scholarly journals In vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration

2021 ◽  
Author(s):  
Mira I. Pronobis ◽  
Susan Zheng ◽  
Sumeet P. Singh ◽  
Kenneth D. Poss
Keyword(s):  
Cell Types ◽  
Myocardial Cell ◽  
Protein Networks ◽  
Transgenic Zebrafish ◽  
Heart Regeneration ◽  
Interacting Protein ◽  
Membrane Compartment ◽  
Rho A ◽  
Erbb2 Signaling

AbstractStrategies have not been available until recently to uncover interacting protein networks specific to key cell types, their subcellular compartments, and their major regulators during complex in vivo events. Here we apply BioID2 proximity labeling to capture protein networks acting within cardiomyocytes during a key model of innate heart regeneration in zebrafish. Transgenic zebrafish expressing a promiscuous BirA2 localized to the entire myocardial cell or membrane compartment were generated, each identifying unique proteomes in adult cardiomyocytes that became altered during regeneration. BioID2 profiling for interactors with ErbB2, a co-receptor for the cardiomyocyte mitogen Nrg1, implicated Rho A as a target of ErbB2 signaling in cardiomyocytes. Blockade of Rho A during heart regeneration, or during cardiogenic stimulation by the mitogenic influences Nrg1, Vegfaa or Vitamin D, disrupted muscle creation. Our findings reveal proximity labeling as a useful resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish.

scholarly journals In vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mira I Pronobis ◽  
Susan Zheng ◽  
Sumeet Pal Singh ◽  
Joseph A Goldman ◽  
Kenneth D Poss
Keyword(s):  
Cell Types ◽  
Myocardial Cell ◽  
Protein Networks ◽  
Transgenic Zebrafish ◽  
Heart Regeneration ◽  
Interacting Protein ◽  
Membrane Compartment ◽  
Rho A ◽  
Erbb2 Signaling

Strategies have not been available until recently to uncover interacting protein networks specific to key cell types, their subcellular compartments, and their major regulators during complex in vivo events. Here, we apply BioID2 proximity labeling to capture protein networks acting within cardiomyocytes during a key model of innate heart regeneration in zebrafish. Transgenic zebrafish expressing a promiscuous BirA2 localized to the entire myocardial cell or membrane compartment were generated, each identifying distinct proteomes in adult cardiomyocytes that became altered during regeneration. BioID2 profiling for interactors with ErbB2, a co-receptor for the cardiomyocyte mitogen Nrg1, implicated Rho A as a target of ErbB2 signaling in cardiomyocytes. Blockade of Rho A during heart regeneration, or during cardiogenic stimulation by the mitogenic influences Nrg1, Vegfaa, or vitamin D, disrupted muscle creation. Our findings reveal proximity labeling as a useful resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish.

scholarly journals Using zebrafish to elucidate glial-vascular interactions during CNS development

2021 ◽  
Author(s):  
Robyn A. Umans ◽  
Carolyn Pollock ◽  
William A. Mills ◽  
Harald Sontheimer
Keyword(s):  
Blood Vessels ◽  
Cell Types ◽  
Transgenic Zebrafish ◽  
Suitable Model ◽  
Developmental Studies ◽  
Brain Vessels ◽  
Brain Vasculature ◽  
Area Of Interest ◽  
Vessel Development

AbstractAn emerging area of interest in Neuroscience is the cellular relationship between glia and blood vessels, as many of the presumptive support roles of glia require an association with the vasculature. These interactions are best studied in vivo and great strides have been made using mice to longitudinally image glial-vascular interactions. However, these methods are cumbersome for developmental studies, which could benefit from a more accessible system. Zebrafish (Danio rerio) are genetically tractable vertebrates, and given their translucency, are readily amenable for daily live imaging studies. We set out to examine whether zebrafish glia have conserved traits with mammalian glia regarding their ability to interact with and maintain the developing brain vasculature. We utilized transgenic zebrafish strains in which oligodendrocyte transcription factor 2 (olig2) and glial fibrillary acidic protein (gfap) identify different glial populations in the zebrafish brain and document their corresponding relationship with brain blood vessels. Our results demonstrate that olig2 and gfap zebrafish glia have distinct lineages and each interact with brain vessels as previously observed in mouse brain. Additionally, we manipulated these relationships through pharmacological and genetic approaches to distinguish the roles of these cell types during blood vessel development. olig2 glia use blood vessels as a pathway during their migration and Wnt signaling inhibition decreases their single-cell vessel co-option. By contrast, the ablation of gfap glia at the beginning of CNS angiogenesis impairs vessel development through a reduction in Vascular endothelial growth factor (Vegf), supporting a role for gfap glia during new brain vessel formation in zebrafish. This data suggests that zebrafish glia, akin to mammalian glia, have different lineages that show diverse interactions with blood vessels, and are a suitable model for elucidating glial-vascular relationships during vertebrate brain development.

scholarly journals A novel Rho GTPase-activating-protein interacts with Gem, a member of the Ras superfamily of GTPases

2002 ◽  
Vol 367 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Sandra ARESTA ◽  
Marie-France de TAND-HEIM ◽  
Florence BÉRANGER ◽  
Jean de GUNZBURG
Keyword(s):  
Rho Gtpase ◽  
Cell Types ◽  
Gtpase Activating Protein ◽  
Interacting Protein ◽  
Rich Domain ◽  
Ras Superfamily ◽  
Extracellular Stimuli ◽  
Rat Fibroblasts

Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half. The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo. The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts. These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein).

scholarly journals RIP1/RIP3/MLKL-mediated necroptosis contributes to vinblastine-induced myocardial damage

2020 ◽  
Author(s):  
Huiling Zhou ◽  
Lijun Liu ◽  
Xiaolong Ma ◽  
Jian Wang ◽  
Jinfu Yang ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Cell Damage ◽  
Animal Experiments ◽  
Myocardial Damage ◽  
Myocardial Cell ◽  
Neonatal Rat ◽  
H9c2 Cells ◽  
Interacting Protein

AbstractVinblastine (VBL) has been considered as a first-line anti-tumor drug for many years. However, vinblastine-caused myocardial damage has been continually reported. The underlying molecular mechanism of the myocardial damage remains unknown. Here, we show that vinblastine induces myocardial damage and necroptosis is involved in the vinblastine-induced myocardial damage both in vitro and in vivo. The results of WST-8 and flow cytometry analysis show that vinblastine causes damage to H9c2 cells, and the results of animal experiments show that vinblastine causes myocardial cell damage. The necrosome components, receptor-interacting protein 1 (RIP1) receptor-interacting protein 3 (RIP3), are significantly increased in vinblastine-treated H9c2 cells, primary neonatal rat ventricular myocytes and rat heart tissues. And the downstream substrate of RIP3, mixed lineage kinase domain like protein (MLKL) was also increased. Pre-treatment with necroptosis inhibitors partially inhibits the necrosome components and MLKL levels and alleviates vinblastine-induced myocardial injury both in vitro and in vivo. This study indicates that necroptosis participated in vinblastine-evoked myocardial cell death partially, which would be a potential target for relieving the chemotherapy-related myocardial damage.

scholarly journals Endoglin regulates PI3-kinase/Akt trafficking and signaling to alter endothelial capillary stability during angiogenesis

2012 ◽  
Vol 23 (13) ◽  
pp. 2412-2423 ◽  
Author(s):  
Nam Y. Lee ◽  
Christelle Golzio ◽  
Catherine E. Gatza ◽  
Arun Sharma ◽  
Nicholas Katsanis ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Cell Function ◽  
Capillary Tube ◽  
Transforming Growth Factor Β ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Interacting Protein ◽  
Endothelial Cell Function

Endoglin (CD105) is an endothelial-specific transforming growth factor β (TGF-β) coreceptor essential for angiogenesis and vascular homeostasis. Although endoglin dysfunction contributes to numerous vascular conditions, the mechanism of endoglin action remains poorly understood. Here we report a novel mechanism in which endoglin and Gα-interacting protein C-terminus–interacting protein (GIPC)–mediated trafficking of phosphatidylinositol 3-kinase (PI3K) regulates endothelial signaling and function. We demonstrate that endoglin interacts with the PI3K subunits p110α and p85 via GIPC to recruit and activate PI3K and Akt at the cell membrane. Opposing ligand-induced effects are observed in which TGF-β1 attenuates, whereas bone morphogenetic protein-9 enhances, endoglin/GIPC-mediated membrane scaffolding of PI3K and Akt to alter endothelial capillary tube stability in vitro. Moreover, we employ the first transgenic zebrafish model for endoglin to demonstrate that GIPC is a critical component of endoglin function during developmental angiogenesis in vivo. These studies define a novel non-Smad function for endoglin and GIPC in regulating endothelial cell function during angiogenesis.

scholarly journals Using Zebrafish to Elucidate Glial-Vascular Interactions During CNS Development

2021 ◽  
Vol 9 ◽  
Author(s):  
Robyn A. Umans ◽  
Carolyn Pollock ◽  
William A. Mills ◽  
Kareem C. Clark ◽  
Y. Albert Pan ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Cell Types ◽  
Transgenic Zebrafish ◽  
Suitable Model ◽  
Developmental Studies ◽  
Brain Vessels ◽  
Brain Vasculature ◽  
Area Of Interest ◽  
Vessel Development

An emerging area of interest in Neuroscience is the cellular relationship between glia and blood vessels, as many of the presumptive support roles of glia require an association with the vasculature. These interactions are best studied in vivo and great strides have been made using mice to longitudinally image glial-vascular interactions. However, these methods are cumbersome for developmental studies, which could benefit from a more accessible system. Zebrafish (Danio rerio) are genetically tractable vertebrates, and given their translucency, are readily amenable for daily live imaging studies. We set out to examine whether zebrafish glia have conserved traits with mammalian glia regarding their ability to interact with and maintain the developing brain vasculature. We utilized transgenic zebrafish strains in which oligodendrocyte transcription factor 2 (olig2) and glial fibrillary acidic protein (gfap) identify different glial populations in the zebrafish brain and document their corresponding relationship with brain blood vessels. Our results demonstrate that olig2+ and gfap+ zebrafish glia have distinct lineages and each interact with brain vessels as previously observed in mouse brain. Additionally, we manipulated these relationships through pharmacological and genetic approaches to distinguish the roles of these cell types during blood vessel development. olig2+ glia use blood vessels as a pathway during their migration and Wnt signaling inhibition decreases their single-cell vessel co-option. By contrast, the ablation of gfap+ glia at the beginning of CNS angiogenesis impairs vessel development through a reduction in Vascular endothelial growth factor (Vegf), supporting a role for gfap+ glia during new brain vessel formation in zebrafish. This data suggests that zebrafish glia, akin to mammalian glia, have different lineages that show diverse interactions with blood vessels, and are a suitable model for elucidating glial-vascular relationships during vertebrate brain development.

scholarly journals In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish

2020 ◽  
Author(s):  
Zherui Xiong ◽  
Harriet P. Lo ◽  
Kerrie-Ann McMahon ◽  
Nick Martel ◽  
Alun Jones ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Vascular Endothelial Cells ◽  
Specific Interaction ◽  
Cell Types ◽  
Interaction Networks ◽  
Transgenic Zebrafish ◽  
Proteomic Profiling ◽  
Cellular Processes ◽  
Biotin Labelling

AbstractProtein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.

scholarly journals In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Zherui Xiong ◽  
Harriet P Lo ◽  
Kerrie-Ann McMahon ◽  
Nick Martel ◽  
Alun Jones ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Vascular Endothelial Cells ◽  
Specific Interaction ◽  
Cell Types ◽  
Interaction Networks ◽  
Transgenic Zebrafish ◽  
Proteomic Profiling ◽  
Cellular Processes ◽  
Biotin Labelling

Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here, we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.

scholarly journals Zebrafish cardiac regeneration—looking beyond cardiomyocytes to a complex microenvironment

2020 ◽  
Author(s):  
Rebecca Ryan ◽  
Bethany R. Moyse ◽  
Rebecca J. Richardson
Keyword(s):  
Cardiac Injury ◽  
Cell Communication ◽  
Cardiac Regeneration ◽  
Cell Types ◽  
Regenerative Process ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Underlying Mechanisms ◽  
Different Cell Types

Abstract The study of heart repair post-myocardial infarction has historically focused on the importance of cardiomyocyte proliferation as the major factor limiting adult mammalian heart regeneration. However, there is mounting evidence that a narrow focus on this one cell type discounts the importance of a complex cascade of cell–cell communication involving a whole host of different cell types. A major difficulty in the study of heart regeneration is the rarity of this process in adult animals, meaning a mammalian template for how this can be achieved is lacking. Here, we review the adult zebrafish as an ideal and unique model in which to study the underlying mechanisms and cell types required to attain complete heart regeneration following cardiac injury. We provide an introduction to the role of the cardiac microenvironment in the complex regenerative process and discuss some of the key advances using this in vivo vertebrate model that have recently increased our understanding of the vital roles of multiple different cell types. Due to the sheer number of exciting studies describing new and unexpected roles for inflammatory cell populations in cardiac regeneration, this review will pay particular attention to these important microenvironment participants.

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

Sign in / Sign up

Export Citation Format

Share Document