Total Internal Reflection Fluorescence Imaging Research Articles

EBP50 promotes focal adhesion turnover and vascular smooth muscle cells migration

The ezrin–radixin–moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild‐type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild‐type cells. Assembly and disassembly of focal adhesion—assessed by live-cell total internal reflection fluorescence imaging—in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions—determined by fluorescence recovery after photobleaching—was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.

Pore Determinants of KCNQ3 K+ Current Expression

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels—the C-terminus and the pore region—have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.

Pore Helix-S6 Interactions Are Critical in Governing Current Amplitudes of KCNQ3 K+ Channels

Two mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the end of the D-helix, where KCNQ3 possesses a histidine. However, H2O2-mediated enhancement of KCNQ4 currents was identical in the C643A mutant, and KCNQ3 H646C produced homomeric or heteromeric (with KCNQ2) currents similar to those of wild-type KCNQ3, ruling out this divergent residue as underlying the small KCNQ3 amplitudes. In KcsA, F103 in S6 is critical for pore-mediated destabilization of the conductive pathway. We found that mutations at the analogous F344 in KCNQ3 dramatically decreased the KCNQ3 currents. Total internal reflection fluorescence imaging revealed only minor differential surface expression among the wild-type and mutant channels. Homology modeling suggests that the effects of the F344 mutants arise from the disruption of the interaction between F344 and A315 in the pore helix. These data support a secondary role of the C-terminus, compared with pore helix-S6 interactions, in governing KCNQ3 current amplitudes.

Differences in adhesion and protrusion properties correlate with differences in migration speed under EGF stimulation

BackgroundCell migration plays an essential role in many biological processes, such as cancer metastasis, wound healing and immune response. Cell migration is mediated through protrusion and focal adhesion (FA) assembly, maturation and disassembly. Epidermal growth factor (EGF) is known to enhance migration rate in many cell types; however it is not known how FA maturation, FA dynamics and protrusion dynamics are regulated during EGF-induced migration. Here we use total internal reflection fluorescence (TIRF) microscopy and image analysis to quantify FA properties and protrusion dynamics under different doses of EGF stimulation.ResultsEGF was found to broaden the distribution of cell migration rates, generating more fast and slow cells. Furthermore, groups based on EGF stimulation condition or cell migration speed were marked by characteristic signatures. When data was binned based on EGF stimulation conditions, FA intensity and FA number per cell showed the largest difference among stimulation groups. FA intensity decreased with increasing EGF concentration and FA number per cell was highest under intermediate stimulation conditions. No difference in protrusion behavior was observed. However, when data was binned based on cell migration speed, FA intensity and not FA number per cell showed the largest difference among groups. FA intensity was lower for fast migrating cells. Additionally, waves of protrusion tended to correlate with fast migrating cells.ConclusionsOnly a portion of the FA properties and protrusion dynamics that correlate with migration speed, correlate with EGF stimulation condition. Those that do not correlate with EGF stimulation condition constitute the most sensitive output for identifying why cells respond differently to EGF. The idea that EGF can both increase and decrease the migration speed of individual cells in a population has particular relevance to cancer metastasis where the microenvironment can select subpopulations based on some adhesion and protrusion characteristics, leading to a more invasive phenotype as would be seen if all cells responded like an “average” cell.

Hydrogen peroxide mediates oxidant‐dependent stimulation of arterial smooth muscle L‐type calcium channels

Changes in calcium and redox status influence multiple cellular processes. We tested the hypothesis that the ubiquitous reactive oxygen species hydrogen peroxide mediates oxidant‐dependent stimulation of arterial smooth muscle L‐type calcium channels. Using a combinatorial approach including conventional electrophysiology and total internal reflection fluorescence imaging, we found that application of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L‐type calcium channels. Similarly, oxidant‐dependent stimulation of L‐type calcium channels by the vasoconstrictor angiotensin II was abolished by intracellular application of catalase. Catalase also prevented angiotensin II from increasing localized subplasmalemmal generation of endogenous reactive oxygen species previously associated with colocalized calcium influx through L‐type channels. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with superoxide dismutase had no effect on angiotensin‐dependent stimulation of L‐type calcium channels. We conclude that hydrogen peroxide is necessary for oxidant‐dependent stimulation of rat arterial smooth muscle L‐type calcium channels.

Quantification of Membrane Protein Inhibition by Optical Ion Flux in a Droplet Interface Bilayer Array

Optical platforms for assaying membrane protein function offer a promising route to scalable high-throughput screening (see picture). For the first time quantitative measurements of membrane protein inhibition are reported in an optically addressable lipid bilayer array. Wide-field total internal reflection fluorescence (TIRF) imaging of Ca2+ flux enables the quantification of α-hemolysin inhibition by γ-cyclodextrin.

Hydrogen peroxide mediates oxidant-dependent stimulation of arterial smooth muscle L-type calcium channels

Changes in calcium and redox homeostasis influence multiple cellular processes. Dysregulation of these signaling modalities is associated with pathology in cardiovascular, neuronal, endocrine, and other physiological systems. Calcium and oxidant signaling mechanisms are frequently inferred to be functionally related. To address and clarify this clinically relevant issue in the vasculature we tested the hypothesis that the ubiquitous reactive oxygen molecule hydrogen peroxide mediates oxidant-dependent stimulation of cerebral arterial smooth muscle L-type calcium channels. Using a combinatorial approach including intact arterial manipulations, electrophysiology, and total internal reflection fluorescence imaging, we found that application of physiological levels of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L-type calcium channels. Similarly, oxidant-dependent stimulation of L-type calcium channels by the vasoconstrictor ANG II was abolished by intracellular application of catalase. Catalase also prevented ANG II from increasing localized subplasmalemmal sites of increased oxidation previously associated with colocalized calcium influx through L-type channels. Furthermore, catalase largely attenuated the contractile response of intact cerebral arterial segments to ANG II. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with SOD had no effect on ANG II-dependent stimulation of L-type calcium channels. From these data we conclude that hydrogen peroxide is important for oxidant-dependent regulation of smooth muscle L-type calcium channels and arterial function. These data also support the emerging concept of hydrogen peroxide as a biologically relevant oxidant second messenger in multiple cell types with a diverse array of physiological functions.

Experimental Studies on Blinking Behavior of Single InP/ZnS Quantum Dots: Effects of Synthetic Conditions and UV Irradiation

In this study, we first synthesized a series of highly fluorescent InP/ZnS core/shell QDs under varied reaction conditions and then systematically investigated the blinking behaviors of QDs by single molecule imaging technique. In the measurement of blinking processes, the PL intensity trajectories of single QDs with different emission peak were recorded by a total internal reflection fluorescence (TIRF) imaging system. We observed that particle size, molar ratio of myristic acid/indium (MA:In), and the overcoating of ZnS shell had significant influence on the blinking behaviors of InP/ZnS QDs. Statistical analysis documented that both the on and off events (mon and moff) followed the power-law distributions P(ton/off) = Bt–mon/off, and the values for mon and moff were in the ranges of 1.3–1.7 and 1.4–1.9, respectively. As the wavelength of emission peak increased from 565 to 646 nm, the on-time fraction changed from 30% to 70%. The QDs prepared under higher MA/In ratio possessed a longer on-time fraction. The optimal overcoating time of the ZnS shell was about 3 h for blinking suppression in this case. Furthermore, we found that UV irradiation of QDs certainly suppressed the off-time fraction by reducing surface traps. We were able to control the blinking behavior of QDs by varying the synthesis parameters, and prepared InP/ZnS QDs with the on-time fraction up to 80% for future biological and photoelectric applications.

Mixed-surface, lipid-tethered quantum dots for targeting cells and tissues

Quantum dots (QDs), with their variable luminescent properties, are rapidly transcending traditional labeling techniques in biological imaging and hold vast potential for biosensing applications. An obstacle in any biosensor development is targeted specificity. Here we report a facile procedure for creating QDs targeted to the cell membrane with the goal of cell-surface protease biosensing. This procedure generates water-soluble QDs with variable coverage of lipid functional groups. The resulting hydrophobicity is quantitatively controlled by the molar ratio of lipids per QD. Appropriate tuning of the hydrophobicity ensures solubility in common aqueous cell culture media, while providing affinity to the lipid bilayer of cell membranes. The reaction and exchange process was directly evaluated by measuring UV absorption spectra associated with dithiocarbamate formation. Cell membrane binding was assessed using flow cytometry and total internal reflection fluorescence imaging with live cells, and tissue affinity was measured using histochemical staining and fluorescence imaging of frozen tissue sections. Increases in cell and tissue binding were found to be regulated by both QD hydrophobicity and surface charge, underlying the importance of QD surface properties in the optimization of both luminescence and targeting capability.

Clathrin-Mediated Endocytosis Introduces a Nonergodic Diffusion Process in the Plasma Membrane

Tracking individual potassium channels in the plasma membrane reveals complex dynamics involving anomalous diffusion. Theoretical models show that anomalous subdiffusion can be caused by several different processes. In particular, transient binding events, modeled by a continuous time random walk (CTRW), may not only induce anomalous subdiffusion but also weak ergodicity breaking, that is, the ensemble and time averages do not coincide. We studied the physical mechanism underlying Kv2.1 and Kv1.4 potassium channel anomalous dynamics by performing time series analysis of extensive single molecule tracking in the membrane of live mammalian cells. We find ample evidence showing that the ensemble and temporal distributions are different. Our data reveal that two anomalous subdiffusion processes simultaneously coexist and only one of them is ergodic. Weak ergodicity breaking is found to be maintained by immobilization events as long as 60 seconds. In the presence of either actin or chlathrin inhibitors, ergodicity is recovered. In order to elucidate the effects of clathrin endocytosis on Kv2.1 trafficking and diffusion, we have performed simultaneous total internal reflection fluorescence imaging of quantum-dot-tagged Kv2.1 and RFP-tagged clathrin. Our results show that Kv2.1 channels are frequently recycled from and to the plasma membrane. Retrieval of Kv2.1 from the membrane is found to be attained via a clathrin-mediated endocytic pathway. These endocytic/insertion processes are needed to maintain a nonergodic CTRW. Furthermore, the Kv2.1 stalling events colocalize with clathrin clusters. These results suggest that abortive endocytic events are responsible for the observed channel immobilization in the plasma membrane.

Modulation of Integrin-Extracellular Matrix Adhesions by RhoA and Src

RhoA and Src are considered potential modulators of the mechanical signaling that promotes actin polymerization and focal adhesion formation. Here, we performed adhesion force spectroscopy measurements on vascular smooth muscle cells (VSMC) expressing RhoA or Src (wild type, constitutively active or dominant negative mutants) using an atomic force microscope (AFM) tip functionalized with fibronectin. Integrin alpha5beta1-fibronectin adhesion force, local cell stiffness at the point of contact, and adhesion probability were determined. The results show that the mechanical properties of the VSMC as well as integrin-fibronectin adhesion force are modulated by RhoA and Src. Inhibition of RhoA activity induces low cytoskeletal tension and decreased integrin-fibronectin adhesion force. Also, VSMC expressing RhoA or Src dominant negative constructs were found to be the softest. Conversely, RhoA and Src activation increased alpha5beta1 integrin-fibronectin adhesion force and cell stiffness. To independently verify the effect of RhoA and Src on cytoskeletal changes, fluorescence imaging experiments were performed to quantify actin fibers (confocal imaging of cells co-expressing RhoA-GFP and actin-mRFP) or protein phosphorylation at focal adhesions (total internal reflection fluorescence imaging of cells co-expressing Src-mCherry and dSH2-YFP), respectively. Consistent with the AFM measurements, we observed an increase in actin fibers, and in protein phosphorylation at focal adhesion sites in cells expressing constitutively active RhoA and Src, respectively. In addition, these two proteins were maximally decreased by expression of dominant negative RhoA and Src. Taken together, these results show that RhoA and Src are critical modulators of the mechanical signaling cross-talk between the cell and the extracellular matrix.

Imaging Fluorescence Correlation Spectroscopy Characterizes Membrane Dynamics and Organization

Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS), a camera based FCS approach, characterizes membrane dynamics by measuring the diffusion coefficient at every point on a membrane patch and accesses membrane organization by using the so-called ΔCCF approach in which the cross-correlation of neighboring pixels is used to characterize membrane heterogeneity. Here we discuss the various technical aspects of ITIR-FCS and ΔCCF and the influence of time resolution, measurement length, and laser power on the reliability of the results. We show that ITIR-FCS can unambiguously determine the point spread function of a system and that this measurement is essential to obtain proper absolute values for diffusion coefficients and concentrations. We apply ITIR-FCS first to a range of supported lipid bilayers with increasing complexity from single lipid bilayers to binary and ternary mixtures consisting of different phospholipids, sphingomyelin, and cholesterol. ITIR-FCS yields very consistent diffusion coefficients over dozens of bilayer preparations. We complement this bottom-up approach, whereby the complexity of an artificial membrane is increased step-by-step to approach the complexity of the cellular plasma membrane, by a top-down approach in which we characterize live cell membranes in their natural state and altered in composition by a range of biochemical approaches. Since a single ITIR-FCS measurement can be binned per software after the recording, it is ideal for testing the FCS diffusion laws, which determine the dependence of the diffusion coefficient on the size of the observation area, and to demonstrate the existence of lipid domains in ternary lipid mixtures and cell membranes.

Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue β-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion.

Optical Recording of Single Channel TRPV1 Activity in Living Cells

The multi-modal cation channel transient receptor potential vanilloid 1 (TRPV1), which is activated by heat, protons and a variety of endogenous and exogenous ligands amongst other stimuli, senses the extracellular environment. To investigate how the cell tunes its sensitivity to this environment, we asked whether the cell imposes functional constraints on TRPV1 by exercising a control over its localization. We and others have previously shown that TRPV1 channels can be regulated by trafficking and that surface expression of the channel is increased by treatment with Nerve Growth Factor (NGF), which induces inflammation. Investigation of fluorescently-labeled single channels by total internal reflection fluorescence (TIRF) imaging reveals a broad distribution of channel movement in the plasma membrane from immobile up to a lateral diffusion of 1.9 micrometer2/sec. We next assayed the functional subset of this large distribution by sparklet analysis, which employs an intracellular fluorescent calcium indicator as a probe for TRPV1 opening events. TRPV1 sparklets appear as both mobile and immobile. Mobile TRPV1 sparklets exhibit an average effective diffusion of 0.072 micrometer2/sec (n=9) with a maximal effective diffusion of .15 micrometer2/sec for one channel. The time dependent behavior of the more mobile subset of sparklets reveals that their mobility decreased, and our ongoing effort to understand the spatio-temporal behavior of TRPV1 is directed at how the channel's activity slows its movement. Funding provided through the University of Washington Training Grant in Cardiovascular Pathology (NIH) and the National Eye Institute (NIH).

TIRFM-N&B Analysis of FGFR1 Clustering in Response to NCAM and FGF2

Neural cell adhesion molecule (NCAM) is a nonconventional ligand for fibroblast growth factor receptor-1 (FGFR1). NCAM exerts a peculiar control on the intracellular trafficking of FGFR1, resulting in a specific cellular response, which is remarkably different from that elicited by the canonical ligand FGF2 (Francavilla et al., JCB 2009).We studied the dynamics and oligomerization of cell-surface FGFR1 using a biologically active FGFR1-mEGFP chimera expressed in HeLa cells. FGFR1-mEGFP was shown to bind FGF2 and NCAM, and to undergo phosphorylation and internalization.The monomer-dimer-oligomer dynamics of FGFR1 in response to NCAM and FGF2 was studied as a function of time by total internal reflection fluorescence (TIRF) microscopy combined with the number and brightness (N&B) analysis.HeLa/FGFR1-mEGFP cells were starved overnight, and time series of TIRF images were collected up to 40-70 min after ligand-mediated stimulation. N&B analysis was carried out using SimFCS SW (E. Gratton) and EMCCD camera calibrated according to Unruh and Gratton (Biophys. J. 2008).At the membrane level, NCAM binding induced a reorganization of FGFR1 molecules different from that elicited by FGF2. FGF2 induced a sustained dimerization of the receptors, followed by internalization and degradation. Conversely, in the presence of NCAM, FGFR1 underwent fast assembly in dimers and few high order oligomers, followed by disassembly back to monomers. Assembly and disassembly cycles were detected for more than 1 hour after NCAM addition, and they might represent internalization and recycling of ligand-free receptors at the cell membrane.Our data provides novel insights into the cell biological basis of the dichotomy between NCAM and FGF2 with regard to the ligand-induced response of FGFR. These findings also point to the novel concept that the degree of clustering at the cell surface dictates the intracellular fate of an endocytic receptor.

Selective Ion Changes during Spontaneous Mitochondrial Transients in Intact Astrocytes

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na+ concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na+ concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca2+ concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg2+ concentration accompanying mitochondrial Na+ spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na+-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

Single-channel Ca2+ imaging implicates Aβ1–42 amyloid pores in Alzheimer’s disease pathology

Oligomeric forms of Aβ peptides are implicated in Alzheimer's disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca(2+)) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca(2+)-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca(2+) influx in Xenopus laevis oocytes. Aβ1-42 oligomers evoked single-channel Ca(2+) fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (P(o)) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high P(o), large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca(2+)-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.

Probing the regulation of TASK potassium channels by PI(4,5)P2 with switchable phosphoinositide phosphatases

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of Gαq is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) as the cause for channel closure or to a direct inhibitory interaction of active Gαq with the channel. Here, we address the role of PI(4,5)P2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell.When expressed in CHO cells, TASK-1- and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P2-5'-phosphatase. Depletion of both PI(4,5)P2 and PI(4)P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P2 and PI(4)P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P2.

Redistribution of caveolae during mitosis

Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis.