Comprehensive profiling of mRNA polyadenylation in specific cell types in vivo by cTag-PAPERCLIP

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

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Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽

10.3390/cells10010066 ◽

2021 ◽

Vol 10 (1) ◽

pp. 66

Author(s):

Rashmita Pradhan ◽

Phuong A. Ngo ◽

Luz d. C. Martínez-Sánchez ◽

Markus F. Neurath ◽

Rocío López-Posadas

Keyword(s):

Intestinal Mucosa ◽

Rho Gtpases ◽

Current Knowledge ◽

Cell Types ◽

Effector Proteins ◽

Special Focus ◽

Specific Cell ◽

Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

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MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816656115 ◽

2018 ◽

Vol 116 (1) ◽

pp. 303-312 ◽

Cited By ~ 24

Author(s):

Erol C. Bayraktar ◽

Lou Baudrier ◽

Ceren Özerdem ◽

Caroline A. Lewis ◽

Sze Ham Chan ◽

...

Keyword(s):

Metabolite Profiling ◽

Cultured Cells ◽

Transgenic Animals ◽

Cell Types ◽

Tissue Level ◽

Specific Cell ◽

Mammalian Tissues ◽

Cell Type Specific ◽

Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

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MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

10.1101/425454 ◽

2018 ◽

Author(s):

Erol Can Bayraktar ◽

Lou Baudrier ◽

Ceren Özerdem ◽

Caroline A. Lewis ◽

Sze Ham Chan ◽

...

Keyword(s):

Metabolite Profiling ◽

Cultured Cells ◽

Transgenic Animals ◽

Cell Types ◽

Tissue Level ◽

Specific Cell ◽

Mammalian Tissues ◽

Cell Type Specific ◽

Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

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In vivotissue-specific chromatin profiling inDrosophila melanogasterusing GFP-tagged nuclei

10.1101/2021.03.23.436625 ◽

2021 ◽

Author(s):

Juan Jauregui-Lozano ◽

Kimaya Bakhle ◽

Vikki M. Weake

Keyword(s):

Cell Types ◽

Chromatin Accessibility ◽

Chromatin State ◽

Specific Cell ◽

Histone Marks ◽

The Many ◽

Chromatin Profiling ◽

Photoreceptor Neurons ◽

Multicellular Organisms

AbstractThe chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasisin vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolateDrosophilanuclei tagged with GFP expressed under Gal4/UAS control. Using this protocol, we profiled chromatin accessibility using Omni-ATAC, and examined the distribution of histone marks using ChIP-seq and CUT&Tag in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types inDrosophila.

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Chemogenetic Tools for Causal Cellular and Neuronal Biology

Physiological Reviews ◽

10.1152/physrev.00009.2017 ◽

2018 ◽

Vol 98 (1) ◽

pp. 391-418 ◽

Cited By ~ 23

Author(s):

Deniz Atasoy ◽

Scott M. Sternson

Keyword(s):

G Protein ◽

Ex Vivo ◽

Cell Types ◽

Cell Populations ◽

Specific Cell ◽

Cellular Processes ◽

Distinct Cell ◽

G Protein Coupled ◽

Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

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Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch

Development ◽

10.1242/dev.121.11.3637 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3637-3650 ◽

Cited By ~ 33

Author(s):

C.P. Austin ◽

D.E. Feldman ◽

J.A. Ida ◽

C.L. Cepko

Keyword(s):

Cell Fate ◽

Ganglion Cells ◽

Notch Pathway ◽

Cell Types ◽

Projection Neurons ◽

Specific Cell ◽

Vitro Assay ◽

Notch Activity

The first cells generated during development of the vertebrate retina are the ganglion cells, the projection neurons of the retina. Although they are one of the most intensively studied cell types within the central nervous system, little is known of the mechanisms that determine ganglion cell fate. We demonstrate that ganglion cells are selected from a large group of competent progenitors that comprise the majority of the early embryonic retina and that differentiation within this group is regulated by Notch. Notch activity in vivo was diminished using antisense oligonucleotides or augmented using a retrovirally transduced constitutively active allele of Notch. The number of ganglion cells produced was inversely related to the level of Notch activity. In addition, the Notch ligand Delta inhibited retinal progenitors from differentiating as ganglion cells to the same degree as did activated Notch in an in vitro assay. These results suggest a conserved strategy for neurogenesis in the retina and describe a versatile in vitro and in vivo system with which to examine the action of the Notch pathway in a specific cell fate decision in a vertebrate.

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HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21

Molecular Biology of the Cell ◽

10.1091/mbc.e08-02-0139 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4062-4075 ◽

Cited By ~ 131

Author(s):

Andrew J. Wilson ◽

Do-Sun Byun ◽

Shannon Nasser ◽

Lucas B. Murray ◽

Kanyalakshmi Ayyanar ◽

...

Keyword(s):

Growth Promotion ◽

Cell Types ◽

Specific Cell ◽

Physical Interaction ◽

Down Regulation ◽

Proliferative Zone ◽

P21 Promoter ◽

Hct116 Cells

The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4–HDAC3–N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21.

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T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse

eLife ◽

10.7554/elife.32417 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 18

Author(s):

Tobias X Dong ◽

Shivashankar Othy ◽

Amit Jairaman ◽

Jonathan Skupsky ◽

Angel Zavala ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Tracking ◽

Cell Types ◽

Cell Receptor ◽

Receptor Activation ◽

Specific Cell ◽

Transgenic Expression ◽

Reporter Mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of ‘Salsa6f’, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients (‘sparkles’) as cells migrate.

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Patterned Optogenetic Modulation of Neurovascular and Metabolic Signals

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.189 ◽

2014 ◽

Vol 35 (1) ◽

pp. 140-147 ◽

Cited By ~ 12

Author(s):

Thomas J Richner ◽

Ryan Baumgartner ◽

Sarah K Brodnick ◽

Mehdi Azimipour ◽

Lisa A Krugner-Higby ◽

...

Keyword(s):

Microelectrode Array ◽

Metabolic Response ◽

Cerebral Arteries ◽

Cell Types ◽

Fluorescence Signal ◽

Specific Cell ◽

Temporal Precision ◽

Optogenetic Stimulation ◽

Neurometabolic Coupling

The hemodynamic and metabolic response of the cortex depends spatially and temporally on the activity of multiple cell types. Optogenetics enables specific cell types to be modulated with high temporal precision and is therefore an emerging method for studying neurovascular and neurometabolic coupling. Going beyond temporal investigations, we developed a microprojection system to apply spatial photostimulus patterns in vivo. We monitored vascular and metabolic fluorescence signals after photostimulation in Thy1-channelrhodopsin-2 mice. Cerebral arteries increased in diameter rapidly after photostimulation, while nearby veins showed a slower smaller response. The amplitude of the arterial response was depended on the area of cortex stimulated. The fluorescence signal emitted at 450/100 nm and excited with ultraviolet is indicative of reduced nicotinamide adenine dinucleotide, an endogenous fluorescent enzyme involved in glycolysis and the citric acid cycle. This fluorescence signal decreased quickly and transiently after optogenetic stimulation, suggesting that glucose metabolism is tightly locked to optogenetic stimulation. To verify optogenetic stimulation of the cortex, we used a transparent substrate microelectrode array to map cortical potentials resulting from optogenetic stimulation. Spatial optogenetic stimulation is a new tool for studying neurovascular and neurometabolic coupling.

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