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. Author manuscript; available in PMC: 2026 Feb 26.
Published in final edited form as: Methods Mol Biol. 2021;2251:91–104. doi: 10.1007/978-1-0716-1142-5_6

Imaging the nanoscale distribution of phosphoinositides in the cell plasma membrane with single-molecule localization super-resolution microscopy

Fan Fan 1, Chen Ji 1,*, Xuelin Lou 1
PMCID: PMC12935277  NIHMSID: NIHMS2141617  PMID: 33481233

Abstract

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity, regulate protein function and organization, and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5 trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.

Keywords: membrane lipid; TIRFM; PALM; dSTORM; PtdIns(4,5)P2; INS-1 cells; insulin secretion; exocytosis; endocytosis; cell signaling

1. Introduction

The nanoscale organization of the plasma membrane (PM) of eukaryotic cells is not fully understood. It is clear that the PM composes a complex topographic structure of proteins and lipids that are mingled with protein clusters [1,2] or lipid rafts [3]. These types of molecule organizations facilitate the efficiency, fidelity, and specificity of cellular signaling in time and space [3]. However, it is unclear whether a similar organization pattern applies to phosphoinositides (PIs) in the PM.

Phosphoinositides (PIs) include a family of seven members that are derived from reversible phosphorylation of the inositol ring at position-3, −4 or/and −5 via PI kinases and phosphatases [4,5]. These PIs are compartmentalized in live cells with dynamic equilibrium. PtdIns(4,5)P2 predominantly locates in the inner leaflet of the PM and participate in a variety of cellular processes [4,6], including signal transduction, actin dynamics, membrane trafficking, and ion channel activity. PtdIns(4,5)P2 is produced through PtdIns4P 5-kinases (PIP5Ks) activation and can be converted into other PIs, as well as the second messenger inositol 1,4,5-trisphosphate (IP3) and diacylglycerol [7]. Our previous work demonstrates important function of PtdIns(4,5)P2 signaling in synaptic transmission [8-10] and insulin secretion [11]. PtdIns4P is synthesized by PI4KIIIα in the PM [12,13]. PtdIns4P and PtdIns(4,5)P2 determine the PM properties and subcellular localization of many PM proteins [14,13]. In contrast, PI(3,4,5)P3 has a lower level in the PM, but it also affects actin dynamics, phagocytosis, and hormone secretion, among other functions [15,16].

PtdIns(4,5)P2 interacts with proteins through its negative charges and structured domains, such as pleckstrin homology (PH), ENTH/ANTH, FERM, FYVE, PX, and TARF domains [17]. The development of high affinity PtdIns(4,5)P2 biosensor (Kd = 2 μM) based on the PHPLC-δ1 domain [18] allows visualizing PtdIns(4,5)P2 distribution and dynamics in live cells. However, the spatial organization of PtdIns(4,5)P2 in the PM appears diverse and controversial. Inconsistent results have been reported using different cells and experimental systems, ranging from uniform distribution [19-21], large patches [22,23], to small dense puncta [24,23,25,26]. At the nanoscale level, the work using stimulated-emission depletion (STED) microscopy reported the presence of abundant dense PtdIns(4,5)P2 clusters (~73 nm in diameter) in PC12 PM sheets [27], and such clusters drive syntaxin-1A clustering. In contrast, another study using the rapid freezing electron microscopy (EM) reported loose microdomains of PtdIns(4,5)P2 but no dense clusters [28,29]. To better understand this issue, we have developed a different method: single-molecule approach. We combine it with the cell PM sheet method adapted from previous method [30,31] to study PIs nanoscale distribution in INS-1 cell membrane [32,33].

Single-molecule localization microscopy (SMLM) significantly improves the spatial resolution of light microscopy. Because it marks accurate coordinates of molecules when their fluorescent probes are photo-activated or photo-converted at well-separated locations and times, and thus offers a better spatial resolution than structured-illuminated microscopy (SIM). The use of genetic proteins allows specific labeling, and the stunning spatial resolution makes SMLM a powerful tool to study cell biology and macromolecule complexes [34-40]. The method offers a new way to study the subcellular organization of PIs using optical microscopy, which avoids harsh sample processing in conventional EM that can distort normal lipid organization [28]. We apply the SMLM super-resolution method to PtdIns(4,5)P2 in cellular PM, a similar method may be adapted to study the nanoscale distribution of other PIs.

2. Materials

2.1. DNA constructs of photo-switchable PI probes for PALM

C1 and N1 cloning vectors (Clontech), PAmCherry1 [41], mEos3.1 [38], and the following plasmids subcloned in our previous work [32] including iRFP-PAmCherry1, iRFP-mEos3.1, iRFP-PAmCherry1-PHPLCδ1, iRFP-PAmCherry1-PHosh2, iRFP-PAmCherry1-PHGRP1.

2.2. Cell culture

1. Insulin-secreting INS-1 832/13 cells (from Dr. Christopher B. Newgard, Duke University), passages 50–65. Other cells grown on coverslips may be used similarly.

2. RPMI-1640 culture medium: 11.1 mM glucose, 10 mM HEPES, 10 mM glutamine, 10 mM sodium pyruvate, 50 μM β-mercaptoethanol, and 10% FBS (Atlanta Biological)

3. Round coverslips (#1.5, 18 mm in diameter)

4. Fibronectin, 30 μg/ml

5. Transfection reagents. We use lipofectamine 3000 (Life Technologies) here, other reagents similar or transfection methods may be used after optimization.

2.3. Membrane sheet preparation

1. 500 μg/ml poly-d-lysine

2. PBS buffer

3. 1 mM EGTA

4. A metal plate: We used a one-foot square metal plate to maintain the low temperature of the plasma membrane sheets, other methods that keep samples at the constant temperature may work.

5. Fixative: 4% paraformaldehyde (PFA), 0.2% glutaraldehyde in PBS

2.4. Primary and secondary antibodies for dSTORM

1. PBS

2. 50 mm NH4Cl in PBS

3. 0.1% sodium borohydride in PBS.

4. Blocking buffer: 5% (v/v) normal goat serum, 5% (v/v) BSA, and 50 mm NH4Cl in PBS

5. Primary antibody for PtdIns(4,5)P2 (Santa Cruz Biotechnology, 1:300 dilution)

6. Secondary F(ab′)2 goat anti-mouse Alexa Fluor 647 (Invitrogen, 1:300)

2.5. Nanoscopy: PALM and dSTORM

1. TetraSpeck beads (200-nm diameter, Invitrogen)

2. Imaging chambers with and without temperature control

3. Dual-channel temperature controller

4. Extracellular buffer for live-cell imaging: 135 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 3 mM glucose and 20 mM HEPES (pH adjusted to 7.3 with NaOH)

5. dSTORM imaging buffer: 50 mM Tris-HCl, 10 mM NaCl, 10% glucose, 0.8 mg/mL glucose oxidase, 40 μg/mL catalase and 10 mM cysteamine (MEA) (Sigma), pH 8.0.

6. SMLM system. We have built the SMLM based on Nikon Ti-E Eclipse inverted microscope. It is capable of TIRFM and spinning disk confocal imaging besides SMLM (PALM and dSTORM). The system has the Ti-ND6-PFS Perfect Focus and multiple objectives (CFI Plan Fluor ×4; NA 0.13, WD 17.2 mm; CFI Plan APO λ ×20, NA 0.75, WD 1.00 mm; Plan APO VC ×60 oil, NA 1.4, WD 0.13 mm; and APO ×100 oil, NA 1.49, WD 0.12 mm), a spinning disk (CSU X-1, 10,000 rpm, Yokogawa), a Ti-TIRF motorized illuminator unit, and an Agilent MLC400 high-power monolithic laser combiner SP with 405-, 488-, 561-, and 642-nm lasers. The EMCCD (iXon X3 DU897, Andor) and Neo-sCMOS cameras (Andor, 6.5 × 6.5 μm pixel size) are installed at the left and right imaging ports to acquire confocal and TIRFM/SMLM images, respectively, under the control of NIS-Elements AR software. Other systems with similar functionalities may be used.

7. MATLAB and MLE_GPU algorithm [32]. Other similar algorithms and software with single-molecule localization function may be used similarly.

3. Methods

3.1. Cloning the dual-color PALM probes

1. PCR cloning. We focused on the PALM probe of PtdIns(4,5)P2 with PHPLCδ1 [18,42] (from Tamas Balla at NIH) here but other single-molecule probes for PI4P and PI(3,4,5)P3 can be also generated.

2. Photo-activatable PAmCherry1 and photo-switchable mEos3.1 are PCR-amplified, which are used to replace enhanced GFP (EGFP) in C1 and N1 cloning vectors (Clontech) with AgeI and BglII and AgeI and NotI, respectively (see Note 1).

3. iRFP is PCR-amplified and inserted before the PAmCherry1 with NheI and AgeI to make the iRFP-PAmCherry1-C1 vector (see Notes 2 and 3).

4. PHPLCδ1, PHosh2, and PHGRP1 domains (from Pietro DeCamilli at Yale) can be PCR-amplified and inserted after iRFP-PAmCherry1 or mEos3.1 vector with BglII and BamHI to generate the new PALM probes iRFP-PAmCherry1-PHPLCδ1, iRFP-PAmCherry1-PHosh2, iRFP-PAmCherry1-PHGRP1, and mEos3.1-PHPLCδ1. We have generated these probes in our previous work [32,33].

3.2. INS-1 cell culture and DNA transfection

1. Grow INS-1 cells on #1.5 (18 mm, round) coverslips pre-coated with 30 μg/mL fibronectin with the standard culture protocol [43]. Wait till cells reach ~60% confluence.

2. Transfect INS-1 cells with iRFP-PAmCherry1-PHPLCδ1 DNA using Lipofectamine-3000 following the manufacture protocol. iRFP-PAmCherry1 with ΔCMV promoter is transfected in parallel as a control probe.

3. Continue growing cells for another 48 hours till fluorescence probes are visible. Use them for live-cell imaging or membrane sheet generation (see Note 4). Figure 1A shows a typical distribution of fluorescence probers in live INS-1 cells under TIRFM.

Figure 1. PI(4,5)P2 spatial organization in INS-1 cell PM.

Figure 1.

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TIRFM images of the PM are taken at four different conditions below: A) Intact PM of two live INS-1 cells expressing EGFP-PHPLCδ1 at low and elevated levels; B) The PM sheet fixed at 4 °C with 4% PFA + 0.2% GA and immunostained with PI(4,5)P2 antibody; C) The PM sheet expressing iRFP-PAmCherry1-PHPLCδ1; D) The PM sheet fixed at room temperature with 4% PFA and immunostained with PI(4,5)P2 antibody. Note the images in B and C (but not D) recapitulate the spatial distribution of fluorescence probes in live cells in A. Scale bars: 5 μm in A, 3 μm in B - D.

3.3. PM sheet generation and fixation

1. To prepare coverslips, coat coverslips on only one-side with ~0.5 - 1 mL of 500 μg/ml Poly-D-Lysine (PDL; diluted in dH2O) for 1 ~ 2 h, drain excessive PDL by placing a tissue paper at coverslip edge, place the coverslips on the metal plate, and keep it at 4 °C in a refrigerator for later use.

2. Wash pre-transfected INS-1 cells three times with ~0.5 - 1 mL cold (4 °C) PBS containing 1 mM EGTA and carefully drain PBS with tissue paper.

3. Place these coverslips with cells on the PDL-coated coverslips on the pre-chilled metal plate with tweezers, keep the cells and plate back in the refrigerator (4 °C) for 8 min (see Note 5) to allow cell attachment (Figure 2).

Figure 2. The scheme of membrane sheet preparation and PALM imaging with the dual-color probe in INS-1 cells.

Figure 2.

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A) The membrane preparation cartoon. Place the cells on the coverslip (with cells facing down) onto a PDL-coated coverslip (a), and after cell attachment for 7 ~ 10 min at 4 °C, peel off the top coverslips and produce the PM sheet (b), followed with fixation and super resolution imaging (c). B) The dual-color PtdIns(4,5)P2 probe expressed in INS-1 cells for TIRFM and PALM. The peak wavelengths of excitation and emission are indicated. iRFP is used to identify the cells with probe expression under TIRFM, without consuming PAmCherry1 molecules before PALM acquisition.

4. Take out the metal plate from the refrigerator and gently peel off the top coverslip using tweezers. This step produces a thin layer of PM sheet on the PDL-coated coverslip.

5. Gently wash the PM sheets with ~0.5 - 1 ml ice-cold PBS, and then fix with ~0.5 - 1 ml ice-cold fixative for 15 min at 4 °C (see Notes 6 and 7).

6. Image the PM sheets or leave them in ~0.5 - 1 ml PBS at 4 °C for short storage in dark (see Note 8).

3.4. PALM imaging of the PM sheets

1. Add TetraSpeck beads (prediluted with PBS containing extra 50 mM MgCl2) to the imaging chamber, let them settle down for 10 minutes on the coverslip, and then wash away those mobile beads with PBS. Other drift-correction markers similar to TetraSpeck beads may work as well.

2. Place the chamber onto the SMLM sample stage, find the PM sheets expressing PH probes in the iRFP-channel (642 nm excitation, 700/75 nm emission) under the TIRF mode (100X lens, NA = 1.49),

3. Adjust the membrane sheets position so that the image area has one or two TetraSpeck beads nearby (see Note 9).

4. Draw an imaging area covering both PM sheets and beads and take an iRFP TIRF image (642 nm excitation, 700/75 nm emission) as the reference of future imaging reconstruction.

5. Switch to PAmCherry1-channel (561 nm excitation, 600/50 nm emission), bleach the background fluorescence from the membrane using the full intensity of 561-laser for 10-20 s.

6. Set imaging parameters such as cameral binning (2x2, effective pixel size 130 x130 nm) and exposure time, and the fast acquisition protocol “ND sequence acquisition”.

7. Start imaging PAmCherry1 (561 nm laser at 100% power, 600/50 nm emission) at 50 ms/frame with simultaneous 405 nm laser excitation at low intensity to activate PAmCherry1. The low 405 activation power is key to achieve better imaging resolution (see Notes 10 and 11). All the images are collected continuously.

8. The acquisition continues till PAmCherry1 signal is no longer activated, with 10,000 – 40,000 images collected from each INS-1 cell.

3.5. PALM imaging in live cells

This is performed as described above for the PM sheets with minor changes.

1. Set up imaging temperature. Maintained cells at 35 °C with the temperature controller under constant perfusion of the extracellular buffer.

2. Add TetraSpeck beads in the imaging chamber for 10 minutes as described above.

3. Find INS-1 cells expressing mEOS3.1-PHPLCδ1 under TIRFM at the green-channel (488 nm excitation, 525/50 nm emission) (see Note 12) and take an image with TetraSpeck beads.

4. Switch to the red-channel (561 nm excitation, 600/50 nm emission), bleach background fluorescence of the membrane with the 561 laser for 10 - 20 s, and acquire PALM images (561 nm excitation, 600/50 nm emission) with optimal 405 excitation (1% of full power) at 10 ms/frame (see Notes 13, 14, and 15).

3.6. Immunostaining the PM sheets and dSTORM imaging

1. Prepare fixed membrane sheets from normal INS-1 cells at 4°C as described above (Section 3.3).

2. Wash the coverslips three times with ice-cold PBS containing 50 mM NH4Cl, quench with 0.1% sodium borohydride in PBS for 7 min and wash with PBS w/o 50 mM NH4Cl.

3. Block the samples with Blocking buffer

4. Add the primary PtdIns(4,5)P2 antibody (1:300 dilution) in blocking solution for 1 h. Wash three times (10 min each) with PBS containing 50 mM NH4Cl.

5. Add the secondary F(ab’)2-goat-anti-mouse, Alexa Fluor 647 ( Invitrogen, 1:300) in blocking buffer for 1 h, and then wash three times. Other far-red fluorophores may be used, but we choose Alexa Fluor 647 here because it has been well-characterized and proven in SMLM application.

6. Fix samples with fixative for 15 min, wash three times (7 min each time), followed with dSTORM imaging. Other samples are temporally kept in PBS at 4°C.

7. To perform dSTORM using the SMLM system next, prepare fresh dSTORM imaging buffer

8. Find the PM sheets and TetraSpeck beads, adjust acquisition parameters and illumination angle (slightly larger than the TIRF angle), and add dSTORM buffer to the samples.

9. Bleach the samples with the full intensity of 642 nm laser, and then adjust UV laser to the optimal intensity for AF-647 photoactivation.

10. Collect images at 40 ms/frame (642 nm excitation, 700/75 nm emission) when individual fluorescent molecules are spatially isolated. ~30,000 images are collected from individual cells for reconstruction.

3.7. Point localization, image process, and reconstruction for super resolution imaging

1. Transfer image files to the imaging analysis station, convert image stacks from “nd2” to “tiff” format. Launch the imaging reconstruction program customer-written in MATLAB as described previously [38,32].

2. Localize individual molecules from each image by a wavelet transform algorithm in a 7x7 pixel area. The precise locations are determined by the local maximum of each fluorescence point with a mask of 5 x 5 pixels after 2-D gaussian fitting using the MLE_GPU algorithm [44]. Optimize the parameters for point detection by visual inspection before the reconstruction (see Note 16). Minor drift is corrected with TetraSpeck beads. A typical PALM image of the fixed membrane sheets is shown in (Figure 3)

Figure 3. The single-molecule localization microscopy reveals the nanoscale distribution of PI(4,5)P2 probes in the INS-1 cell PM.

Figure 3.

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A-B) TIRFM and PALM images of the INS-1 cell PM sheet expressing iRFP-PAmCherry1-PHPLCδ1. C1-C2) Enlarged views in B. Note the homogeneous PI(4,5)P2 probe distribution in the major PM area and some sparse micro-domains (arrows) enriched with PI(4,5)P2 probes. D) enlarged view of the boxed area in (C1). D) The histogram of PI(4,5)P2 micro-domain size in INS-1 membrane sheets (383.3 ± 14.3 nm in diameter). Scale bar: A-B: 3 μm; C1-C2: 500 nm; D: 200 nm (adapted from Ji et al, 2015).

3. For PALM imaging of the fixed PM sheets expressing iRFP-PAmCherry1-PHPLCδ1, all the image stacks from the same sample are reconstructed and combined into a single super resolution image. For live-cell PALM, the image stack of mEOS3.1-PHPLCδ1 is separated into several 1000-frame stacks, and each stack is reconstructed as a single PALM image (see Note 17). This produces a temporal resolution of 10 s in the time series of PALM images.

4. After the image reconstruction, quantify molecule density and micro-domain density and size with the home-written program in MATLAB [32]. Cluster analysis is performed with pair-correlation [45].

4. Notes

  1. Both PAmCherry1 and mEos3.1 are used as PALM probes for different purposes. The former shows a negligible level of photo-blinking and excellent monomeric property in cells [46], and thus it is a good option for quantitative PALM. The dual-color PAmCherry1 probe we generated shows typical monomeric behavior with little photo-blinking in cells [32], suggesting that this probe in cellular environment preserves the photophysical and biochemical properties of single PAmCherry1 alone [46,36,47] and thus avoids overcounting and probe oligomerization [32].

  2. iRFP identifies the cells with PALM probe expression. This is performed at the 647-channel to avoid activating PAmCherry1 or converting mEos3.1 (Figure 2B) and reserve all the PALM probe molecules for quantitative PALM imaging.

  3. We have used a truncated CMV promoter (ΔCMV) [48] to express iRFP-PAmCherry1 at a low level to avoid the probe molecule oligomerization (as the single-molecule control). It is suitable to characterize the single-molecule property (e.g., photo blinking and monomer distribution) of the dual-color probe in a cellular environment.

  4. Live-cell imaging must be acquired at a speed before the objectives of interest move during acquisition. We used 200 ms/frame to image the probes under TIRFM.

  5. The incubation time is 7 - 10 min for cells to attach on the PDL-coated surface. A longer duration should be avoided since the refrigerator environment is dry.

  6. PtdIns(4,5)P2 is sensitive to fixation conditions, such as fixation composition and temperature [32]. The sample fixation conditions commonly used in immunocytochemistry can disrupt PtdIns(4,5)P2 distribution and compromise data interpretation (Figure 1D). By comparing different fixation conditions in INS-1 cells, we find that the fixation condition we used here (4% PFA + 0.2% GA at 4 °C) better preserves PtdIns(4,5)P2 distribution (Figure 1B and 1C), where cells recapitulate their patterns in live cells (Figure 1A) and no adverse effect is seen. In addition, the use of membrane sheets avoids detergents (e.g., Triton X-100), the latter can induce artificial molecular clustering in the PM [49]

  7. Paraformaldehyde and glutaraldehyde are toxic. Handle them with caution in a fume hood with the protection of skin and eyes.

  8. We recommend performing imaging experiments on the same day after the PM sheet fixation for the optimal results. Despite fixed, the bound PH probes on the PM may gradually dissociate from the membrane after long-term storage (days or weeks).

  9. This is needed later for drift correction during imaging reconstruction. The mechanical stability of the imaging system is critical for better resolution. Even with special attention and effort, the drift correction is often needed to achieve the best results.

  10. We use 405-laser at 0.1 - 1% of full intensity. This number may vary depending on the exact imaging system and fluorescence probes used. Ideally, it should activate probes at optimal illumination so that the activated molecule density is high but still isolated spatially from each other in each image. Increase this number gradually to compensate for the decease of available probes for photoactivation/conversion.

  11. The density of the PALM probe is critical for the nanoscale resolution. Changing the expression level of the PALM probe shows comparable results in the PM sheets, suggesting the excessive number of PALM probes in INS-1 cells. As a general drawback of the PH domain-based probes, however, it remains possible that the probes cannot access all the targets (e.g., those bound PIs by some sequestering proteins) due to space hindrance or competition in cells.

  12. The green fluorescence from mEos3.1 is used for cell identification, without inducing photoconversion. We tune TIRF laser to a narrow illumination angle (controlled by the TIRF angle panel) for live-cell imaging, and the resulting short penetration depth under TIRFM minimizes the fluorescence from a small number of unbound mEos3 probes in the cytosol.

  13. The high-speed imaging is critical for live-cell imaging. Despite it is not an issue for confocal, TIRFM or instant SIM imaging, but it is challenging for PALM, especially if objects move fast. PALM needs a sufficient number of images before object moves.

  14. Since the number of unconverted mEOS3.1-PHPLCδ1 molecules decreases during acquisition, one can increase the 405 nm laser power gradually to bring up the single-molecule density.

  15. The duration of the acquisition depends on the imaging system, probe density, and dynamics, as well as cell tolerance to photo-toxicity. We saw no sign of photodamage (e.g., cell morphology changes) during 5 min PALM acquisition. We limit each image stack to less than 4 GB, a limit for the later MATLAB analysis.

  16. The point detection is critical for SMLM, both for the best resolution and avoiding overcount artifact. The program includes multiple parameters to optimize this process. We set the Neighborhood-distance as 65 nm (half effective pixel width), the fluorescence points within this distance are combined and fitted as a single molecule. We set Gap-frame as 26 frames (1.3 s) for PAmCherry1 imaging [50], so that individual events that occur within the Gap-frame and Neighboring-distance are combined as a single molecule. Proper choosing these parameters not only impact imaging resolution but also avoid data misinterpretation such as the artificial clusters resulting from overcounting [47]. The exact parameters depend primarily on the single-molecule photo-physics of probes.

  17. During high-speed PALM imaging in live cells, a small number of activated probes may move or dissociate from PtdIns(4,5)P2 in the PM before bleaching and thus lead to oversampling. To account for this issue, we set Neighboring-distance as 130 nm, instead of 65 nm, for imaging analysis, which combines individual molecule signals in this region as a single event.

Acknowledgments

This work is supported partially by the National Institutes of Health (NIH) (R01DK093953 and 1R21NS101584) and the grant AAB1425-135-A5362. We thank Tamas Balla at NIH and Pietro De Camilli at Yale for the PH-domain constructs and Dr. Christopher Newgard at Duke for INS-1 832/13 cells.

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