Plasma Membrane Of Intact Cells Research Articles

Cholesterol-Induced Deformation of Gramicidin A Channel Inhibits Potassium Ion Binding and Transport

Abstract Gramicidin A (gA) is a kind of antibiotic peptide produced by Bacillus brevis and it can dimerize across lipid bilayers to form a monovalent cation channel. In this work, we investigate the impact of cholesterol in the lipid bilayer on the binding of potassium ions with gA channel and the transport of the ions across the channel. The results indicate that cholesterol can significantly influence the conformational stability of the gA channel and cause the channel deformation which inhibits the potassium ion binding with the channel and transport across the channel. The work provides some molecular insights into understanding the influence of lipids on the activity of gA channel in both model membranes and plasma membranes of intact cells.

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals.

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Förster resonance energy transfer-based pH and K+ biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv–GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K+ efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv–pH-Lemon and TAv–GEPII 1.0 spots for simultaneous pH and K+ measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

Membrane nanodomains homeostasis during propofol anesthesia as function of dosage and temperature

Some anesthetics bind and potentiate γ-aminobutyric-acid-type receptors, but no universal mechanism for general anesthesia is known. Furthermore, often encountered complications such as anesthesia induced amnesia are not understood. General anesthetics are hydrophobic molecules easily dissolving into lipid bilayers. Recently, it was shown that general anesthetics perturb phase separation in vesicles extracted from fixed cells. Unclear is whether under physiological conditions general anesthetics induce perturbation of the lipid bilayer, and whether this contributes to the transient loss of consciousness or anesthesia side effects. Here we show that propofol perturbs lipid nanodomains in the outer and inner leaflet of the plasma membrane in intact cells, affecting membrane nanodomains in a concentration dependent manner: 1 μM to 5 μM propofol destabilize nanodomains; however, propofol concentrations higher than 5 μM stabilize nanodomains with time. Stabilization occurs only at physiological temperature and in intact cells. This process requires ARP2/3 mediated actin nucleation and Myosin II activity. The rate of nanodomain stabilization is potentiated by GABAA receptor activity. Our results show that active nanodomain homeostasis counteracts the initial disruption causing large changes in cortical actin. Significance statementGeneral anesthesia is a routine medical procedure with few complications, yet a small number of patients experience side-effects that persist for weeks and months. Very young children are at risk for effects on brain development. Elderly patients often exhibit subsequent amnesia. Here, we show that the general anesthetic propofol perturbs the ultrastructure of the lipid bilayer of the cell membrane in intact cells. Initially propofol destabilized lipid nanodomains. However, with increasing incubation time and propofol concentration, the effect is reversed and nanodomains are further stabilized. We show that this stabilization is caused by the activation of the actin cortex under the membrane. These perturbations of membrane bilayer and cortical actin may explain how propofol affects neuronal plasticity at synapses.

Application of Higher Density Iron Oxide Nanoparticle Pellicles to Enrich the Plasma Membrane and Its Proteome from Cells in Suspension.

Enrichment of the plasma membrane represents one valuable method to characterize the surfaceome, along with other plasma membrane and structural proteins. Currently, the overlapping densities of many subcellular organelles hinder enrichment of the plasma membrane by centrifugation. However, external access to the plasma membrane of intact cells allows the attachment of a nanoparticle pellicle to enhance its density and facilitate enrichment. We describe the synthesis of iron oxide nanoparticles, attachment of the pellicle to suspended cells, and recovery of plasma membrane proteins for proteomic analysis.

The dual role of group V secretory phospholipase A2 in pancreatic β-cells.

Group X (GX) and group V (GV) secretory phospholipase A2 (sPLA2) potently release arachidonic acid (AA) from the plasma membrane of intact cells. We previously demonstrated that GX sPLA2 negatively regulates glucose-stimulated insulin secretion (GSIS) by a prostaglandin E2 (PGE2)-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS. GSIS and pancreatic islet-size were assessed in wild-type (WT) and GV sPLA2-knock out (GV KO) mice. GSIS was also assessed ex vivo in isolated islets and in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing. GSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice and in MIN6 cells with siRNA-mediated GV sPLA2 suppression. MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. Though the amount of AA released into the media by MIN6-GV cells was significantly higher, PGE2 production was not enhanced or cAMP content decreased compared to control MIN6 cells. Surprisingly, GV KO mice exhibited a significant increase in plasma insulin levels following i.p. injection of glucose compared to WT mice. This increase in GSIS in GV KO mice was associated with a significant increase in pancreatic islet size and number of proliferating cells in β-islets compared to WT mice. Deficiency of GV sPLA2 results in diminished GSIS in isolated pancreatic beta-cells. However, the reduced GSIS in islets lacking GV sPLA2 appears to be compensated by increased islet mass in GV KO mice.

N-glycosylation critically regulates function of oxalate transporter SLC26A6.

The brush border Cl--oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking, and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6 but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 is critically important for chloride-dependent oxalate transport and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation and that exoglycosidase-mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.

Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles.

Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.

Abstract 483: Non-redundant and Opposing Roles of Group X and Group V Secretory Phospholipase A2s on Pancreatic Beta-cell Function

Background: Group X and group V secretory phospholipase A2s (GX and GV sPLA2s) potently release arachidonic acid (AA) from the plasma membrane of intact cells. AA is an activator of glucose-stimulated insulin secretion (GSIS) by β-islet cells. However, the AA metabolite prostaglandin E2 (PGE2) is a known inhibitor of GSIS. Both GX and GV sPLA2s are expressed in mouse pancreatic islet cells. We previously demonstrated that GX sPLA2 negatively regulates GSIS by a PGE2-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS. Methods and Results: GSIS was measured in pancreatic islet cells isolated from WT and GV sPLA2-deficient (GV KO) mice. To complement these studies, GSIS was also assessed in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing. In marked contrast to our findings in GX KO mice, GSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice. Similarly, there was a significant decrease in GSIS in MIN6 cells with siRNA-mediated GV sPLA2 suppression. Consistent with these findings, MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. As expected, the amount of AA released into the media by MIN6-GV cells was significantly increased compared to control MIN6 cells. However, unlike MIN6 cells overexpressing GX sPLA2, MIN6-GV cells did not exhibit enhanced PGE2 production or decreased cAMP content compared to control MIN6 cells, despite similar amounts of sPLA2 activity produced by the two cell lines. Conclusions: We conclude that GX and GV sPLA2s play opposing and non-redundant roles in pancreatic β-cell function. Whereas GV sPLA2 activates GSIS, GX sPLA2 suppresses this process. This functional difference appears to be due to the extent to which AA generated by the respective sPLA2’s is coupled to PGE2 production.

Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm

We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.

General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles

A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand gated ion channels. Here we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (Tc) of these GPMVs without strongly altering the ratio of the two liquid phases found below Tc. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC50) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC50 we see a 4{\deg}C downward shift in Tc, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critical temperatures are also lowered to a similar extent by propofol, phenylethanol, and isopropanol when added at anesthetic concentrations, but not by tetradecanol or 2,6 diterbutylphenol, two structural analogs of general anesthetics that are hydrophobic but have no anesthetic potency. We propose that liquid general anesthetics provide an experimental tool for lowering critical temperatures in plasma membranes of intact cells, which we predict will reduce lipid-mediated heterogeneity in a way that is complimentary to increasing or decreasing cholesterol. Also, several possible implications of our results are discussed in the context of current models of anesthetic action on ligand gated ion channels.

Liquid General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles

A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand-gated ion channels. In this study we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell-derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (Tc) of these GPMVs without strongly altering the ratio of the two liquid phases found below Tc. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC50) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC50 we see a 4°C downward shift in Tc, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critical temperatures are also lowered to a similar extent by propofol, phenylethanol, and isopropanol when added at anesthetic concentrations, but not by tetradecanol or 2,6 diterbutylphenol, two structural analogs of general anesthetics that are hydrophobic but have no anesthetic potency. We propose that liquid general anesthetics provide an experimental tool for lowering critical temperatures in plasma membranes of intact cells, which we predict will reduce lipid-mediated heterogeneity in a way that is complimentary to increasing or decreasing cholesterol. Also, several possible implications of our results are discussed in the context of current models of anesthetic action on ligand-gated ion channels.

Use of mutant 125 I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells

Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment. In this study, we describe a high-throughput shRNA screen to identify host factors required for anthrax lethal toxin-induced cell death. The cytosolic chaperonin complex chaperonin containing t-complex protein 1 (CCT) was identified, and subsequent studies showed that CCT is required for efficient delivery of LF and related fusion proteins into the cytosol. We further show that knockdown of CCT inhibits the acid-induced delivery of LF and the fusion protein LFN-Bla (N terminal domain of LF fused to β-lactamase) across the plasma membrane of intact cells. Together, these results suggest that CCT is required for efficient delivery of enzymatically active toxin to the cytosol and are consistent with a direct role for CCT in translocation of LF through the protective antigen pore.

Super-resolution characterization of TCR-dependent signaling clusters.

Multi-molecular signaling complexes drive the earliest events of immune cell activation via immunoreceptors with unexplained specificity and speed. Fluorescence microscopy has shown that these complexes form microclusters at the plasma membrane of activated T cells upon engagement of their antigen receptors (TCRs). Although crucial for cell function, much remains to be learned about the molecular content, fine structure, formation mechanisms, and function of these microclusters. Recent advancements in super-resolution microscopy have enabled the study of signaling microclusters at the single molecule level with resolution down to approximately 20 nm. These techniques have now helped to characterize the size distributions of signaling clusters at the plasma membrane of intact cells and to shed light on the formation mechanisms that govern their assembly. Surprisingly, dynamic and functional nanostructures have been identified within the signaling clusters. We expect that these novel methodologies, combined with older techniques, will shed new light on the nature of signaling clusters and their critical role in T-cell activation.

Abstract 143: Group X Secretory Phospholipase A2 Regulates Insulin Secretion by Mouse Pancreatic Beta Cells

Background: Group X secretory phospholipase A2 (GX sPLA2) potently releases arachidonic acid (AA) from the plasma membrane of intact cells. AA is a precursor of bioactive prostaglandins that are known to modulate insulin secretion by β-islet cells. C57BL/6 mice deficient in GX sPLA2 (GX KO) are protected from age-related defect in glucose tolerance. GX sPLA2 is expressed in mouse pancreatic islet cells. In this study we tested the hypothesis that GX sPLA2 regulates pancreatic insulin secretion. Methods and results: Glucose stimulated insulin secretion (GSIS) was measured in vivo in WT and GX KO mice and ex vivo using pancreatic islet cells isolated from WT and GX KO mice. To complement these studies, GSIS was also assessed in vitro using Min6 pancreatic beta cell lines with or without GX sPLA2 overexpression. GSIS was significantly increased in GX KO mice compared to WT mice, and in islet cells isolated from GX KO mice compared to WT mice. Consistent with this finding, Min6 cells overexpressing GX sPLA2 demonstrated significantly decreased GSIS compared to control cells. Expression of ABCA1 and ABCG1 mRNAs were significantly upregulated in islet cells from GX KO mice compared to WT mice, consistent with our previous report that GX sPLA2 negatively regulates LXR activity. However, there was no significant difference in cholesterol content between islets from WT and GX KO mice suggesting that altered LXR activity may not be the mechanism. Min6 cells overexpressing GX sPLA2 secrete significantly increased levels of PGE2- a known negative regulator of GSIS, in the medium compared to control cells. Treatment of these cells with NS398- an inhibitor of COX2 enzyme, abolished the decrease in GSIS shown by these cells. Conclusions: We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion most likely by enhancing PGE2 production.

Chemical Imaging of the Lipid and Cholesterol Distribution in the Plasma Membranes of Intact Cells

Domains enriched with cholesterol and sphingolipids, often referred to as lipid rafts, are believed to be present in the plasma membranes of eukaryotic cells and mediate important cellular functions, including cell signaling and virus budding. Changes in the abundances of cholesterol and sphingolipids influence many cellular processes. Yet, the distributions of cholesterol and sphingolipids within the plasma membrane have not been established. The inability to directly visualize lipids and cholesterol without the use of labels that may induce clustering renders characterizing the precise distribution of cholesterol and sphingolipids within the plasma membrane especially challenging. To address this problem, we have previously combined a chemically specific and spatially well-resolved imaging technique, high-resolution secondary ion mass spectrometry (SIMS), with metabolic labeling in order to image the distributions of sphingolipids within the plasma membranes of intact cells with at least 100 nm lateral resolution. We now report the use of this approach to image the sphingolipid and cholesterol distributions in parallel on the surface of intact fibroblast cells. We also quantitatively assess whether the cholesterol and sphingolipids are heterogeneously distributed and co-localized within the membrane. The results of these studies and their implications on models of membrane organization will be discussed.

Chemical Imaging of Lipid Organization in the Plasma Membranes of Intact Cells with High Lateral Resolution

Compartmentalization of the plasma membrane into domains of differing composition is required for proper cell function, but the extent and origins of this organization is poorly understood. The non-random distributions of specific membrane proteins has been established through the use of functionalized immunolabels and genetically encoded fluorescent protein constructs. Cholesterol- and sphingolipid-enriched microdomains, which are sometimes called lipid rafts, are also postulated to be present in the plasma membrane and mediate cell signaling, virus budding, and many other disease-related cellular processes. Advanced high-resolution fluorescence microscopy techniques have revealed the presence of biophysically distinct microdomains and cholesterol-dependent nanoclusters within the plasma membranes of living cells. However a chemically specific and spatially well-resolved imaging technique is required to unambiguously establish how specific lipid species are organized within the plasma membrane. Imaging mass spectrometry is a promising approach for visualizing lipid distribution within membranes with chemical specificity and submicron lateral resolution. Previously, the distributions of two isotopically labeled lipids within phase-separated model lipid membranes were successfully imaged with 100-nm-lateral resolution by using high-resolution secondary ion mass spectrometry (SIMS). Here we extend this approach to actual cells and chemically image the distributions of specific lipids within the plasma membrane with better than 100 nm lateral resolution.

Partitioning and Single-Molecule Diffusion Dynamics of Bodipy-FTY720 in Biomembranes

FTY720, a synthetic analog of sphingosine that has immunosuppressive properties, is the first oral drug to be approved by the U.S. FDA for treatment of multiple sclerosis (under the trade name Gilenya). We have examined the partitioning and diffusion properties of a fluorescent (Bodipy) FTY720 analog (Bdp-FTY720) in the plasma membrane (PM) of epithelial cells (HTB126) and model membranes. Based on two-channel confocal co-localization imaging with DiI-C12, a liquid-disordered phase marker, Bdp-FTY720 has an affinity for the liquid-disordered lipid phase in both binary and ternary phase GUVs. Bdp-FTY720 resides in both the cytoplasm and the plasma membrane of HTB126 cells. The translational diffusion of single Bdp-FTY720 molecules was investigated in the PM of intact cells and giant PM vesicles (GPMVs) using fluorescence correlation spectroscopy. In GPMVs, the two-dimensional anomalous (α=0.9) diffusion of Bdp-FTY720 is 3×10−8 cm2/s. The corresponding cytosolic Bdp-FTY720 diffuses mostly as a fast species (3.5×10−7 cm2/s); a small population (∼2%) diffuses at a slow rate (1.5×10−8 cm2/s). On the nanosecond time scale, the Bdp-FTY720 anisotropy in GPMVs decayed as a biexponential (f1=40 ± 20 ns, β1 = 0.10 ± 0.04, f2=2.4 ± 0.9 ns, β2=0.06 ± 0.02) with an estimated order parameter of 0.62 in the lipid bilayer. In the cytoplasm, the Bdp-FTY720 rotational motion is significantly slower (f1=120 ± 50 ns, β2 = 0.13 ± 0.01, f2=4.4 ± 0.7 ns, and β1 = 0.088 ± 0.009). These results indicate heterogeneous Bdp-FTY720 interactions in the cytoplasm compared with a hindered diffusion in the PM. These findings elucidate the biophysical characteristics of this new FTY720 analog and set the stage for its application in cell biology.

Activated Rac1 GTPase Translocates Protein Phosphatase 5 to the Cell Membrane and Stimulates Phosphatase Activity in Vitro

Physiological studies of ion channel regulation have implicated the Ser/Thr protein phosphatase 5 (PP5) as an effector of Rac1 GTPase signaling, but direct biochemical evidence for PP5 regulation by Rac1 is lacking. In this study we used immunoprecipitation, in vitro binding, cellular fractionation, and immunofluorescence techniques to show that the tetratricopeptide repeat domain of PP5 interacts specifically and directly with active Rac1. Consequently, activation of Rac1 promoted PP5 translocation to the plasma membrane in intact cells and stimulated PP5 phosphatase activity in vitro. In contrast, neither constitutively active RhoA-V14 nor dominant negative Rac1N17, which preferentially binds GDP and retains an inactive conformation, bound PP5 or stimulated its activity. In addition, Rac1N17 and Rac1(PBRM), a mutant lacking the C-terminal polybasic region required for Rac1 association with the membrane, both failed to cause membrane translocation of PP5. Mutation of predicted contact residues in the PP5 tetratricopeptide repeat domain or within Rac1 also disrupted co-immunoprecipitation of Rac1-PP5 complexes and membrane translocation of PP5. Specific binding of PP5 to activated Rac1 provides a direct mechanism by which PP5 can be stimulated and recruited to participate in Rac1-mediated signaling pathways.

PI(4,5)P2 Degradation Promotes the Formation of Cytoskeleton‐Free Model Membrane Systems

Giant plasma membrane vesicles (GPMVs) are cell-derived model membrane systems that undergo large-scale lipid phase separation when cooled below room temperature. Because of their presumably more physiological lipid composition, they are increasingly used as alternatives to synthetic model membranes. However, the exact mechanism of GPMV formation, and thus, effects of this process on the physiological integrity of the membrane are still unclear. Herein, we identify the key steps of GPMV formation and characterize their differences with respect to the plasma membrane of intact cells. Addition of GPMV-inducing reagents triggers a steady Ca2+ influx that is accompanied by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] degradation and phosphatidyl serine (PS) externalization before detachment of the cytoskeleton and the onset of vesicle formation. When comparing GPMVs to other cell-derived model systems, PI(4,5)P2 is not detectable in phase-separating plasma membrane spheres (PMSs) either, but is present in non-phase-separating blebs. GPMVs differ from the physiological state of the plasma membrane in the presence of specific lipids, which limits their use as model systems. Furthermore, we propose that PI(4,5)P2 influences the phase-separation behavior.