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1.
Nat Commun ; 15(1): 1799, 2024 Feb 28.
Article in English | MEDLINE | ID: mdl-38418824

ABSTRACT

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal and pericentral axis. How the mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combine intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We find that periportal and pericentral mitochondria are morphologically and functionally distinct; beta-oxidation is elevated in periportal regions, while lipid synthesis is predominant in the pericentral mitochondria. In addition, comparative phosphoproteomics reveals spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifts mitochondrial phenotypes in the periportal and pericentral regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.


Subject(s)
Liver , Mitochondria , Liver/metabolism , Oxidation-Reduction , Mitochondria/metabolism
2.
Curr Opin Cell Biol ; 86: 102292, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38064779

ABSTRACT

The unique architecture of the liver allows for spatial compartmentalization of its functions, also known as liver zonation. In contrast to organelles and cells, this compartment is devoid of a surrounding membrane, rendering traditional biochemical tools ineffective for studying liver zonation. Recent advancements in tissue imaging and single-cell technologies have provided new insights into the complexity of tissue organization, rich cellular composition, and the gradients that shape zonation. Hepatocyte gene expression profiles and metabolic programs differ based on their location. Non-parenchymal cells further support hepatocytes from different zones through local secretion of factors that instruct hepatocyte activities. Collectively, these elements form a cohesive and dynamic network of cell-cell interactions that vary across space, time, and disease states. This review will examine the cell biology of hepatocytes in vivo, presenting the latest discoveries and emerging principles that govern tissue-level and sub-cellular compartmentalization.


Subject(s)
Hepatocytes , Liver , Cell Communication , Liver/metabolism , Transcriptome
3.
J Invest Dermatol ; 144(6): 1311-1321.e7, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38103827

ABSTRACT

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and GPCRs play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation. We identified 3 key receptors-HCAR3, LTB4R, and GPR137-and found that knockdown of these receptors led to changes in numerous gene networks that are important for maintaining cell identity and promoting proliferation while inhibiting differentiation. Our study also revealed that the metabolite receptor HCAR3 regulates keratinocyte migration and cellular metabolism. Knockdown of HCAR3 led to reduced keratinocyte migration and respiration, which could be attributed to altered metabolite use and aberrant mitochondrial morphology caused by the absence of the receptor. This study contributes to understanding the complex interplay between GPCR signaling and epithelial cell fate decisions.


Subject(s)
Cell Movement , Cell Proliferation , Cell Respiration , Keratinocytes , Receptors, G-Protein-Coupled , Humans , Keratinocytes/metabolism , Keratinocytes/cytology , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Cell Respiration/physiology , Signal Transduction , Cell Differentiation , Cells, Cultured , Receptors, Leukotriene B4/metabolism , Receptors, Leukotriene B4/genetics , Epithelial Cells/metabolism , Receptors, Nicotinic
4.
bioRxiv ; 2023 May 31.
Article in English | MEDLINE | ID: mdl-37398171

ABSTRACT

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and G protein-coupled receptors (GPCRs) play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation. We identified three key receptors, hydroxycarboxylic acid-receptor 3 (HCAR3), leukotriene B4-receptor 1 (LTB4R), and G Protein-Coupled Receptor 137 (GPR137) and found that knockdown of these receptors led to changes in numerous gene networks that are important for maintaining cell identity and promoting proliferation while inhibiting differentiation. Our study also revealed that the metabolite receptor HCAR3 regulates keratinocyte migration and cellular metabolism. Knockdown of HCAR3 led to reduced keratinocyte migration and respiration, which could be attributed to altered metabolite use and aberrant mitochondrial morphology caused by the absence of the receptor. This study contributes to understanding the complex interplay between GPCR signaling and epithelial cell fate decisions.

5.
bioRxiv ; 2023 Oct 26.
Article in English | MEDLINE | ID: mdl-37333328

ABSTRACT

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal (PP) and pericentral (PC) axis. How these mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combined intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We found that PP and PC mitochondria are morphologically and functionally distinct; beta-oxidation was elevated in PP regions, while lipid synthesis was predominant in the PC mitochondria. In addition, comparative phosphoproteomics revealed spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifted mitochondrial phenotypes in the PP and PC regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

6.
Blood ; 141(20): 2520-2536, 2023 05 18.
Article in English | MEDLINE | ID: mdl-36735910

ABSTRACT

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.


Subject(s)
Proteomics , Spermidine , Humans , Spermidine/metabolism , Peptide Initiation Factors/genetics , Cell Differentiation , Eukaryotic Translation Initiation Factor 5A
7.
Am J Physiol Gastrointest Liver Physiol ; 324(4): G245-G249, 2023 04 01.
Article in English | MEDLINE | ID: mdl-36749570

ABSTRACT

Tumor heterogeneity is a hallmark of cancer but a challenging problem to dissect mechanistically. Less recognized is that cells within normal tissues are also remarkably diverse. Hepatocytes are a great example because their spatial positioning and the local microenvironment govern their genetic heterogeneity. Recent studies show that primary liver tumors display heterogeneity similar to that observed in the normal tissue providing clues to the cellular precursor of the tumor and how variations in the lobule microenvironment support tumor formation and aggressiveness. Identifying the principles that control cellular diversity in a healthy liver may highlight potential mechanisms driving hepatic tumor heterogeneity.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver/pathology , Hepatocytes/pathology , Tumor Microenvironment
8.
J Biol Chem ; 299(1): 102766, 2023 01.
Article in English | MEDLINE | ID: mdl-36470425

ABSTRACT

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.


Subject(s)
ErbB Receptors , Neoplasms , Proto-Oncogene Proteins c-cbl , Animals , Humans , Mice , Epidermal Growth Factor/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , HEK293 Cells , HeLa Cells , Mice, Nude , Neoplasms/genetics , Neoplasms/physiopathology , Phosphorylation , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Ubiquitination , Membrane Proteins/metabolism , Proteolysis , Gene Expression Regulation, Neoplastic
9.
Hepatol Commun ; 6(8): 1949-1961, 2022 08.
Article in English | MEDLINE | ID: mdl-35357082

ABSTRACT

As important as the fasting response is for survival, an inability to shut it down once nutrients become available can lead to exacerbated disease and severe wasting. The liver is central to transitions between feeding and fasting states, with glucagon being a key initiator of the hepatic fasting response. However, the precise mechanisms controlling fasting are not well defined. One potential mediator of these transitions is liver kinase B1 (LKB1), given its role in nutrient sensing. Here, we show LKB1 knockout mice have a severe wasting and prolonged fasting phenotype despite increased food intake. By applying RNA sequencing and intravital microscopy, we show that loss of LKB1 leads to a dramatic reprogramming of the hepatic lobule through robust up-regulation of periportal genes and functions. This is likely mediated through the opposing effect that LKB1 has on glucagon pathways and gene expression. Conclusion: Our findings show that LKB1 acts as a brake to the glucagon-mediated fasting response, resulting in "periportalization" of the hepatic lobule and whole-body metabolic inefficiency. These findings reveal a mechanism by which hepatic metabolic compartmentalization is regulated by nutrient-sensing.


Subject(s)
AMP-Activated Protein Kinases , Fasting , Glucagon , Liver , AMP-Activated Protein Kinases/genetics , Animals , Glucagon/metabolism , Liver/metabolism , Mice , Mice, Knockout
10.
Front Physiol ; 12: 732929, 2021.
Article in English | MEDLINE | ID: mdl-34566696

ABSTRACT

Despite the ever-increasing prevalence of non-alcoholic fatty liver disease (NAFLD), the etiology and pathogenesis remain poorly understood. This is due, in part, to the liver's complex physiology and architecture. The liver maintains glucose and lipid homeostasis by coordinating numerous metabolic processes with great efficiency. This is made possible by the spatial compartmentalization of metabolic pathways a phenomenon known as liver zonation. Despite the importance of zonation to normal liver function, it is unresolved if and how perturbations to liver zonation can drive hepatic pathophysiology and NAFLD development. While hepatocyte heterogeneity has been identified over a century ago, its examination had been severely hindered due to technological limitations. Recent advances in single cell analysis and imaging technologies now permit further characterization of cells across the liver lobule. This review summarizes the advances in examining liver zonation and elucidating its regulatory role in liver physiology and pathology. Understanding the spatial organization of metabolism is vital to further our knowledge of liver disease and to provide targeted therapeutic avenues.

11.
Curr Protoc ; 1(5): e139, 2021 May.
Article in English | MEDLINE | ID: mdl-34033261

ABSTRACT

The liver is central in maintaining glucose homeostasis. Indeed, impaired hepatic glucose uptake has been implicated in the development of hyperglycemia in type II diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). However, current approaches to evaluate glucose mobilization rely on indirect measurements that do not provide spatial and temporal information. Here, we describe confocal-based intravital microscopy (IVM) of the liver that allows the identification of hepatocyte spatial organization and glucose transport. Specifically, we describe a method to fluorescently label hepatic landmarks to identify different compartments within the liver. In addition, we outline an in vivo fluorescent glucose uptake assay to quantitatively measure glucose mobilization in space and time. These protocols allow direct investigation of hepatic glycemic control and can be further applied to murine models of liver disease. © Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Mouse surgical procedure and positioning for liver intravital imaging Basic Protocol 2: Fluorescent labeling and intravital imaging of mouse hepatic compartments Basic Protocol 3: Mouse hepatic glucose uptake assay and intravital imaging analysis.


Subject(s)
Glucose/metabolism , Intravital Microscopy , Liver/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/metabolism , Animals , Deoxyglucose/analogs & derivatives , Deoxyglucose/metabolism , Fluorescent Dyes/chemistry , Hepatocytes/metabolism , Imaging, Three-Dimensional , Mice
12.
Nat Cell Biol ; 21(12): 1578-1589, 2019 12.
Article in English | MEDLINE | ID: mdl-31792379

ABSTRACT

Yes-associated protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a select set of enhancers for target gene activation. How YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized the YAP transcription factor TEAD1 and other YAP-related co-activators, including TAZ, and subsequently induced the transcription of YAP-specific proliferation genes. Super-resolution imaging using assay for transposase-accessible chromatin with photoactivated localization microscopy revealed that the YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA polymerase II, the accessible chromatin domains later acquired RNA polymerase II, transcribing RNA. The removal of the intrinsically-disordered YAP transcription activation domain prevented the formation of YAP condensates and diminished downstream YAP signalling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cell Cycle Proteins/genetics , Gene Expression Regulation/genetics , Genome/genetics , Animals , Cell Line , Cell Nucleus/genetics , Chromatin/genetics , Female , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Nuclear Proteins/genetics , RNA Polymerase II/genetics , Signal Transduction/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics , Transcriptional Activation/genetics , YAP-Signaling Proteins
13.
iScience ; 11: 440-449, 2019 Jan 25.
Article in English | MEDLINE | ID: mdl-30661001

ABSTRACT

Mitochondria are dynamic organelles undergoing fission, fusion, and translocation. These processes have been studied in cultured cells; however, little is known about their regulation in cells within tissues in vivo. We applied four-dimensional intravital microscopy to address this in secretory cells of the salivary gland. We found that mitochondria are organized in two populations: one juxtaposed to the basolateral plasma membrane and the other dispersed in the cytosol. Under basal conditions, central mitochondria exhibit microtubule-dependent motility and low fusion rate, whereas basolateral mitochondria are static and display high fusion rate. Increasing cellular energy demand by ß-adrenergic stimulation of regulated exocytosis selectively enhanced motility and fusion of central mitochondria. Inhibition of microtubule polymerization led to inhibition of central mitochondrial motility and fusion and a marked reduction in exocytosis. This study reveals a conserved heterogeneity in mitochondrial positioning and dynamics in exocrine tissues that may have fundamental implications in organ pathophysiology.

14.
Oncotarget ; 9(26): 18454-18479, 2018 Apr 06.
Article in English | MEDLINE | ID: mdl-29719618

ABSTRACT

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

15.
Cell Syst ; 4(3): 277-290.e9, 2017 03 22.
Article in English | MEDLINE | ID: mdl-28330614

ABSTRACT

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.


Subject(s)
Bile Canaliculi/metabolism , Bile Canaliculi/physiology , Biliary Tract/diagnostic imaging , Animals , Bile/metabolism , Biliary Tract/metabolism , Biliary Tract/physiology , Chemical and Drug Induced Liver Injury/metabolism , Cholestasis/metabolism , Computer Simulation , Forecasting , Hepatocytes/metabolism , Hydrodynamics , Liver/metabolism , Mice , Mice, Inbred C57BL
16.
Hepatology ; 64(4): 1317-29, 2016 10.
Article in English | MEDLINE | ID: mdl-27396550

ABSTRACT

UNLABELLED: Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. CONCLUSION: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317-1329).


Subject(s)
Hepatocytes/physiology , Hepatocytes/ultrastructure , Protein Serine-Threonine Kinases/physiology , Tight Junctions/physiology , Tight Junctions/ultrastructure , AMP-Activated Protein Kinases , Animals , Female , Male , Mice , Mice, Knockout
17.
Cell Rep ; 9(2): 514-21, 2014 Oct 23.
Article in English | MEDLINE | ID: mdl-25373899

ABSTRACT

Little is known about the spatiotemporal coordination of mitochondrial metabolism in multicellular organisms in situ. Using intravital microscopy in live animals, we report that mitochondrial metabolism undergoes rapid and periodic oscillations under basal conditions. Notably, mitochondria in vivo behave as a network of functionally coupled oscillators, which maintain a high level of coordination throughout the tissue via the activity of gap junctions. These findings reveal a unique aspect of the relationship between tissue architecture and self-organization of mitochondrial metabolism in vivo.


Subject(s)
Acinar Cells/metabolism , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Animals , Gap Junctions/metabolism , Male , Rats , Rats, Sprague-Dawley
18.
PLoS One ; 8(11): e81987, 2013.
Article in English | MEDLINE | ID: mdl-24282620

ABSTRACT

Clathrin-independent endocytosis (CIE) is a form of bulk plasma membrane (PM) endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs) utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen.


Subject(s)
Clathrin/metabolism , Endocytosis , Endosomes/metabolism , Cells, Cultured , Collagen/metabolism , Fluorescent Dyes/metabolism , Humans , Integrin beta1/immunology , Integrin beta1/metabolism , Receptors, Transferrin/metabolism
19.
J Vis Exp ; (79)2013 Sep 01.
Article in English | MEDLINE | ID: mdl-24022089

ABSTRACT

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.


Subject(s)
Microscopy, Fluorescence/methods , Salivary Glands/ultrastructure , Secretory Vesicles/ultrastructure , Acinar Cells/metabolism , Acinar Cells/ultrastructure , Animals , Exocytosis , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Male , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Salivary Glands/metabolism , Secretory Vesicles/metabolism , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructure
20.
J Cell Biol ; 201(7): 969-79, 2013 Jun 24.
Article in English | MEDLINE | ID: mdl-23798727

ABSTRACT

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.


Subject(s)
Microscopy, Fluorescence/methods , Time-Lapse Imaging/methods , Animals , Cell Biology/trends , Cells, Cultured , Cytological Techniques , Green Fluorescent Proteins/analysis , Humans , Mice , Microscopy, Fluorescence/instrumentation , Time-Lapse Imaging/instrumentation
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