scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

scholarly journals The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices

2002 ◽  
Vol 115 (6) ◽  
pp. 1123-1136 ◽  
Author(s):  
Kayla J. Bayless ◽  
George E. Davis
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Dominant Negative ◽  
Extracellular Matrices ◽  
Wild Type ◽  
Lumen Formation ◽  
Clostridium Difficile Toxin B ◽  
Green Fluorescent ◽  
Intracellular Vacuole

Here we show a requirement for the Cdc42 and Rac1 GTPases in endothelial cell (EC) morphogenesis in three-dimensional extracellular matrices. Cdc42 and Rac1 specifically regulate EC intracellular vacuole and lumen formation in both collagen and fibrin matrices. Clostridium difficile toxin B(which blocks all three Rho GTPases) completely inhibited the ability of ECs to form both vacuoles and lumens, whereas C3 transferase, a selective inhibitor of Rho, did not. Expression of either dominant-negative (N17) or constitutively active (V12) Cdc42 using recombinant adenoviruses dramatically inhibited EC vacuole and lumen formation in both collagen and fibrin matrices. Both vacuole and lumen formation initiated in ECs expressing dominant-negative(N17) Rac1 but later collapsed, indicating a role for Rac1 during later stages of vessel development. Analysis of cultures using confocal microscopy revealed green fluorescent protein-V12Rac1, -Rac1 wild-type and -Cdc42 wild-type chimeric proteins targeted to intracellular vacuole membranes during the lumen formation process. Also, expression of the verprolin-cofilin-acidic domain of N-WASP, a downstream Cdc42 effector, in ECs completely interfered with vacuole and lumen formation. These results collectively reveal a novel role for Cdc42 and Rac1 in the process of EC vacuole and lumen formation in three-dimensional extracellular matrices.

Subcellular redistribution of the renal betaine transporter during hypertonic stress

2003 ◽  
Vol 285 (5) ◽  
pp. C1091-C1100 ◽  
Author(s):  
Stephen A. Kempson ◽  
Vaibhave Parikh ◽  
Lixuan Xi ◽  
Shaoyou Chu ◽  
Marshall H. Montrose
Keyword(s):  
Plasma Membrane ◽  
Posttranscriptional Regulation ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Live Cells ◽  
Hypertonic Medium ◽  
Hypertonic Stress ◽  
Antibody Staining ◽  
Renal Inner Medulla ◽  
Green Fluorescent

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.

High-Order Photobleaching of Green Fluorescent Protein inside Live Cells in Two-Photon Excitation Microscopy

2002 ◽  
Vol 291 (5) ◽  
pp. 1272-1275 ◽  
Author(s):  
Tong-Sheng Chen ◽  
Shao-Qun Zeng ◽  
Qing-Ming Luo ◽  
Zhi-Hong Zhang ◽  
Wei Zhou
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
High Order ◽  
Live Cells ◽  
Two Photon ◽  
Photon Excitation ◽  
Green Fluorescent ◽  
Two Photon Excitation

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

2002 ◽  
Vol 156 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Francesca Santini ◽  
Ibragim Gaidarov ◽  
James H. Keen
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Membrane Skeleton ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
Cortical Actin ◽  
Endocytic Machinery ◽  
Green Fluorescent ◽  
G Protein Coupled

Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing β2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

scholarly journals Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

2002 ◽  
Vol 156 (3) ◽  
pp. 511-518 ◽  
Author(s):  
Pierre Barbero ◽  
Lenka Bittova ◽  
Suzanne R. Pfeffer
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Late Endosome ◽  
Live Cells ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network ◽  
Protein Variants

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

scholarly journals Phosphatidylserine dynamics in cellular membranes

2012 ◽  
Vol 23 (11) ◽  
pp. 2198-2212 ◽  
Author(s):  
Jason G. Kay ◽  
Mirkka Koivusalo ◽  
Xiaoxiao Ma ◽  
Thorsten Wohland ◽  
Sergio Grinstein
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Annexin V ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Optical Methods ◽  
Live Cells ◽  
Cortical Actin ◽  
Limited Mobility ◽  
Green Fluorescent

Much has been learned about the role of exofacial phosphatidylserine (PS) in apoptosis and blood clotting using annexin V. However, because annexins are impermeant and unable to bind PS at low calcium concentration, they are unsuitable for intracellular use. Thus little is known about the topology and dynamics of PS in the endomembranes of normal cells. We used two new probes—green fluorescent protein (GFP)–LactC2, a genetically encoded fluorescent PS biosensor, and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phospho-l-serine (TopFluor-PS), a synthetic fluorescent PS analogue—to examine PS distribution and dynamics inside live cells. The mobility of PS was assessed by a combination of advanced optical methods, including single-particle tracking and fluorescence correlation spectroscopy. Our results reveal the existence of a sizable fraction of PS with limited mobility, with cortical actin contributing to the confinement of PS in the plasma membrane. We were also able to measure the dynamics of PS in endomembrane organelles. By targeting GFP-LactC2 to the secretory pathway, we detected the presence of PS in the luminal leaflet of the endoplasmic reticulum. Our data provide new insights into properties of PS inside cells and suggest mechanisms to account for the subcellular distribution and function of this phospholipid.

scholarly journals Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin

2013 ◽  
Vol 304 (5) ◽  
pp. F553-F564 ◽  
Author(s):  
Richard Bouley ◽  
Paula Nunes ◽  
Billy Andriopoulos ◽  
Margaret McLaughlin ◽  
Matthew J. Webber ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Target Cells ◽  
Intracellular Camp ◽  
Coated Pits ◽  
Parathyroid Hormone Receptor ◽  
Body Fluid Homeostasis ◽  
Green Fluorescent ◽  
G Protein Coupled

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

scholarly journals CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization in Aspergillus nidulans

2006 ◽  
Vol 17 (4) ◽  
pp. 2021-2034 ◽  
Author(s):  
Vladimir P. Efimov ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Live Cells ◽  
Physiological Conditions ◽  
Physical Interactions ◽  
Cytoplasmic Microtubules ◽  
Growth Promoting ◽  
Green Fluorescent ◽  
Hyphal Tip

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32°C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150Glued dynactin, and NUDF were all seen as plus-end comets at 32°C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32°C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42°C).

scholarly journals A Visual Screen of Protein Localization during Sporulation Identifies New Components of Prospore Membrane-Associated Complexes in Budding Yeast

2014 ◽  
Vol 13 (3) ◽  
pp. 383-391 ◽  
Author(s):  
Chien Lam ◽  
Ethan Santore ◽  
Elizabeth Lavoie ◽  
Leor Needleman ◽  
Nicholas Fiacco ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Leading Edge ◽  
Ring Structure ◽  
Content Type ◽  
Prospore Membrane ◽  
Membrane Compartment ◽  
Green Fluorescent

ABSTRACT During ascospore formation in Saccharomyces cerevisiae , the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1 , but not ADY3 . Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.

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