scholarly journals Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

10.3791/2429 ◽  
2010 ◽  
Author(s):  
Shyny Koshy ◽  
Parema Alizadeh ◽  
Lubov T. Timchenko ◽  
Christine Beeton
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Quantitative Measurement ◽  
Glut4 Translocation

Glycoconjugates on the surface of spores of the pathogenic fungus Phytophthora cinnamomi studied using fluorescence and electron microscopy and flow cytometry

1990 ◽  
Vol 36 (3) ◽  
pp. 183-192 ◽  
Author(s):  
A. R. Hardham ◽  
E. Suzaki
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Phytophthora Cinnamomi ◽  
Pathogenic Fungus ◽  
Fluorescein Isothiocyanate ◽  
Pathogenic Fungi ◽  
Surface Flow ◽  
Soybean Agglutinin ◽  
Binding Material

Glycoconjugates on the surface of zoospores and cysts of the pathogenic fungus Phytophthora cinnamomi have been studied using fluorescein isothiocyanate labelled lectins for fluorescence microscopy and flow cytometry, and ferritin- and gold-labelled lectins for ultrastructural analysis. Of the five lectins used, only concanavalin A (ConA) binds to the surface of the zoospores, including the flagella and water expulsion vacuole. This suggests that of accessible saccharides, glucosyl or mannosyl residues predominate on the outer surface of the zoospore plasma membrane. Early in encystment, a system of flat disc-like cisternae, which underlie the zoospore plasma membrane, vesiculate. These and other small peripheral vesicles quickly disappear. After the induction of encystment, ConA is no longer localised close to the plasma membrane but binds to material loosely associated with the cell surface. Quantitative measurements by flow cytometry indicate that the ConA-binding material is gradually lost from the cell surface. The cyst wall is weakly labelled, but the site of germ tube emergence stains intensely. During the first 2 min after the induction of encystment, material that binds soybean agglutinin, Helix pommatia agglutinin, and peanut agglutinin appears on the surface of the fungal cells. The distribution of this material, rich in galactosyl or N-acetyl-D-galactosaminosyl residues, is initially patchy, but by 5 min the material evenly coats most of the cell surface. Labelling of zoospores in which intracellular sites are accessible indicates that the soybean agglutinin binding material is stored in vesicles that lie beneath the plasma membrane. Quantitation of soybean agglutinin labelling shows that maximum binding occurs 2–3 min after the induction of encystment. Key words: cell surface, flow cytometry, lectins, pathogenic fungi, Phytophthora cinnamomi.

scholarly journals Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5

2008 ◽  
Vol 19 (7) ◽  
pp. 2718-2728 ◽  
Author(s):  
Irfan J. Lodhi ◽  
Dave Bridges ◽  
Shian-Huey Chiang ◽  
Yanling Zhang ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Critical Role ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain–containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.

8 FLOW CYTOMETRIC MONITORING OF CHOLERA TOXIN B SUBUNIT BINDING TO BOVINE SPERMATOZOA

2008 ◽  
Vol 20 (1) ◽  
pp. 84
Author(s):  
M. Boilard ◽  
M. Beaulieu ◽  
P. Blondin
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Molecular Probes ◽  
Cholera Toxin B Subunit ◽  
Enhanced Fluorescence ◽  
Toxin B ◽  
B Subunit ◽  
Bovine Spermatozoa ◽  
High Fluorescence

In order to become able to fertilize, mammalian spermatozoa must undergo a series of biochemical modifications. This process called capacitation involves several changes of the content and the ultrastucture of the plasma membrane. Among these changes, loss of cholesterol from the plasma membrane is required. Lipid rafts are detergent-insoluble plasma membrane domains rich in cholesterol and sphingolipids. Some proteins are confined to lipid rafts while others are excluded. It has been hypothesized in the past that the loss of cholesterol could destabilize and relocate lipid rafts and would thus affect protein interactions in the plasma membrane, thereby leading to downstream events involved in the capacitation process. Thus, quantification of lipid rafts within the membrane of spermatozoa would become useful to monitor sperm functions and maturation level. The present study aimed to quantify lipid rafts in bovine spermatozoa using the Vibrant Lipid Raft detection kit from Molecular Probes (Invitrogen Canada, Inc., Burlingame, Ontario, Canada) and flow cytometry. The Vibrant kit uses the cholera toxin B subunit (CT-B) and claims to detect ganglioside Gm1 that sublocalizes within lipid rafts. Briefly, freshly ejaculated and frozen/thawed spermatozoa were washed once by centrifugation at 250g for five min in sp-Talp and were then re-suspended in sp-Talp containing 1 �g mL–1 CT-B. Then, cells were incubated at 4�C for 10 min, washed in chilled sp-Talp, incubated for 15 min in the presence of an anti-CT-B antibody coupled to the Alexa Fluor� 488 dye (Molecular Probes), and washed again to remove excess antibody. Spermatozoa were then analyzed with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Two populations showing different fluorescence levels were observed in all samples. Greater proportions of spermatozoa displayed the high fluorescence pattern in cryopreserved samples (37.9%) when compared to freshly ejaculated spermatozoa (8.2%) (P < 0.01). Also, when compared to freshly ejaculated spermatozoa, increased proportions of high fluorescence was detected following a 6-h incubation in sp-Talp containing bicarbonate and BSA. These results suggest that capacitation and cryopreservation both promote exposure of CT-B binding molecules in bovine spermatozoa. Microscopic observation of labeled cryopreserved spermatozoa did not yield the expected raft labeling patterns, but rather 5 different patterns of labeling. In the past, some of these patterns were recognized to be associated with capacitation and acrosome reaction. At this point, more work is needed to confirm which of the fluorescent patterns observed in microscopy corresponds to the enhanced fluorescence sperm population observed by flow cytometry and to directly associate this enhanced fluorescence to capacitation or the acrosome reaction. In conclusion, it appears that the Vibrant kit from Molecular Probes cannot be used to quantify lipid rafts by flow cytometry. Nevertheless, it might be an interesting tool to use in flow cytometry to monitor membrane changes associated with capacitation or cryo-damage.

scholarly journals Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

2013 ◽  
Vol 24 (16) ◽  
pp. 2544-2557 ◽  
Author(s):  
L. Amanda Sadacca ◽  
Joanne Bruno ◽  
Jennifer Wen ◽  
Wenyong Xiong ◽  
Timothy E. McGraw
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Receptor Activation ◽  
Glut4 Translocation ◽  
Insulin Stimulation ◽  
Transport Vesicles ◽  
Glucose Transporter Glut4

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

scholarly journals Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes

1999 ◽  
Vol 344 (2) ◽  
pp. 511-518 ◽  
Author(s):  
Paru B. OATEY ◽  
Kanamarlapudi VENKATESWARLU ◽  
Alan G. WILLIAMS ◽  
Laura M. FLETCHER ◽  
Emily J. FOULSTONE ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Glucose Uptake ◽  
Confocal Imaging ◽  
Glut4 Translocation ◽  
Nucleotide Exchange ◽  
Subcellular Targeting ◽  
Adp Ribosylation ◽  
Nucleotide Exchange Factors ◽  
Adp Ribosylation Factor

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P3 is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P3 generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P3, were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P3 generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P3 was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3,4,5)P3 than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln67 → Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

Platelet Microparticle Production Is Regulated By STIM1 Dependent Entry Of Extracellular Ca2+ Through Orai1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1059-1059
Author(s):  
Matthew T Duvernay ◽  
Heidi Hamm
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Receptor Agonist ◽  
Conflicts Of Interest ◽  
Lipid Vesicles ◽  
Cell Types ◽  
Published Data ◽  
Receptor Stimulation ◽  
Intense Staining ◽  
Collagen Receptor

Abstract Microparticles are submicron lipid vesicles packed with protein and nucleic acid. They are found in abundance circulating in the vasculature and can be generated from a number of cell types including endothelial cells, leukocytes, and platelets. The microparticles generated from platelets far out number those generated from other cell types and their incidence is correlated with a myriad of cardiovascular diseases. We recently demonstrated that stimulation of gel filtered human platelets through Protease activated receptor (PAR) 4 leads to the generation of 4-5 times more platelet microparticles (PMP) than PAR1 stimulation. Platelet microparticle (PMP) production was demonstrated to be downstream of a Rho kinase-dependent signaling pathway. Consistently, more myosinIIa phosphorylation was observed downstream of PAR4 stimulation. PMP generation was quantified by flow cytometry using polystyrene microbeads of standardized size for appropriate size gating, and CD41a (aIIb) and CD62p (P-selectin) staining for positive identification of platelet derived membranes. Submicron particles positive for both CD41a and CD62p were classified as true PMP. Recently, we have expanded these observations to platelet stimulation with thrombin, convulxin (a GPVI collagen receptor agonist), and dual PAR/collagen receptor stimulation. Thrombin and PAR4-activating peptide (AP) stimulation leads to equivalent levels of PMP production, confirming that PAR4 is the major thrombin receptor responsible for PMP generation, with PAR1 playing only a minor role. Collagen receptor stimulation with convulxin lead to a comparable level of PMP generation as thrombin stimulation. However, co-stimulation with convulxin and thrombin or convulxin and PAR1-AP or PAR4-AP lead to PMP production exceeding the sum of PMP by convulxin and PAR agonist alone by as much as 350%, suggesting a synergistic response. It is well documented that PMP generation does not occur in the absence of extracellular Ca2+. Recently published data collected with Orai1 knockout mice indicate that the majority of extracellular Ca2+ entry into the platelet is mediated by the plasma membrane Ca2+ channel Orai1. STIM1 is an ER calcium sensor that migrates near the cell surface upon depletion of intracellular Ca2+ stores to oligomerize and activate Orai1. In an attempt to further elucidate the signaling pathways responsible for PMP generation we treated platelets with the STIM1 inhibitor SKF 96365. Preincubation with SKF 96365 nearly abolished PMP production induced by PAR4-AP, thrombin, convulxin, or the combination of PAR and collagen receptor agonist. Scanning Electron Microscopy of PAR4-AP stimulated platelets in suspension revealed extended filipodia and bag-like structures protruding from the platelet core which correlated with the size of PMPs as analyzed by flow cytometry. Pretreatment with the SKF 96365 or exclusion of extracellular Ca2+ prevented the formation of the microparticle-like extensions and blunted filipodia extension. Finally, confocal analysis of Orai1 staining on platelets spread on a collagen matrix and co-stimulated with PAR4-AP revealed Orai1 throughout the plasma membrane with intense staining of the microparticle-like structures. These data suggest that PMP generation is nucleated by STIM1 dependent Orai1 Ca2+ entry. Current efforts are focused on elucidating the mechanism by which PAR and collagen receptor agonists differentially regulate STIM1 or Orai1 activity to mediate PMP generation. Disclosures: No relevant conflicts of interest to declare.

Conditional Knockout-First Gene Disruption of Dematin Causes Precipitous Loss of Erythrocyte Membrane Stability and Severe Hemolytic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 157-157
Author(s):  
Yunzhe Lu ◽  
Toshihiko Hanada ◽  
Athar H. Chishti
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Hemolytic Anemia ◽  
Erythrocyte Membrane ◽  
Half Life ◽  
Gene Disruption ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Wild Type

Abstract Dematin is an actin binding and bundling protein originally identified as a component of the erythrocyte membrane junctional complex. A widely expressed member of the villin-family of adaptor proteins, dematin regulates RhoA activity and cell shape in fibroblasts. Actin binding and bundling activity of dematin is regulated by phosphorylation of its headpiece domain by the cAMP-dependent protein kinase. Despite its extensive biochemical characterization, the physiological function of dematin in mature erythrocytes remains unknown. We used a conditional gene disruption strategy by generating a targeting construct that has the potential for full body gene knockout as well as tissue-specific deletion of dematin gene using the Cre-lox gene deletion system. Wild type, heterozygous, and homozygous progeny were obtained in a typical Mendelian ratio of 1:2:1. Dramatic splenomegaly in 7-week old full length dematin knockout (FLKO) mice was observed with the average spleen weight 10-fold higher than those of the wild type littermates. Flow cytometry showed a ~16-fold increase in reticulocytes (Fig.1A), which was also seen in the blood smear (Fig.1B,C). Severe hemolytic anemia is most likely the cause of relative pallor observed in FLKO mice at day 1 after birth. The adult FLKO mice continue to show relatively smaller body size as compared to wild type and heterozygous mice. These findings are consistent with severe anemia and compensatory erythropoiesis. FLKO mice exhibit typical signs of anisocytosis, microcytosis, macrocytosis, and polychromasia, which are indicative of tremendous variation in RBC cell size and the premature release of reticulocytes from the bone marrow. Moreover, additional RBC abnormalities, including poikilocytosis, acanthocytosis, fragmented RBC, and spherocytes, are consistent with severe hemolytic disease. By scanning EM, the FLKO erythrocytes showed dramatic variation in shape and size. The spherocytes, microcytic vesiculation, and the protruding structures are observed in FKLO mice, as well as extensive intravascular hemolysis (Fig. 1D,E). RBC half-life measurements in vivo by NHS-biotin labeling and flow cytometry showed mutant cells almost immediately cleared from the circulation in FLKO mice. A seven-week chase experiment showed that the half-life of RBCs was reduced from 22 days in wild type and heterozygous mice to less than 3 days in FLKO mice. The hematological phenotype of FLKO mice indicated reduced RBC count, hemoglobin, and hematocrit with increase in the RBC distribution width. Collectively, these findings indicate that the mechanical strength of RBC membrane strictly relies on the presence of full length dematin. We employed membrane fractionation, in vitro protein domain mapping, transmission/scanning electron microscopy, and dynamic deformability measurements to investigate the underlying mechanisms of extreme membrane fragility in FLKO erythrocytes. We also examined the protein profile of RBC ghosts. Surprisingly, the major cytoskeletal proteins remained unchanged in the FLKO ghosts; however, a marked reduction of spectrin, adducin, and actin was observed. When normalized against band 3, these proteins were reduced by 60%, 90%, and 90%, respectively. Since these membrane proteins are essential for RBC stability, our findings suggest a specific role of dematin in recruiting or maintaining a stable association of essential cytoskeletal proteins in the plasma membrane. These results raise the possibility that dematin may directly interact with adducin, and together anchor the spectrin molecules to the plasma membrane. Our findings provide the first in vivo evidence that dematin is essential for the maintenance of erythrocyte shape and membrane mechanical properties by regulating the integrity of the spectrin-actin junctions. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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