MDCKII) cells are widely used to study epithelial morphogenesis. To better understand this process, we performed time course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome that occurs after the first cell cycle and coincides with lumen establishment. This change appears to be linked to translocation of β-catenin, supported by analyses with AVL9- and DENND5A-knockdown clones, and regulation by HNF1B, supported by ATAC-seq study. These findings indicate a qualitative change model for transcriptome remodeling during epithelial morphogenesis, leading to cell proliferation decrease and cell polarity establishment. Furthermore, our study reveals that active mitochondria are retained and chromatin accessibility decreases in 3D cysts but not in 2D polarized cells. This indicates that 3D culture is a better model than 2D culture for studying epithelial morphogenesis., Graphical Abstract, Highlights • The transcriptome switches after the first cell cycle and during MDCKII lumenogenesis • The transcriptome switch is linked to β-catenin translocation and HNF1B activation • Chromatin accessibility decreases during MDCKII cystogenesis • Active mitochondria are maintained in 3D, but not 2D, epithelial morphogenesis, Developmental Biology; Embryology; Transcriptomics
- Published
- 2020
20. Fibroblast-derived HGF drives acinar lung cancer cell polarization through integrin-dependent RhoA-ROCK1 inhibition
- Author
-
Emma Sandilands, Keith E. Mostov, Anirban Datta, and David M. Bryant
- Subjects
0301 basic medicine ,RHOA ,Lung Neoplasms ,Medical Physiology ,Acinar Cell ,0302 clinical medicine ,Cell polarity ,Guanine Nucleotide Exchange Factors ,HGF ,Lung ,Cancer ,0303 health sciences ,rho-Associated Kinases ,Tumor ,biology ,Chemistry ,Hepatocyte Growth Factor ,Integrin beta1 ,Cell Polarity ,Hedgehog signaling pathway ,Cell biology ,Extracellular Matrix ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Met ,Epithelia ,Morphogen ,Signal Transduction ,Biochemistry & Molecular Biology ,Stromal cell ,1.1 Normal biological development and functioning ,Integrin ,Morphogenesis ,Adenocarcinoma of Lung ,Adenocarcinoma ,Article ,Cell Line ,03 medical and health sciences ,Underpinning research ,ROCK1 ,Cell Line, Tumor ,Paracrine Communication ,medicine ,Humans ,Fibroblast ,030304 developmental biology ,Carcinoma, Acinar Cell ,Carcinoma ,RhoA ,Cell Biology ,Fibroblasts ,Repressor Proteins ,030104 developmental biology ,biology.protein ,Biochemistry and Cell Biology ,rhoA GTP-Binding Protein - Abstract
The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate β1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways.
- Published
- 2017
21. Afadin orients cell division to position the tubule lumen in developing renal tubules
- Author
-
Lei, Gao, Zhufeng, Yang, Chitkale, Hiremath, Susan E, Zimmerman, Blake, Long, Paul R, Brakeman, Keith E, Mostov, David M, Bryant, Katherine, Luby-Phelps, and Denise K, Marciano
- Subjects
Male ,Microfilament Proteins ,Epithelial Cells ,Nephrons ,Spindle Apparatus ,Kidney Diseases, Cystic ,Madin Darby Canine Kidney Cells ,Mice ,Dogs ,Kidney Tubules ,Morphogenesis ,Animals ,Female ,Cell Division ,Cells, Cultured ,Research Article - Abstract
In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.
- Published
- 2017
22. Developing renal tubules orient cell division via Afadin to position the tubule lumen
- Author
-
Chitkale Hiremath, Paul Brakeman, Zhufeng Yang, Lei Gao, Keith E. Mostov, David M. Bryant, Denise K. Marciano, Susan E. Zimmerman, Blake Long, and Katherine Luby-Phelps
- Subjects
0301 basic medicine ,Cell division ,Morphogenesis ,Kidney development ,Lumen (anatomy) ,Nephron ,Biology ,Spindle pole body ,Spindle apparatus ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Cell cortex ,medicine ,Molecular Biology ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.
- Published
- 2017
23. A Molecular Switch for the Orientation of Epithelial Cell Polarization
- Author
-
Dennis J. Eastburn, Minji Kim, Andrew J. Ewald, Arend W. Overeem, Wei Yu, David M. Bryant, Keith E. Mostov, Anirban Datta, Julie Roignot, Xiao Peng, and Zena Werb
- Subjects
RHOA ,Medical and Health Sciences ,Epithelium ,Madin Darby Canine Kidney Cells ,Cell membrane ,Extracellular matrix ,chemistry.chemical_compound ,Ezrin ,Cell polarity ,Morphogenesis ,Protein Phosphatase 2 ,RNA, Small Interfering ,Phosphorylation ,rho-Associated Kinases ,biology ,Integrin beta1 ,GTPase-Activating Proteins ,Cell Polarity ,Biological Sciences ,Extracellular Matrix ,Cell biology ,medicine.anatomical_structure ,Transcytosis ,Podocalyxin ,RNA Interference ,Signal Transduction ,Sodium-Hydrogen Exchangers ,Sialoglycoproteins ,1.1 Normal biological development and functioning ,Small Interfering ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Dogs ,Underpinning research ,Protein Kinase C beta ,medicine ,Animals ,Molecular Biology ,Cell Membrane ,Epithelial Cells ,Cell Biology ,Apical membrane ,Phosphoproteins ,Cytoskeletal Proteins ,chemistry ,Focal Adhesion Kinase 1 ,biology.protein ,RNA ,rhoA GTP-Binding Protein ,Developmental Biology - Abstract
SummaryThe formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to downregulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.
- Published
- 2014
24. cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner
- Author
-
Holger Gerhardt, Pavel I. Nedvetsky, Xiaocheng Zhao, Irene M. Aspalter, Fabio Stanchi, Keith E. Mostov, Ross J. Metzger, and Thomas Mathivet
- Subjects
0301 basic medicine ,Notch ,Angiogenesis ,Neovascularization, Physiologic ,Motility ,Biology ,Signal transduction ,Cardiovascular ,Polymerase Chain Reaction ,Medical and Health Sciences ,Retina ,03 medical and health sciences ,Mice ,Cell Movement ,Receptors ,Animals ,Protein kinase A ,Physiologic ,Molecular Biology ,Zebrafish ,Neovascularization ,Pediatric ,Receptors, Notch ,Kinase ,Endothelial Cells ,Vascular development ,Biological Sciences ,biology.organism_classification ,Embryonic stem cell ,Cyclic AMP-Dependent Protein Kinases ,Mice, Mutant Strains ,Cell biology ,Endothelial stem cell ,Mutant Strains ,030104 developmental biology ,Cardiovascular and Metabolic Diseases ,Immunology ,Sprouting ,Research Article ,Signal Transduction ,Developmental Biology - Abstract
cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior.
- Published
- 2016
25. Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity
- Author
-
Keith E. Mostov, Travis R. Ruch, Joanne N. Engel, and David M. Bryant
- Subjects
0301 basic medicine ,rac1 GTP-Binding Protein ,Cell Culture Techniques ,Cell Cycle Proteins ,Biology ,Madin Darby Canine Kidney Cells ,Tight Junctions ,Cell membrane ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Dogs ,Cell Movement ,Cell polarity ,medicine ,Animals ,Guanine Nucleotide Exchange Factors ,Humans ,T-Lymphoma Invasion and Metastasis-inducing Protein 1 ,Phosphatidylinositol ,Molecular Biology ,Epithelial polarity ,Adaptor Proteins, Signal Transducing ,Cell Membrane ,Cell Polarity ,Membrane Proteins ,Cell Biology ,Apical membrane ,Cell biology ,Transport protein ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,Membrane protein ,chemistry ,Phosphatidylinositol 3-kinase signaling ,Brief Reports ,Phosphatidylinositol 3-Kinase ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
The polarity regulator Par3 directs the reorganization of the apical membrane in polarized cells through the action of phosphatidylinositol 3-kinase and the Rac1 exchange factor Tiam1. This mechanism may be exploited by pathogens to bypass the epithelial barrier., Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens.
- Published
- 2016
26. Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25
- Author
-
Naoki Tanimizu, Toshihiro Mitaka, Atsushi Miyajima, Keith E. Mostov, and Kazunori Senga
- Subjects
Morphogenesis ,Biology ,Cell junction ,Epithelium ,Tight Junctions ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Claudin-3 ,Humans ,Cell Interactions ,Claudin-4 ,Claudin ,Molecular Biology ,Transcription factor ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Tight junction ,Quinolinium Compounds ,CLDN3 ,Proteins ,Articles ,Cell Biology ,Cell biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Liver ,030220 oncology & carcinogenesis ,Transcriptome ,Transcription Factors - Abstract
Grainyhead-like 2 (Grhl2) is a transcription factor that regulates the size of the luminal space surrounded by polarized epithelial cells. Grhl2 promotes epithelial barrier function and the formation of large lumen by up-regulating Cldn3, Cldn4, and Rab25. The results reveal a molecular network regulating epithelial lumen formation., During development, epithelial progenitors establish intercellular junctions, including tight junctions (TJs), and form three-dimensional (3D) tissue structures, which are often associated with luminal structures. Here we identify grainyhead-like 2 (Grhl2) as a transcription factor that regulates the size of luminal space surrounded by polarized epithelial cells. We show that HPPL, a liver progenitor cell line, transfected with Grhl2 cDNA forms remarkably larger cysts than the control cells in 3D cultures. We find that Grhl2 up-regulates claudin (Cldn) 3 and Cldn4, and their functions are necessary for the formation of large cysts. Overexpression of Cldn3 alone induces the cyst expansion. In contrast, expression of Cldn4 alone does not induce expansion, as it is not localized at TJs. Of interest, Rab25, another Grhl2 target, not only increases the Cldn4 protein, but also enhances its localization at TJs. Taken together, the results indicate that Grhl2 regulates epithelial morphogenesis through transcriptional up-regulation of Cldn3 and Cldn4, as well as of Rab25, which increases the Cldn4 protein and its localization at TJs. The results reveal a molecular network regulating epithelial lumen formation organized by Grhl2.
- Published
- 2012
27. Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells
- Author
-
Mitsunori Fukuda, David M. Bryant, Natalie Spivak, Ilenia Bernascone, Keith E. Mostov, Alejo E. Rodriguez-Fraticelli, Christiaan L. Slim, Fernando Martín-Belmonte, Anirban Datta, Takao Yasuda, Inmaculada Bañón-Rodríguez, Kitty Young, Silvia Vergarajauregui, Paul Brakeman, and Manuel Gálvez-Santisteban
- Subjects
Fluorescent Antibody Technique ,Biology ,Polymerase Chain Reaction ,Synaptotagmin 1 ,Vesicle tethering ,Article ,Cell Line ,Cell membrane ,03 medical and health sciences ,Synaptotagmins ,0302 clinical medicine ,Cell polarity ,medicine ,Animals ,Humans ,030304 developmental biology ,Epithelial polarity ,0303 health sciences ,Microscopy, Confocal ,Vesicle ,Cell Membrane ,Rab GTPases ,epithelial morphogenesis ,Cell Polarity ,Epithelial Cells ,Cell Biology ,phosphoinositides ,Membrane transport ,Apical membrane ,Microarray Analysis ,synaptotagmin-like proteins ,Cell biology ,medicine.anatomical_structure ,vesicle trafficking ,030217 neurology & neurosurgery ,lumen formation - Abstract
The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.
- Published
- 2012
28. Cyclic AMP regulates formation of mammary epithelial acini in vitro
- Author
-
Pavel I. Nedvetsky, Jayanta Debnath, Sang Ho Kwon, and Keith E. Mostov
- Subjects
Programmed cell death ,Morphogenesis ,Cell Culture Techniques ,Apoptosis ,Acinar Cells ,Biology ,Integrin alpha6 ,Cell Line ,Extracellular matrix ,03 medical and health sciences ,0302 clinical medicine ,Acinus ,medicine ,Cyclic AMP ,Humans ,Protein kinase A ,Mammary Glands, Human ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Cell Biology ,Articles ,Cyclic AMP-Dependent Protein Kinases ,In vitro ,Signaling ,Cell biology ,Extracellular Matrix ,medicine.anatomical_structure ,Proto-Oncogene Proteins c-bcl-2 ,Cell culture ,030220 oncology & carcinogenesis ,Female - Abstract
Cyclic AMP–dependent protein kinase A (PKA) is required for MCF10A mammary epithelial acinus formation in vitro. PKA plays a dual role by facilitating polarization of cells attached to the extracellular matrix and apoptosis of detached cells., Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.
- Published
- 2012
29. p114RhoGEF governs cell motility and lumen formation during tubulogenesis through a ROCK–myosin-II pathway
- Author
-
Andrew J. Ewald, Zena Werb, Annette M. Shewan, Minji Kim, and Keith E. Mostov
- Subjects
RHOA ,Epithelial tube morphogenesis ,Motility ,Biology ,Medical and Health Sciences ,Madin Darby Canine Kidney Cells ,Dogs ,Tube ,Cell Movement ,Myosin ,medicine ,Animals ,Madin–Darby canine kidney cells ,Rho-associated protein kinase ,Migration ,Hepatocyte growth factor ,Myosin Type II ,rho-Associated Kinases ,Cell migration ,Epithelial Cells ,Cell Biology ,Anatomy ,Biological Sciences ,Madin-Darby canine kidney cells ,Cell biology ,Kidney Tubules ,Generic Health Relevance ,biology.protein ,Epithelia ,Rho Guanine Nucleotide Exchange Factors ,Developmental Biology ,Lumen (unit) ,medicine.drug ,Research Article - Abstract
© 2015. Published by The Company of Biologists Ltd. Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here, we utilized tubulogenesis induced by hepatocyte growth factor (HGF) in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of p114RhoGEF (also known as ARHGEF18), a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by HGF. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho kinase (ROCK) or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live-cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a new pathway that regulates directed cell migration and formation of a single lumen during epithelial tube morphogenesis.
- Published
- 2015
30. Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism
- Author
-
Arlinet Kierbel, Keith E. Mostov, Jessica Rossello, Anirban Datta, Paola Lepanto, and David M. Bryant
- Subjects
Host cell membrane ,Pseudomonas aeruginosa ,media_common.quotation_subject ,Immunology ,Biology ,SRC Family Tyrosine Kinase ,medicine.disease_cause ,Microbiology ,Bacterial cell structure ,Cell biology ,LYN ,Interaction with host ,Cell culture ,Virology ,medicine ,Internalization ,media_common - Abstract
Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell-cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)-enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3-enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells.
- Published
- 2011
31. p120 catenin is required for normal renal tubulogenesis and glomerulogenesis
- Author
-
Dennis J. Eastburn, David M. Bryant, Denise K. Marciano, Gerard M.J. Beaudoin, Keith E. Mostov, Chao Zong Lee, Louis F. Reichardt, Ilse Hofmann, Natalie Spivak, and Paul Brakeman
- Subjects
Male ,rho GTP-Binding Proteins ,Delta Catenin ,animal structures ,RHOA ,Kidney Glomerulus ,Kidney development ,Models, Biological ,Cell Line ,Mice ,Cystic kidney disease ,Dogs ,Pregnancy ,Morphogenesis ,medicine ,Polycystic kidney disease ,Animals ,RNA, Small Interfering ,Molecular Biology ,Research Articles ,Cytoskeleton ,Cell Proliferation ,Armadillo Domain Proteins ,Mice, Knockout ,Base Sequence ,biology ,Cadherin ,Cell Polarity ,Catenins ,Nephrons ,Kidney Diseases, Cystic ,Cadherins ,Phosphoproteins ,medicine.disease ,Cell biology ,Kidney Tubules ,Phenotype ,Tubule ,Gene Knockdown Techniques ,Catenin ,biology.protein ,Female ,Erratum ,rhoA GTP-Binding Protein ,Cell Adhesion Molecules ,Developmental Biology - Abstract
Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.
- Published
- 2011
32. Molecular Regulation of Lumen Morphogenesis
- Author
-
Anirban Datta, Keith E. Mostov, and David M. Bryant
- Subjects
0303 health sciences ,Cell signaling ,Body Patterning ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Cellular differentiation ,030302 biochemistry & molecular biology ,Morphogenesis ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Epithelium ,Cell biology ,Extracellular matrix ,03 medical and health sciences ,medicine.anatomical_structure ,Cell polarity ,medicine ,General Agricultural and Biological Sciences ,030304 developmental biology ,Lumen (unit) - Abstract
The asymmetric polarization of cells allows specialized functions to be performed at discrete subcellular locales. Spatiotemporal coordination of polarization between groups of cells allowed the evolution of metazoa. For instance, coordinated apical-basal polarization of epithelial and endothelial cells allows transport of nutrients and metabolites across cell barriers and tissue microenvironments. The defining feature of such tissues is the presence of a central, interconnected luminal network. Although tubular networks are present in seemingly different organ systems, such as the kidney, lung, and blood vessels, common underlying principles govern their formation. Recent studies using in vivo and in vitro models of lumen formation have shed new light on the molecular networks regulating this fundamental process. We here discuss progress in understanding common design principles underpinning de novo lumen formation and expansion.
- Published
- 2011
- Full Text
- View/download PDF
33. Transcriptional Profiling Identifies TNS4 Function in Epithelial Tubulogenesis
- Author
-
Keith E. Mostov, Sang Ho Kwon, and Pavel I. Nedvetsky
- Subjects
Transcription, Genetic ,Green Fluorescent Proteins ,Cell Culture Techniques ,Epithelium ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,Dogs ,0302 clinical medicine ,Downregulation and upregulation ,Tensins ,medicine ,Animals ,Tensin ,STAT3 ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,Agricultural and Biological Sciences(all) ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Hepatocyte Growth Factor ,Gene Expression Profiling ,Microfilament Proteins ,Molecular biology ,Cell biology ,Gene expression profiling ,Kidney Tubules ,medicine.anatomical_structure ,Cell culture ,030220 oncology & carcinogenesis ,biology.protein ,Hepatocyte growth factor ,General Agricultural and Biological Sciences ,medicine.drug - Abstract
Summary Hepatocyte growth factor (HGF) plays central roles in tubulogenesis and metastasis [1–4]. HGF treatment of Madin-Darby canine kidney (MDCK) cells grown as cysts in three-dimensional culture induces tubulogenesis [5, 6], which like most tubulogenic processes proceeds through distinct intermediate phases. Identification of genes associated with these phases is central to understanding the molecular mechanisms of tubulogensis; however, because of inefficient, asynchronous tubule formation, isolating such genes has been unfeasible. Here we developed a synchronous, efficient tubulogenesis system and used time-course transcriptional profiling to identify genes temporally regulated in developmental intermediates. Knockdown (KD) of tensin 4 (TNS4), a particularly highly upregulated gene, leads to a decrease in formation of extensions and tubules, two sequential intermediates in tubulogenesis. Exogenous expression of TNS4 marks invasive cells in an epithelial sheet. A mutation in the SH2 domain of TNS4 prevents the transition from extension formation to invasive migration during tubule formation and leads to increased basal activation of STAT3. Exogenous expression of a constitutively active STAT3 mimics the defect by the mutation. Our study highlights the role of the TNS4-STAT3 axis in epithelial sheet invasion and tubulogenesis.
- Published
- 2011
34. A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor
- Author
-
Scott M. Ulrich, Frédéric Luton, Kirk C. Hansen, Keith E. Mostov, Alma L. Burlingame, David M. Bryant, Dennis J. Eastburn, Tao Su, Marcel Verges, Kevan M. Shokat, Anirban Datta, Department of Anatomy, University of California [San Francisco] (UCSF), University of California-University of California, Department of Biochemistry and Biophysics, Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Cardiovascular Genetics Centre (IdIBGi), Universitat de Girona (UdG), Department of Cellular and Molecular Pharmacology [San Francisco] (CMP), Department of Chemistry, Ithaca College, Department of Pharmaceutical Chemistry, Proteomics Core, and University of Colorado Anschutz [Aurora]
- Subjects
MESH: Signal Transduction ,MAPK/ERK pathway ,MESH: Receptors, Polymeric Immunoglobulin ,MESH: Transcytosis ,MESH: Proto-Oncogene Proteins c-yes ,MESH: Amino Acid Sequence ,Mice ,0302 clinical medicine ,MESH: Animals ,Proto-Oncogene Proteins c-yes ,0303 health sciences ,Receptors, Polymeric Immunoglobulin ,Cell biology ,ErbB Receptors ,Liver ,Transcytosis ,Phosphorylation ,Signal transduction ,Signal Transduction ,MESH: Rats ,Endosome ,Molecular Sequence Data ,MESH: Sequence Alignment ,MESH: Receptor, Epidermal Growth Factor ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Endosomes ,Biology ,Article ,03 medical and health sciences ,Animals ,Humans ,MESH: Immunoglobulin A ,Amino Acid Sequence ,Protein kinase A ,MESH: Mice ,MESH: Protein Kinases ,030304 developmental biology ,MESH: Humans ,MESH: Molecular Sequence Data ,Cell Biology ,Immunoglobulin A ,Rats ,MESH: rab GTP-Binding Proteins ,MESH: Endosomes ,rab GTP-Binding Proteins ,Polymeric immunoglobulin receptor ,Protein Kinases ,Sequence Alignment ,RAB11A ,030217 neurology & neurosurgery ,MESH: Liver - Abstract
International audience; Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes-EGFR-ERK cascade, decreased pIgA-pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA-pIgR transcytosis. Our results reveal a novel Yes-EGFR-ERK-FIP5 signalling network for regulation of pIgA-pIgR transcytosis.
- Published
- 2010
35. Cse1l Is a Negative Regulator of CFTR-Dependent Fluid Secretion
- Author
-
Michel Bagnat, Sara Herbstreith, Jan Huisken, Silvia Curado, Sherif E. Gabriel, Keith E. Mostov, Didier Y.R. Stainier, Adam Navis, and Koroboshka Brand-Arzamendi
- Subjects
medicine.medical_specialty ,congenital, hereditary, and neonatal diseases and abnormalities ,Green Fluorescent Proteins ,Cystic Fibrosis Transmembrane Conductance Regulator ,Genes, Recessive ,medicine.disease_cause ,Cystic fibrosis ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Dogs ,Cellular Apoptosis Susceptibility Protein ,Internal medicine ,Genetic model ,medicine ,Polycystic kidney disease ,Animals ,Homeostasis ,Immunoprecipitation ,Secretion ,Zebrafish ,Mutation ,Microscopy, Confocal ,biology ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Zebrafish Proteins ,biology.organism_classification ,medicine.disease ,Cystic fibrosis transmembrane conductance regulator ,Body Fluids ,Cell biology ,Gastrointestinal Tract ,Endocrinology ,biology.protein ,General Agricultural and Biological Sciences - Abstract
Summary Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8–10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo.
- Published
- 2010
- Full Text
- View/download PDF
36. Pseudomonas aeruginosa -Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium
- Author
-
Joanne N. Engel, Keith E. Mostov, and Iwona Bucior
- Subjects
Immunology ,Plasma protein binding ,Biology ,medicine.disease_cause ,Microbiology ,Bacterial Adhesion ,Epithelium ,Cell Line ,Epithelial Damage ,Dogs ,Organ Culture Techniques ,Cell polarity ,medicine ,Animals ,Humans ,Receptor ,Glycoproteins ,chemistry.chemical_classification ,Cellular Microbiology: Pathogen-Host Cell Molecular Interactions ,Pseudomonas aeruginosa ,Cell Polarity ,Epithelial Cells ,Cell biology ,carbohydrates (lipids) ,Infectious Diseases ,medicine.anatomical_structure ,chemistry ,Cell culture ,Parasitology ,Heparitin Sulfate ,Glycoprotein ,Protein Binding - Abstract
Pseudomonas aeruginosa , an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa -mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa .
- Published
- 2010
37. STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis
- Author
-
Keith E. Mostov, Lucy Erin O'Brien, Sang Ho Kwon, and Minji Kim
- Subjects
Cell Physiology ,Epithelial-Mesenchymal Transition ,Cellular differentiation ,Organogenesis ,Green Fluorescent Proteins ,Immunoblotting ,Cell Culture Techniques ,Kidney ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Dogs ,medicine ,Animals ,STAT1 ,Epithelial–mesenchymal transition ,Phosphorylation ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Hepatocyte Growth Factor ,Reverse Transcriptase Polymerase Chain Reaction ,Tyrosine phosphorylation ,Cell Differentiation ,Epithelial Cells ,Cell Biology ,Articles ,Molecular biology ,Cell biology ,Enzyme Activation ,Proto-Oncogene Proteins c-raf ,Tamoxifen ,Kidney Tubules ,STAT1 Transcription Factor ,chemistry ,Cell culture ,030220 oncology & carcinogenesis ,Mutation ,biology.protein ,Tyrosine ,Hepatocyte growth factor ,RNA Interference ,Signal transduction ,medicine.drug ,Protein Binding - Abstract
Signal transducers and activators of transcription (STAT)1 is the key to the sequential control of Madin-Darby canine kidney tubulogenesis. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 is sufficient to restore cord formation but not mature lumens. These data suggest that STAT1 is necessary for the redifferentiation phase of tubulogenesis and that mature lumenogenesis requires a distinct signal., Tubule formation in vitro using Madin-Darby canine kidney (MDCK) epithelial cells consists mainly of two processes. First, the cells undergo a partial epithelial–mesenchymal transition (pEMT), losing polarity and migrating. Second, the cells redifferentiate, forming cords and then tubules with continuous lumens. We have shown previously that extracellular signal-regulated kinase activation is required for pEMT. However, the mechanism of how the pEMT phase is turned off and the redifferentiation phase is initiated is largely unknown. To address the central question of the sequential control of these two phases, we used MDCK cells grown as cysts and treated with hepatocyte growth factor to model tubulogenesis. We show that signal transducer and activator of transcription (STAT)1 controls the sequential progression from the pEMT phase to the redifferentiation phase. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 allows redifferentiation to occur even when cells are otherwise prevented from progressing beyond the pEMT phase by exogenous activation of Raf. Moreover, tyrosine phosphorylation defective STAT1 partially restored cord formation in such cells, suggesting that STAT1 functions in part as nonnuclear protein mediating signal transduction in this process. Constitutively active or inactive forms of STAT1 did not promote lumen maturation, suggesting this requires a distinct signal.
- Published
- 2010
38. Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity
- Author
-
Mirjam M. P. Zegers, Qi-Wen Fan, David M. Bryant, William A. Weiss, Dennis J. Eastburn, Annette M. Shewan, Keith E. Mostov, Wei Yu, Paul Brakeman, and Anirban Datta
- Subjects
RHOA ,Blotting, Western ,Scientific Report ,Biology ,Transfection ,Biochemistry ,Cell Line ,Extracellular matrix ,Small hairpin RNA ,Cell polarity ,Myosin ,Genetics ,Animals ,Humans ,RNA, Small Interfering ,Molecular Biology ,Rho-associated protein kinase ,Epithelial polarity ,Myosin Type II ,rho-Associated Kinases ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Polarity ,Epithelial Cells ,Cell biology ,Cell culture ,biology.protein ,rhoA GTP-Binding Protein - Abstract
In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.
- Published
- 2008
39. Regulation of cell polarity during epithelial morphogenesis
- Author
-
Fernando Martín-Belmonte and Keith E. Mostov
- Subjects
Vesicle ,Morphogenesis ,Cell Polarity ,Cell Biology ,Biology ,Models, Biological ,Epithelium ,Cell biology ,Extracellular matrix ,chemistry.chemical_compound ,Membrane ,chemistry ,Cell polarity ,Animals ,Humans ,Phosphatidylinositol ,Lumen (unit) ,Epithelial polarity - Abstract
Epithelial cells have an apical surface facing a lumen or outside of the organism, and a basolateral surface facing other cells and extracellular matrix. The identity of the apical surface is determined by phosphatidylinositol 4,5-bisphosphate, while phosphatidylinositol 3,4,5-trisphophosphate determines the identity of the basolateral surface. The Par3/Par6/atypical protein kinase C complex, as well as the Crumbs and Scribble complexes, controls epithelial polarity. Par4 and AMP kinase regulate polarity during conditions of energy depletion. Lumens are formed in hollow cysts and tubules by fusions of apical vesicles, such as the vacuolar apical compartment, with the plasma membrane. The polarity of individual cells is oriented and coordinated with other cells by interactions with the extracellular matrix.
- Published
- 2008
40. Phosphoinositides Control Epithelial Development
- Author
-
Fernando Martín-Belmonte and Keith E. Mostov
- Subjects
Phosphatidylinositol 4,5-Diphosphate ,Scaffold protein ,Cellular polarity ,Cell Polarity ,Cell Biology ,CDC42 ,GTPase ,Biology ,Apical membrane ,Phosphatidylinositols ,Models, Biological ,Epithelium ,Cell biology ,chemistry.chemical_compound ,chemistry ,Cell polarity ,Animals ,Humans ,Phosphatidylinositol ,cdc42 GTP-Binding Protein ,Molecular Biology ,Annexin A2 ,Developmental Biology ,Epithelial polarity - Abstract
Epithelial organs consist on layers of cubical cells that separate different compartments. They form a physical barrier that allows the regulated transports of certain molecules and ions. To perform this and other functions epithelial cells require to be highly polarized. The molecular mechanisms that integrate cellular polarity with epithelial architecture are poorly understood. Using a three-dimensional model of epithelial morphogenesis, we have recently reported a molecular mechanism for the formation of the apical membrane and the central lumen.(1) This molecular pathway is initiated by the membrane segregation of phosphoinositides at the apical domain. Apically localized phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] recruits the scaffolding protein Annexin2 and the GTPase Cdc42 to generate the apical plasma membrane domain and the central lumen.
- Published
- 2007
41. Polarity proteins PAR6 and aPKC regulate cell death through GSK-3β in 3D epithelial morphogenesis
- Author
-
Minji Kim, Paul Brakeman, Wei Yu, Anirban Datta, and Keith E. Mostov
- Subjects
Programmed cell death ,Glycogen Synthase Kinase 3 beta ,Morphogenesis ,Cell Polarity ,Gene Expression ,Apoptosis ,Epithelial Cells ,Cell Biology ,Biology ,Cell Line ,Cell biology ,Glycogen Synthase Kinase 3 ,Dogs ,GSK-3 ,Cell culture ,Caspases ,Cell polarity ,Animals ,Ectopic expression ,GSK3B ,Protein Kinase C ,Adaptor Proteins, Signal Transducing - Abstract
Epithelial cells are polarized, with an apical surface facing a lumen or outer surface and a basolateral surface facing other cells and extracellular matrix (ECM). Hallmarks of epithelial carcinogenesis include loss of polarity, as well as uncontrolled proliferation and resistance to apoptosis. Are these features controlled by a common molecular mechanism? The partitioning-defective 3 (PAR3)-PAR6-atypical PKC (aPKC) complex is a master regulator that controls polarization in many animal cells. Here we show that PAR6 is involved in apoptosis by regulating aPKC and glycogen synthase kinase 3beta (GSK-3beta) activity. During epithelial morphogenesis in 3D culture of Madin-Darby canine kidney (MDCK) cells, expression of an N-terminally deleted PAR6 (PAR6DeltaN) leads to a significant increase in caspase-dependent cell death by downregulating aPKC activity. Accordingly, inhibition of aPKC in wild-type (WT) MDCK cells with either a cell-permeable PKCzeta pseudosubstrate or RNAi promotes apoptosis, which suggests that PAR6 regulates apoptosis via an aPKC-mediated pathway. GSK-3beta, a substrate of aPKC, is hyper-activated by expressing PAR6DeltaN. GSK-3beta inhibitors block PAR6DeltaN-induced apoptosis while expression of constitutively active GSK-3beta (S9A) promotes apoptosis, which is rescued by ectopic expression of aPKC. We conclude that a PAR6-aPKC-GSK-3beta mechanism links cell polarity and apoptosis.
- Published
- 2007
42. EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin
- Author
-
Shannon R. Joseph, Luke A. Hammond, Markus C. Kerr, Jennifer L. Stow, David M. Bryant, Keith E. Mostov, and Rohan D. Teasdale
- Subjects
rac1 GTP-Binding Protein ,Endosome ,media_common.quotation_subject ,education ,Vesicular Transport Proteins ,Endosomes ,Biology ,Cell Line, Tumor ,Cell polarity ,Humans ,RNA, Small Interfering ,Macropinosome ,Transport Vesicles ,Internalization ,Sorting Nexins ,media_common ,Epidermal Growth Factor ,Cadherin ,Pinocytosis ,Membrane Proteins ,Epithelial Cells ,Cell Biology ,Cadherins ,Endocytosis ,Cell biology ,Protein Transport ,Sorting nexin ,Intercellular Junctions ,Intracellular ,Signal Transduction - Abstract
In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling.
- Published
- 2007
43. Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells
- Author
-
Joanne N. Engel, Arlinet Kierbel, Keith E. Mostov, Fernando Martín-Belmonte, Martin B.A. ter Beest, Ama Gassama-Diagne, and Wei Yu
- Subjects
Polarity (international relations) ,Phosphatidylinositol (3,4,5)-trisphosphate ,Cell Membrane ,Cell Polarity ,Membrane Proteins ,Epithelial Cells ,Cell Biology ,Basolateral plasma membrane ,Biology ,Fusion protein ,Cell Line ,Green fluorescent protein ,Cell biology ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Dogs ,Membrane ,Phosphatidylinositol Phosphates ,chemistry ,Mutation ,Animals ,Cell Surface Extensions ,Phosphatidylinositol ,Protein kinase B ,Phosphoinositide-3 Kinase Inhibitors - Abstract
Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.
- Published
- 2006
44. The Role of Syntaxins in the Specificity of Vesicle Targeting in Polarized Epithelial Cells
- Author
-
Steven J. Chapin, Keith E. Mostov, Dana Avrahami, and Martin B.A. ter Beest
- Subjects
endocrine system ,Qa-SNARE Proteins ,urogenital system ,Cell Polarity ,Epithelial Cells ,Articles ,Cell Biology ,Transfection ,Biology ,environment and public health ,Syntaxin 3 ,Cell Line ,Cell biology ,Chimera (genetics) ,Dogs ,Secretory protein ,Membrane ,nervous system ,Cell culture ,Cell polarity ,Animals ,Syntaxin ,biological phenomena, cell phenomena, and immunity ,Molecular Biology - Abstract
In polarized epithelial cells syntaxin 3 is at the apical plasma membrane and is involved in delivery of proteins from the trans-Golgi network to the apical surface. The highly related syntaxin 4 is at the basolateral surface. The complementary distribution of these syntaxins suggests that they play a role in the specificity of membrane traffic to the two surfaces. We constructed a chimeric syntaxin where we removed the N-terminal 29 residues of syntaxin 3 and replaced it with the corresponding portion of syntaxin 4. When expressed in polarized epithelial cells, this chimera was exclusively localized to the basolateral surface. This indicates that the N-terminal domain of syntaxin 3 contains information for its polarized localization. In contrast to the apical localization of syntaxin 3, the basolateral localization of syntaxin 4 was not dependent on its N-terminal domain. Syntaxin 3 normally binds to Munc18b, but not to the related Munc18c. Overexpression of the chimera together with overexpression of Munc18b caused membrane and secretory proteins that are normally sent primarily to the apical surface to exhibit increased delivery to the basolateral surface. We suggest that syntaxins may play a role in determining the specificity of membrane targeting by permitting fusion with only certain target membranes.
- Published
- 2005
45. The Phosphoinositol-3-Kinase–Protein Kinase B/Akt Pathway Is Critical forPseudomonas aeruginosaStrain PAK Internalization
- Author
-
Joanne N. Engel, Keith E. Mostov, Arlinet Kierbel, and Ama Gassama-Diagne
- Subjects
media_common.quotation_subject ,macromolecular substances ,Protein Serine-Threonine Kinases ,Biology ,Cell Line ,Wortmannin ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Dogs ,Phosphatidylinositol Phosphates ,Proto-Oncogene Proteins ,Animals ,Humans ,LY294002 ,Phosphatidylinositol ,RNA, Small Interfering ,Internalization ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,media_common ,Kinase ,Epithelial Cells ,Articles ,Cell Biology ,Molecular biology ,Cell biology ,Enzyme Activation ,chemistry ,Pseudomonas aeruginosa ,Signal transduction ,Proto-Oncogene Proteins c-akt ,HeLa Cells ,Signal Transduction - Abstract
Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.
- Published
- 2005
46. Epithelial Cell Polarity Alters Rho-GTPase Responses toPseudomonas aeruginosa
- Author
-
Keith E. Mostov, Joanne N. Engel, and B. I. Kazmierczak
- Subjects
rho GTP-Binding Proteins ,RHOA ,Bacterial Toxins ,Clostridium difficile toxin B ,GTPase ,Biology ,medicine.disease_cause ,Tight Junctions ,Dogs ,Cell polarity ,medicine ,Animals ,Humans ,cdc42 GTP-Binding Protein ,Molecular Biology ,Cells, Cultured ,Actin ,Epithelial polarity ,Clostridioides difficile ,Pseudomonas aeruginosa ,Cell Polarity ,Epithelial Cells ,Articles ,Cell Biology ,Actins ,Epithelium ,Cell biology ,medicine.anatomical_structure ,biology.protein ,rhoA GTP-Binding Protein ,HeLa Cells - Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.
- Published
- 2004
47. An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling
- Author
-
Tzu-Ling Cheng, Shin-Hun Juang, Hsin-Yuan Cheng, Keith E. Mostov, Jen-Feng Lai, Tzuu-Shuh Jou, and Yi-Mei Hung
- Subjects
Proteomics ,rac1 GTP-Binding Protein ,Ecdysone ,Physiology ,Green Fluorescent Proteins ,RAC1 ,Biology ,Kidney ,Green fluorescent protein ,chemistry.chemical_compound ,Genes, Reporter ,Gene expression ,Animals ,Humans ,Anoikis ,Small GTPase ,Molecular Biology ,Gene ,Cell Biology ,Flow Cytometry ,Cell biology ,Luminescent Proteins ,Gene Expression Regulation ,chemistry ,Tetracyclines ,Indicators and Reagents ,Function (biology) ,Signal Transduction - Abstract
Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.
- Published
- 2003
48. Polarized epithelial membrane traffic: conservation and plasticity
- Author
-
Martin B.A. ter Beest, Tao Su, and Keith E. Mostov
- Subjects
Membrane Traffic ,Cellular differentiation ,Cell Membrane ,Cell Polarity ,Epithelial Cells ,Cell Communication ,Cell Biology ,Plasticity ,Biology ,Endocytosis ,Cell biology ,Protein Transport ,Animals ,Humans ,Adaptation ,Transport Vesicles - Abstract
Most cells are polarized and have distinct plasma membrane domains, which are the result of polarized trafficking of proteins and lipids. Great progress has been made in elucidating the highly conserved polarized targeting machinery. A pre-eminent challenge now is to understand the plasticity of polarized traffic, how it is altered by differentiation and dedifferentiation during development, as well as the adaptation of differentiated cells to meet changing physiological needs.
- Published
- 2003
49. Parasympathetic innervation regulates tubulogenesis in the developing salivary gland
- Author
-
Sarah M. Knox, Andreas Ettinger, Keith E. Mostov, Emily Northrup, Elaine Emmerson, Pavel I. Nedvetsky, Matthew P. Hoffman, Candace L. Haddox, Jennifer K. Finley, Craig A. Hodges, Noel Cruz-Pacheco, and Jan Prochazka
- Subjects
medicine.medical_specialty ,Organogenesis ,1.1 Normal biological development and functioning ,Vasoactive intestinal peptide ,Lumen (anatomy) ,Cystic Fibrosis Transmembrane Conductance Regulator ,Apoptosis ,Medical and Health Sciences ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Underpinning research ,Internal medicine ,medicine ,Cyclic AMP ,Salivary Ducts ,Animals ,Protein kinase A ,Molecular Biology ,Tubular network ,Mice, Inbred ICR ,biology ,Salivary gland ,Parasympathetic ,Ganglia, Parasympathetic ,Epithelial Cells ,Cell Biology ,Biological Sciences ,Inbred ICR ,Cyclic AMP-Dependent Protein Kinases ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Endocrinology ,medicine.anatomical_structure ,biology.protein ,Ganglia ,Digestive Diseases ,Vasoactive Intestinal Peptide ,Developmental Biology - Abstract
SummaryA fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(−) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.
- Published
- 2014
50. Phosphoinositide 3-kinase p110δ promotes lumen formation through the enhancement of apico-basal polarity and basal membrane organization
- Author
-
Amel Rayal, Juan Peng, Ama Gassama-Diagne, Romina Moyano, Didier Samuel, Keith E. Mostov, Aline Awad, Sokhavuth Sar, Bart Vanhaesebroeck, Ola Hamze Komaiha, and Annette M. Shewan
- Subjects
Epithelial cell morphogenesis ,Molecular Sequence Data ,General Physics and Astronomy ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Madin Darby Canine Kidney Cells ,Cell membrane ,Extracellular matrix ,Focal adhesion ,Phosphatidylinositol 3-Kinases ,Dogs ,Laminin ,Cell polarity ,medicine ,Cell Adhesion ,Animals ,Humans ,Protein Isoforms ,RNA, Small Interfering ,Cell adhesion ,Epithelial polarity ,Multidisciplinary ,Base Sequence ,Cell Membrane ,Cell Polarity ,Epithelial Cells ,General Chemistry ,Vinculin ,Cell biology ,Extracellular Matrix ,Class Ia Phosphatidylinositol 3-Kinase ,medicine.anatomical_structure ,Microscopy, Fluorescence ,biology.protein ,Collagen ,Paxillin ,HeLa Cells ,Signal Transduction - Abstract
Signalling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signalling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin-Darby canine kidney epithelial cells grown in 3D culture, we show that the p110δ isoform of phosphoinositide 3-kinase co-localizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead-mediated inhibition of p110δ impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110δ also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110δ in epithelial cells in the orientation of the apico-basal axis and lumen formation.
- Published
- 2014
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.