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 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 Intracellular Segregation of Phosphatidylinositol-3,4,5-Trisphosphate by Insulin-Dependent Actin Remodeling in L6 Skeletal Muscle Cells

2003 ◽  
Vol 23 (13) ◽  
pp. 4611-4626 ◽  
Author(s):  
Nish Patel ◽  
Assaf Rudich ◽  
Zayna A. Khayat ◽  
Rami Garg ◽  
Amira Klip
Keyword(s):  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Regulatory Subunit ◽  
Live Cells ◽  
Cortical Actin ◽  
Actin Remodeling ◽  
L6 Myotubes ◽  
Kinase C ◽  
Insulin Dependent ◽  
Green Fluorescent

ABSTRACT Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular pool to the cell surface through a mechanism that is dependent on phosphatidylinositol (PI) 3-kinase (PI3-K) and cortical actin remodeling. Here we test the hypothesis that insulin-dependent actin filament remodeling determines the location of insulin signaling molecules. It has been shown previously that insulin treatment of L6 myotubes leads to a rapid rearrangement of actin filaments into submembrane structures where the p85 regulatory subunit of PI3-K and organelles containing GLUT4, VAMP2, and the insulin-regulated aminopeptidase (IRAP) colocalize. We now report that insulin receptor substrate-1 and the p110α catalytic subunit of PI3-K (but not p110β) also colocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase C λ. Transiently transfected green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of general receptor for phosphoinositides-1 (GRP1) or Akt (ligands of phosphatidylinositol-3,4,5-trisphosphate [PI-3,4,5-P3]) migrated to the periphery of the live cells; in fixed cells, they were detected in the insulin-induced actin structures. These results suggest that PI-3,4,5-P3 is generated on membranes located within the actin mesh. Actin remodeling and GLUT4 externalization were blocked in cells highly expressing GFP-PH-GRP1, suggesting that PI-3,4,5-P3 is required for both phenomena. We propose that PI-3,4,5-P3 leads to actin remodeling, which in turn segregates p85α and p110α, thus localizing PI-3,4,5-P3 production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P3 and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.

scholarly journals A genetically-encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

2019 ◽  
Author(s):  
David L. Prole ◽  
Colin W. Taylor
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cell Signalling ◽  
Intracellular Signalling ◽  
Live Cells ◽  
Green Fluorescent Proteins ◽  
Membrane Contact Sites ◽  
Green Fluorescent

AbstractBackgroundIntrabodies enable targeting of proteins in live cells, but it remains a huge task to generate specific intrabodies against the thousands of proteins in a proteome. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags.ResultsWe generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerizing or heterodimerizing modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria, and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane.ConclusionsThis toolkit greatly expands the utility of intrabodies for studying cell signalling in live cells.

Isolation and Characterization of Nrf1p, a Novel Negative Regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Janet M Murray ◽  
Douglas I Johnson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fluorescent Protein ◽  
Amino Acid Identity ◽  
Negative Regulator ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Isolation And Characterization ◽  
Nucleotide Exchange Factor ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1+, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Δnrf1 mutant was viable but overexpression of nrf1+ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1+ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Planta ◽  
1999 ◽  
Vol 208 (3) ◽  
pp. 392-400 ◽  
Author(s):  
Petra Boevink ◽  
Barry Martin ◽  
Karl Oparka ◽  
Simon Santa Cruz ◽  
Chris Hawes
Keyword(s):  
Green Fluorescent Protein ◽  
Secretory Pathway ◽  
Cold Shock ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Tobacco Leaves ◽  
Green Fluorescent

Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy

2000 ◽  
Vol 113 (22) ◽  
pp. 3921-3930 ◽  
Author(s):  
R.H. Kohler ◽  
P. Schwille ◽  
W.W. Webb ◽  
M.R. Hanson
Keyword(s):  
Active Transport ◽  
Fluorescence Correlation Spectroscopy ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Long Distance ◽  
Fluorescence Correlation ◽  
Photon Excitation ◽  
Green Fluorescent

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

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 The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

2020 ◽  
Vol 295 (15) ◽  
pp. 5036-5050 ◽  
Author(s):  
Tess A. Stanly ◽  
Marco Fritzsche ◽  
Suneale Banerji ◽  
Dilip Shrestha ◽  
Falk Schneider ◽  
...  
Keyword(s):  
Lymphatic Vessel ◽  
Leukocyte Adhesion ◽  
Lymphatic Vessels ◽  
Super Resolution ◽  
Direct Interaction ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Actin Network ◽  
Cortical Actin ◽  
Limited Mobility

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

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