Cell Permeable Ratiometric Fluorescent Sensors ... - ACS Publications

1 downloads 0 Views 8MB Size Report
Apr 15, 2016 - was selective toward phosphoinositides (Figure 4c). SUVs ... amino acid sequences when compared to the corresponding ... Error bars represent standard deviation obtained from three ... Temperature dependent uptake studies show lower cytoplasmic .... Z series were obtained with a spacing of 1.00 μm.
Articles pubs.acs.org/acschemicalbiology

Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides Samsuzzoha Mondal,† Ananya Rakshit,† Suranjana Pal,‡ and Ankona Datta*,† †

Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai-400005, India Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai-400005, India



S Supporting Information *

ABSTRACT: Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.

P

cell membrane and in subcellular membranes.1 Also, the entire class of phosphoinositides constitutes only about 5% of the total cellular phospholipids in eukaryotes.22,23 Hence, the requirement is a sensitive, cell permeable sensor that can report the dynamic intracellular changes in phosphoinositides. Among the seven phosphoinositides, phosphatidylinositol4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P) constitute the majority.1 Each phosphoinositide has a different subcellular localization and also mediates different signaling events.24 For example, PI4P is mainly present on the membrane of the Golgi complex and plays an essential role in maintaining Golgi structure and function.25−27 PI(4,5)P2 is concentrated on the plasma membrane and along with mediating signal transduction regulates quintessential membrane mediated processes including exo- and endocytosis, cell motility, cell adhesion, and ion transport.1,7 A classical PI(4,5)P2 mediated signal transduction process involves its hydrolysis to the soluble inositol (1,4,5)-trisphosphate (IP3), which plays a critical role in intracellular Ca2+ signaling.28,29 A phosphoinositide selective optical sensor should therefore be able to distinguish phosphoinositides from (i) other cellular

hosphoinositides play singularly important functional roles as mediators of cell signaling processes.1,2 The identity of this class of phospholipids is marked by a unique phosphorylation fingerprint on the inositol headgroup. This leads to seven structurally distinct phosphoinositides.3 The locations of the phosphoinositides within cellular and subcellular membranes and the dynamic changes in their distributions form the crux of cell signaling events.4,5 The distribution of phosphoinositides can therefore be considered as an initiating flag for a signal. When the distribution changes, so does the signal.6,7 Visualizing phosphoinositide dynamics is therefore the key link to understanding, manipulating, and engineering cell signaling events.8−13 In this exciting context, developing optical sensors for imaging phosphoinositides becomes imperative. Phospholipid mediated signals at the membrane−cytosol interface are transmitted via the specific binding of phospholipid headgroups with either peripheral or cytosolic proteins.14 Chemists have strategically engineered these phospholipid-interacting proteins to develop chemical tools that can elucidate cell-signaling pathways.15−21 The major challenge, however, remains in the ability to develop tools that can readily permeate the cell membrane and provide information on phospholipid dynamics. Cell permeability becomes a critical parameter for phosphoinositide probes since these phospholipids are present on the inner leaflet of the © 2016 American Chemical Society

Received: January 20, 2016 Accepted: April 15, 2016 Published: April 15, 2016 1834

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology

Figure 1. Ratiometric sensing scheme for phosphoinositide imaging by using cell-permeable 2-dimethylamino-6-acyl-naphthalene (DAN)-labeled peptide-based sensors.

phospholipids, (ii) soluble inositol phosphates, and (iii) cytosolic phosphates. The ultimate and the most challenging task would be to distinguish between different phosphoinositides. By considering all of the aforementioned bottlenecks in phosphoinositide probe development, we have designed peptide-based ratiometric optical sensors that can permeate the cell membrane and specifically light-up PI(4,5)P2 (Figure 1). The most popular choices of receptors for phosphoinositide sensing have been the pleckstrin homology (PH) domains and epsin N-terminal homology (ENTH) domains of phosphoinositide binding proteins.18,30,31 While these domains have exquisite selectivity for PI(4,5)P2 and PI4P over other phospholipids, they are cell-impermeable.18 Hence, they have to be incorporated into cells via either microinjection or transient transfection. It is also exceedingly difficult to mimic the binding sites of these domains to create smaller constructs. This is because the amino acid binding partners for phosphoinositide headgroups reside in physically distinct strands of these domains.32,33 We therefore scanned for phosphoinositide binding proteins that would present the amino acid binding partners in close vicinity, preferably in a short peptide sequence. During this search, we narrowed down to the protein gelsolin, which has a 20 amino acid sequence that has been reported to bind to both PI(4,5)P2 and PI4P with a higher selectivity toward PI(4,5)P2.34 An extensive body of work by Paul Janmey and co-workers also indicated that a shorter 10 amino acid phosphoinositide binding sequence from this protein could be rendered cell-permeable by the attachment of a positively charged dye.35,36 Hence, we focused on developing an optical probe by choosing short peptide sequences from the phosphoinositide binding motif of gelsolin as our receptor units (Figure 1). Ratiometric sensing is ideal for

monitoring biological substrates since this method allows the distinction between bound and unbound probes, and permits quantification.18,19,37 We incorporated ratiometric sensing into our probes by strategically attaching a polarity sensitive dye to the peptide receptors. Polarity sensitive sensing would afford the critical distinction between membrane phosphoinositides and soluble inositol phosphates. By systematically evaluating the peptide receptors, we have developed sensors that enter mammalian cells within 15 min of incubation and report phosphoinositide localization via multiphoton confocal microscopy.



RESULTS AND DISCUSSION Phosphoinositide Selective Ratiometric Sensor Design. To develop a phosphoinositide sensor, the first requirement is a receptor unit that can selectively bind to phosphoinositides. Our main criterion for choosing the receptor unit was the presence of key features that could render the probe cell-permeable. The actin regulating protein, gelsolin, has two PI(4,5)P2 binding domains that control its actin binding and regulating activities.38−40 Unlike other PI(4,5)P2 binding proteins in which the phospholipid binding residues reside on physically distinct domains, gelsolin holds the PI(4,5)P2 interacting residues on two short polypeptide chains (Gel135−142 and Gel150−169). The Gel150−169 polypeptide (KHVVPNEVVVQRLFQVKGRR) has been reported to bind to phosphoinositides in vitro.34,41 The salient feature of this peptide sequence lies in closely spaced positively charged residues which we reckon will favor cell-permeability. We therefore used the Gel150−169 peptide as the initial receptor scaffold on which we could build a phosphoinositide sensor (Figure 2). Molecular dynamics simulations on the Gel150−169 peptide indicate that the C-terminal half (160−169) having multiple 1835

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology

The choice of the polarity sensitive dye for ratiometric sensing was critical since the selectivity of the receptor unit should not be compromised upon dye attachment. Furthermore, a neutral dye that will not affect the overall charge of the peptide unit is preferable. 2-Dimethylamino-6-acyl-naphthalene (DAN) is a neutral polarity sensitive fluorophore that affords a considerable 60−70 nm shift in the emission from the green to blue region of the visible spectrum upon polarity change.44 The dye can also be excited via two-photon excitation in the NIR region (780 nm) where biological samples have minimal absorption and autofluorescence.18 The peptide receptors depicted in Figure 2 were therefore conjugated to the DAN moiety via reaction with acrylodan, a cysteine attachable DAN precursor.45 The labeled peptide receptors, henceforth referred to as probes, were then evaluated for phosphoinositide sensing in vitro and in live cells. Fluorescence Response of the Probes with Phosphoinositides. The fluorescence responses of the four probes were examined using small unilamellar vesicles (SUVs) which served as membrane mimics. Phosphatidylcholine (PC) is a major structural component of biological membranes.46,47 Hence, mixed vesicles containing increasing proportions of phosphoinositides (PI(4,5)P2 and PI4P) in PC were prepared. Upon excitation at 380 nm all four probes exhibited visible emission in the green region with maxima between 521 and 525 nm (Figure 3a and Supporting Information Figure S1). Upon the addition of 10% PI(4,5)P2-PC vesicles, the spectra exhibited a blue shift with maxima between 463 and 470 nm, depending upon the probe used (Figure 3a and Supporting Information Figure S1). These data indicate that the probes afford a ratiometric response upon interacting with PI(4,5)P2 containing vesicles. The ratios of the fluorescence intensities for the probes at 450 to 520 nm (F450/F520) were plotted against increasing PI(4,5)P2 concentrations, as depicted for the DAN-20aa probe in Figure 3b. The data were then fitted to a model considering single site binding to calculate the apparent dissociation constant (Kd) value. All ratiometric data mentioned henceforth were also plotted similarly and analyzed using the single site binding fitting model. The Kd values obtained for the binding of the probes to 10% PI(4,5)P2-PC were in the range of 0.6 μM to 4 μM (Table 1, column 1). Importantly, all the probes responded to SUVs containing lower PI(4,5)P2 content (5% PI(4,5)P2-PC), and the F450/F520 values increased upon increasing PI(4,5)P2 content as shown in Figure 4a and b. We noted that three of the probes, DAN-20aa, DAN-20aa-F163W, and DAN-13aa, depicted a decrease in Kd values upon increasing PI(4,5)P2 content within the SUVs (Supporting Information Table S1). These data might imply a PI(4,5)P2 density dependent enhancement in the membrane affinity. The probes based on the 13 amino acid peptide sequence displayed stronger affinities for the PI(4,5)P2-PC vesicles when compared to the probes based on the 20 amino acid sequence (Table 1, columns 1 and 2). The 13 amino acid probes also afforded a higher F450/F520 response at lower levels of PI(4,5)P2 (100 14 ± 1 42 ± 2

n.b.a n.b.a >100

0.6 ± 0.1

0.6 ± 0.1

0.9 ± 0.1

3.4 ± 0.2

>100

No binding was detected.

PC were prepared. In order to maintain uniformity, all mixed vesicles within the comparison set contained 20% of the phospholipid being evaluated. All four probes afforded the highest response toward 20% PI(4,5)P2-PC. Also, the presence of an additional phospholipid like PE (4:21:75, PI(4,5)P2:PE:PC) did not alter the ratiometric response toward PI(4,5)P2 when compared to PI(4,5)P2-PC (4:96) vesicles (Supporting Information Figure S4). An important aspect of the selectivity studies was the response of the probes toward PI4P. The parent sequence 1837

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology

Figure 4. Ratiometric response of the probes with PI(4,5)P2 and other PLs. Response of DAN-20aa and DAN-20aa-F163W (a) and response of DAN-13aa and DAN-13aa-F163W (b) to SUVs containing an increasing mol % of PI(4,5)P2 in PC. (c) Bar plots representing ratiometric response of the probes to different PLs (20% mixed vesicles in PC) and IP3. Probe concentration, 1 μM; total PL concentration and IP3 concentration, 35 μM. Error bars represent standard deviation calculated from three experiments.

PI(4,5)P2. Cell-impermeability has been a major drawback for existing phosphoinositide sensors. Introducing cell permeability was an important criterion in our sensor design. We therefore tested the probes in live HEK293T cells to assess cellular incorporation. Confocal images of live cells incubated with our probes, taken within 15 min of incubation, indicated green fluorescence within the cytoplasm, plasma membrane, and perinuclear regions (Figure 6, green channel). This result indicates that the probes readily permeate mammalian cells. Temperature dependent uptake studies show lower cytoplasmic fluorescence intensity at 4 °C, hinting at an active transport pathway (Supporting Information Figure S6). Intensity in the blue channel should indicate the presence of PI(4,5)P2. The DAN-13aa probe clearly highlights the cell membrane where PI(4,5)P2 is known to be predominantly localized (Figure 6, DAN-13aa blue channel). Ratiometric analysis of the images can be used to generate 3D intensity plots highlighting regions that show high F450/F520 values (Figure 6, bottom). The 3D ratiometric intensity plot for cells treated with DAN-13aa indicates highest intensity in the plasma membrane, confirming that the probe can detect PI(4,5)P2 in live cells. Certain regions within the cell near the perinuclear space also show intensity in the blue channel, which may be

clearly light up the boundaries of GUVs containing PI(4,5)P2 in both blue and green channels, indicating probe binding on the vesicle surface (Figure 5a). An increase in signal intensities, quantified by intensity analysis of the confocal imaging data (Figure 5b), was clearly observed with increasing PI(4,5)P2 content. Most importantly, 2% PI(4,5)P2-PC vesicles could be visualized using both the DAN-20aa and DAN-13aa probes. This result shows that the probes have high sensitivity and can be applied for imaging low concentrations of PI(4,5)P2 in biological systems. Control GUVs containing 100% PC afforded no signal intensity, indicating that the probes responded selectively to phosphoinositides. The DAN-20aa probe afforded signal enhancement only for PI(4,5)P2, while the DAN-13aa probe also afforded signal intensity for PI4P and PS. The DAN-13aa probe, however, responded to PI4P and PS only at higher percentages of these phospholipids as corroborated by the intensity analysis data (Figure 5b). The GUV imaging data distinctly confirm the applicability of the probes for imaging phosphoinositides using multiphoton confocal microscopy. Confocal Imaging of Cellular Phosphoinositides with DAN Probes. The DAN-20aa and DAN-13aa probes afforded excellent in vitro selectivity toward phosphoinositides, especially 1838

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology

Figure 5. Confocal fluorescence images showing the response of the probes to GUVs of different PL composition using 2-photon excitation (λex 780 nm). (a) Blue channel images for GUVs composed of 0−10 mol % of PI(4,5)P2-PC, 5% PI4P-PC, and 20% PS-PC in the presence of DAN probes (1 μM). Scale bar, 10 μm; calibration bar, 0−255. (b) Bar plots representing the average intensity along the perimeter of the vesicles. Mean intensities were derived from calculations on at least three vesicles, and error bars represent standard deviation.

with the EGFP-PH domain were allowed to recover. Confocal microscopy images indicated that EGFP-PH highlighted the plasma membrane (Figure 7a). The EGFP-PH containing cells were further incubated with either the DAN-20aa or the DAN13aa probe. The DAN-13aa probe (blue channel, λex 780 nm) clearly lighted up the cell membrane, indicating PI(4,5)P2 binding (Figure 7a). In vitro PI(4,5)P2 titration data in the presence of a 1:1 EGFP-PH domain: DAN-13aa was similar to the titration data in the presence of only DAN-13aa (Supporting Information Figure S5). These data show that the EGFP-PH domain does not alter the binding of the DAN13aa probe to P1(4,5)P2, implying that they might not compete for identical PI(4,5)P2 pools. Finally, we wanted to investigate the enhancement in the intensity observed near the perinuclear region with our probes. PI4P and putatively PI(4,5)P2 are known to be localized within the membrane of the Golgi complex, which is present near the perinuclear space.1,25,49 We therefore performed colocalization studies of the DAN-13aa probe with BODIPY-TR-Ceramide, a Golgi targeting dye,50 to check if the increase in intensity in the blue channel observed near the perinuclear space could be attributed to phosphoinositides in the Golgi. We found clear colocalization of the DAN-13aa blue channel intensity with the BODIPY emission near the perinuclear region (Figure 7b). The blue channel intensity in the membrane remained distinct, indicating that the DAN-13aa probe could highlight phosphoi-

attributed to the presence of phosphoinositides in intracellular organelle membranes. The DAN-20aa probe shows punctated staining both on the membrane and near the perinuclear region in the blue channel. The 3D ratiometric intensity plot for the probe also indicates certain high intensity spots in the membrane and close to the perinuclear region. While both DAN-20aa and DAN-13aa afforded a ratiometric response to phosphoinositides in vitro, the DAN-20aa probe seemed more promising due to slightly better selectivity. However, the in cellulo response of the DAN13aa probe reflects the biologically expected PI(4,5)P2 distribution more accurately than the DAN-20aa probe. This might be attributed to differences in the cellular availability and localization of the probes. The DAN-13aa probe also afforded a lower Kd value for PI(4,5)P2 when compared to the DAN-20aa probe (Table 1). Furthermore, the DAN-13aa probe afforded a higher ratiometric response at lower PI(4,5)P2 concentrations (Figure 4b) which would be reflected in cellular imaging. Thus, when we compare the DAN-20aa and DAN-13-aa probes, the latter affords a better in cell response and therefore can be applied for cellular imaging of PI(4,5)P2. The cellular response of the DAN-13aa probe was also compared to the in cell response of the EGFP-tagged PH domain. This domain is used for imaging the plasma membrane PI(4,5)P2.30 The EGFP-PH domain was introduced into the HEK293T cells via electroporation. The cells electroporated 1839

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology

Figure 6. PI(4,5)P2 imaging with DAN-20aa and DAN-13aa in live HEK293T cells incubated with the probes (5 μM) for 15 min. Confocal microscopy images were recorded using 2-photon excitation (λex 780 nm). Images were collected at the blue (435−465 nm) and green channels (510−540 nm) simultaneously by a multichannel detector (top panel). Ratiometric analyses were performed on the highlighted regions (red boxes), and 3D intensity plots were generated from the blue/green ratio (bottom panel). Scale bar, 10 μm.

introduced to fine-tune the PI(4,5)P2 selectivity of the probe, and current efforts are focused toward this direction.

nositides both in the plasma membrane and near the perinuclear region. Since the Golgi membrane is rich in PI4P, the increase in the intensity might be attributed to the interaction of DAN-13aa with PI4P. The ratiometric response near the perinuclear region is however lower than the intensity in the plasma membrane (Figure 6, bottom, DAN-13aa). This is consistent with the in vitro response of the DAN-13aa probe, which is also higher for PI(4,5)P2 than PI4P (Figure 5 and Supporting Information Figure S3). This result conclusively shows that the DAN-13aa probe can be applied as a membranepermeable sensor for imaging cellular phosphoinositides. Conclusions. In this work, we have designed and developed a short peptide based ratiometric sensor for phosphoinositides, which are crucial cell signaling molecules. A major factor that needs to be considered when developing chemical probes and sensors is the ability of the probe to function in living systems. The ability of our sensor to readily enter live mammalian cells within 15 min of incubation and selectively highlight cellular phosphoinositides is the key feature of our probe. The ease of application and the simple design of the probe make it a valuable tool for unravelling cell signaling mechanisms. The sensor will be particularly useful in high-throughput assays involving quantitative PI(4,5)P2 imaging because it allows ratiometric sensing and can readily enter into live cells. Additional modifications in the peptide sequence can be



METHODS

Synthesis of DAN Probes. Peptides were synthesized by using solid phase peptide synthesis strategy on Fmoc protected Rink Amide resins (100−200 mesh, loading 0.74 mmol/g resin) using an automated peptide synthesizer (PS3, Protein Technologies Inc.). Peptides were characterized by MALDI-TOF-MS (Bruker UltrafleXtreme, Bruker Daltonics). To attach the DAN moiety, crude peptides were dissolved in DMF and stirred with Acrylodan (1 equiv) at RT for 12 h. After completion of the reaction, DMF was evaporated under reduced pressure, and the residue was purified by HPLC. DAN probes were characterized by LCESI-MS and MALDI-TOF-MS. Detailed procedures for peptide synthesis and characterization are given in the Supporting Information (sections 3.1, 3.2, and 4). Fluorescence Measurements with DAN Probes in the Presence of SUVs. All measurements were performed in a buffer (20 mM Hepes, 100 mM NaCl, pH 7.4) at 25 °C. Probes were dissolved in a buffer, and concentrations of the stock solutions were measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.) by monitoring the DAN absorbance at 391 nm (molar absorptivity of DAN at 391 nm = 20 000 M−1 cm−1). SUVs with different phospholipid compositions were prepared (see Supporting Information, section 3.4). Probe solutions (1 μM, 200 μL) were mixed with the SUVs, and fluorescence spectra were recorded in a FluoroLog-3 spectrofluorimeter (Horiba Jobin Yvon, Inc.) by 1840

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology

Figure 7. (a) In cell response of DAN probes in the presence of enhanced green fluorescent protein (EGFP) tagged PH domain of PLC-δ1. EGFP fluorescence (λex 488 nm, left panel), DAN-13aa (blue channel, mid panel), and DAN-20aa (blue channel, right panel). Scale bar, 10 μm. (b) Confocal images of colocalization studies of DAN-13aa with Golgi-complex targeting BODIPY TR Ceramide in HEK293T cells. The left panel shows the blue channel image (435−465 nm) of DAN-13aa. The mid panel shows the BODIPY channel (600−650 nm), indicating the distribution of BODIPY TR Ceramide. The right panel shows a merged image of the blue channel (green) and BODIPY channel (red) to depict colocalization near perinuclear space (yellow). λex, 780 nm; scale bar, 10 μm. excitation at 380 nm. To determine dissociation constants (Kd), probes were titrated against SUVs composed of different phospholipids, and F450/F520 values were plotted against corresponding phospholipid concentrations. The response curves were fitted to the equation (F450/F520) = (F450/F520)max/(1 + Kd/[phospholipid]) and (F450/F520)max, and Kd values were obtained from the fits. For comparison studies, fluorescence spectra were recorded with the probes (1 μM) in the presence of either SUVs, inositol phosphates, or other phospho-anions. Confocal Fluorescence Imaging of GUVs. All microscopy experiments were performed at 25 °C in an LSM 710 confocal microscope (Zeiss) coupled with a tunable multiphoton excitation laser (Mai-Tai, Spectra-Physics), and images were collected in the internal detector channels using a 40× water immersion objective (Zeiss). GUVs of desired phospholipid compositions containing Texas Red as a label were prepared (see Supporting Information, section 3.4). For imaging GUVs, solutions containing the probes (160 μL) were taken into the wells of LabTek chambers, and 40 μL of the GUV solutions were carefully added to the probe solutions. The GUVs were allowed to settle down to the bottom of the chamber and were located by monitoring Texas Red fluorescence (λex 543 nm). The probes were excited at 780 nm via two photon excitation, and fluorescence was collected simultaneously at two separate channels, the blue channel (435−465 nm) and green channel (510−540 nm). Laser power and detector gain were adjusted in order to avoid signal saturation at the highest PI(4,5)P2 concentration (10% PI(4,5)P2-PC) imaged. All GUV images of a particular probe were taken under identical laser power and detector gain. Confocal images were analyzed using ImageJ

(NIH, USA) software, and the intensities along the perimeter of the confocal images of the GUVs were measured using a Concentric Circles plugin obtained from http://rsb.info.nih.gov/ij/plugins. Cellular Phosphoinositide Imaging with DAN Peptides. HEK293T cells were serum starved for 6−8 h. The medium was removed and cells were washed with 1× PBS (phosphate buffered saline; pH = 7.4) and incubated with 5 μM of the DAN peptides in 1× PBS for 15 min. After incubation, the cells were imaged under a confocal microscope using similar instrument settings as described in the previous section. Z series were obtained with a spacing of 1.00 μm. Confocal images were analyzed in ImageJ software. Ratiometric image analyses were performed using the Ratio Plus plugin, where the ratios of the blue channel intensity to the green channel intensity were obtained. Ratio images were then converted to 3D intensity plots by using a 3D surface plot plugin. Studies with EGFP-PH Domain. The purified EGFP-PH domain was generously provided by Dr. Thomas Pucadyil’s laboratory, IISER Pune. HEK293T cells were grown in T25 culture plates. The protein was incorporated into the cells by electroporation method (see Supporting Information, section 3.7). After electroporation, cells were plated onto glass coverslip bottomed Petri plates and allowed to recover in the incubator (37 °C and 5% CO2) for 6 h. Following incubation, the medium was removed and cells were washed with 1× PBS. The distribution of the PH domain was imaged using 488 nm laser excitation in the confocal microscope. Either DAN-20aa or DAN13aa in 1× PBS was then added to the EGFP-PH domain containing cells, and the cells were imaged using 780 nm, 2-photon excitation for observing the distribution of the DAN peptides. 1841

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology Colocalization Studies with BODIPY TR Ceramide. HEK293T cells plated on glass coverslip bottomed Petri plates were incubated with BODIPY TR Ceramide (5 μM, Molecular Probes) complexed with BSA in 1× PBS for 30 min at 4 °C and washed with ice cold medium. The cold medium was replaced with a medium at RT, and cells were incubated at 37 °C for at least 30 min. Following incubation, cells were washed with 1× PBS, and DAN-13aa (5 μM) in 1× PBS was added to the cells. Both BODIPY and DAN fluorescence were excited by 2-photon excitation at 780 nm. DAN fluorescence in the blue (435−465 nm) channel and BODIPY fluorescence in the red (600− 650 nm) channel were collected simultaneously.



(10) Idevall-Hagren, O., Dickson, E. J., Hille, B., Toomre, D. K., and De Camilli, P. (2012) Optogenetic control of phosphoinositide metabolism. Proc. Natl. Acad. Sci. U. S. A. 109, E2316−E2323. (11) Idevall-Hagren, O., and De Camilli, P. (2015) Detection and manipulation of phosphoinositides. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1851, 736−745. (12) Vibhute, A. M., Konieczny, V., Taylor, C. W., and Sureshan, K. M. (2015) Triazolophostins: a library of novel and potent agonists of IP3 receptors. Org. Biomol. Chem. 13, 6698−6710. (13) Tóth, J. T., Gulyás, G., Tóth, D. J., Balla, A., Hammond, G. R. V., Hunyady, L., Balla, T., and Várnai, P. (2016) BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1861, 177−187. (14) Heo, W. D., Inoue, T., Park, W. S., Kim, M. L., Park, B. O., Wandless, T. J., and Meyer, T. (2006) PI(3,4,5)P-3 and PI(4,5)P-2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458−1461. (15) Lemmon, M. A. (2008) Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 9, 99−111. (16) Gupta, S., Mondal, S., Mhamane, A., and Datta, A. (2013) Smart “lanthano” proteins for phospholipid sensing. Inorg. Chem. 52, 12314− 12316. (17) Kim, H., Afsari, H. S., and Cho, W. (2013) High-throughput fluorescence assay for membrane-protein interaction. J. Lipid Res. 54, 3531−3538. (18) Yoon, Y., Lee, P. J., Kurilova, S., and Cho, W. (2011) In situ quantitative imaging of cellular lipids using molecular sensors. Nat. Chem. 3, 868−874. (19) Cicchetti, G., Biernacki, M., Farquharson, J., and Allen, P. G. (2004) A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts. Biochemistry 43, 1939−1949. (20) Smith, B. A., and Smith, B. D. (2012) Biomarkers and molecular probes for cell death imaging and targeted therapeutics. Bioconjugate Chem. 23, 1989−2006. (21) Takatori, S., Mesman, R., and Fujimoto, T. (2014) Microscopic methods to observe the distribution of lipids in the cellular membrane. Biochemistry 53, 639−653. (22) Ingolfsson, H. I., Melo, M. N., van Eerden, F. J., Arnarez, C., Lopez, C. A., Wassenaar, T. A., Periole, X., de Vries, A. H., Tieleman, D. P., and Marrink, S. J. (2014) Lipid organization of the plasma membrane. J. Am. Chem. Soc. 136, 14554−14559. (23) Carlton, J. G., and Cullen, P. J. (2005) Coincidence detection in phosphoinositide signaling. Trends Cell Biol. 15, 540−547. (24) Hammond, G. R. V., Fischer, M. J., Anderson, K. E., Holdich, J., Koteci, A., Balla, T., and Irvine, R. F. (2012) PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity. Science 337, 727−730. (25) Roth, M. G. (2004) Phosphoinositides in constitutive membrane traffic. Physiol. Rev. 84, 699−730. (26) Hammond, G. R. V., Machner, M. P., and Balla, T. (2014) A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J. Cell Biol. 205, 113−126. (27) Graham, T. R., and Burd, C. G. (2011) Coordination of Golgi functions by phosphatidylinositol 4-kinases. Trends Cell Biol. 21, 113− 121. (28) Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., and Iino, M. (1999) Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science 284, 1527− 1530. (29) Clapham, D. E. (2007) Calcium signaling. Cell 131, 1047−1058. (30) Balla, T. (2007) Imaging and manipulating phosphoinositides in living cells. J. Physiol. 582, 927−937. (31) Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Pleckstrin homology domains bind to phosphatidylinositol-4,5bisphosphate. Nature 371, 168−170. (32) Ferguson, K. M., Lemmon, M. A., Schlessinger, J., and Sigler, P. B. (1995) Structure of the high-affinity complex of inositol

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00067. Supporting figures and tables; detailed methods for peptide synthesis and purification, SUV and GUV preparation, competition experiments with inositol phosphates and other phospho-anions, cell culture, and electroporation; purity analysis for DAN probes (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS



REFERENCES

A.D. acknowledges financial support from the Department of Atomic Energy, India. The authors acknowledge S. Maiti, S. Bakthavatsalam, B. K. Maity, and A. Rawat, TIFR, for help with cell culture and confocal imaging and T. Pucadyil, D. Andhare, and S. Dar, IISER Pune, for help with GUV preparation and providing the purified EGFP-PH domain.

(1) Di Paolo, G., and De Camilli, P. (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651−657. (2) Holthuis, J. C. M., and Menon, A. K. (2014) Lipid landscapes and pipelines in membrane homeostasis. Nature 510, 48−57. (3) Balla, T. (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019−1137. (4) Hammond, G. R., and Balla, T. (2015) Polyphosphoinositide binding domains: Key to inositol lipid biology. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1851, 746−758. (5) Kutateladze, T. G. (2010) Translation of the phosphoinositide code by PI effectors. Nat. Chem. Biol. 6, 507−513. (6) Honigmann, A., van den Bogaart, G., Iraheta, E., Risselada, H. J., Milovanovic, D., Mueller, V., Mullar, S., Diederichsen, U., Fasshauer, D., Grubmuller, H., Hell, S. W., Eggeling, C., Kuhnel, K., and Jahn, R. (2013) Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat. Struct. Mol. Biol. 20, 679−686. (7) McLaughlin, S., and Murray, D. (2005) Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605−611. (8) Wu, M., Dul, B. E., Trevisan, A. J., and Fiedler, D. (2013) Synthesis and characterization of non-hydrolysable diphosphoinositol polyphosphate second messengers. Chem. Sci. 4, 405−410. (9) Frank, J. A., Moroni, M., Moshourab, R., Sumser, M., Lewin, G. R., and Trauner, D. (2015) Photoswitchable fatty acids enable optical control of TRPV1. Nat. Commun. 6, 7118. 1842

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843

Articles

ACS Chemical Biology trisphosphate with a phospholipase-c pleckstrin homology domain. Cell 83, 1037−1046. (33) Ford, M. G. J., Mills, I. G., Peter, B. J., Vallis, Y., Praefcke, G. J. K., Evans, P. R., and McMahon, H. T. (2002) Curvature of clathrincoated pits driven by epsin. Nature 419, 361−366. (34) Janmey, P. A., Lamb, J., Allen, P. G., and Matsudaira, P. T. (1992) Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin. J. Biol. Chem. 267, 11818−11823. (35) Bucki, R., Janmey, P. A., Vegners, R., Giraud, F., and Sulpice, J. C. (2001) Involvement of phosphatidylinositol 4,5-bisphosphate in phosphatidylserine exposure in platelets: Use of a permeant phosphoinositide-binding peptide. Biochemistry 40, 15752−15761. (36) Cunningham, C. C., Vegner, R., Bucki, R., Funaki, M., Korde, N., Hartwig, J. H., Stossel, T. P., and Janmey, P. A. (2001) Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly. J. Biol. Chem. 276, 43390−43399. (37) Liu, S. L., Sheng, R., O’Connor, M. J., Cui, Y., Yoon, Y., Kurilova, S., Lee, D., and Cho, W. (2014) Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors. Angew. Chem., Int. Ed. 53, 14387−14391. (38) Janmey, P. A., Iida, K., Yin, H. L., and Stossel, T. P. (1987) Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin-filaments blocked by gelsolin. J. Biol. Chem. 262, 12228−12236. (39) Janmey, P. A., and Stossel, T. P. (1987) Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature 325, 362− 364. (40) Sun, H. Q., Yamamoto, M., Mejillano, M., and Yin, H. L. (1999) Gelsolin, a multifunctional actin regulatory protein. J. Biol. Chem. 274, 33179−33182. (41) Xian, W. J., Vegners, R., Janmey, P. A., and Braunlin, W. H. (1995) Spectroscopic studies of a phosphoinositide-binding peptide from gelsolin: Behavior in solutions of mixed solvent and anionic micelles. Biophys. J. 69, 2695−2702. (42) Liepina, I., Czaplewski, C., Janmey, P., and Liwo, A. (2003) Molecular dynamics study of a gelsolin-derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5-bisphosphate. Biopolymers 71, 49−70. (43) Wimley, W. C., and White, S. H. (1996) Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 3, 842−848. (44) Li, X.-H., Culver, J. A., and Rhoades, E. (2015) Tau binds to multiple tubulin dimers with helical structure. J. Am. Chem. Soc. 137, 9218−9221. (45) Prendergast, F. G., Meyer, M., Carlson, G. L., Iida, S., and Potter, J. D. (1983) Synthesis, spectral properties, and use of 6acryloyl-2-dimethylaminonaphthalene (acrylodan)- a thiol-selective, polarity-sensitive fluorescent-probe. J. Biol. Chem. 258, 7541−7544. (46) van Meer, G. (2005) Cellular lipidomics. EMBO J. 24, 3159− 3165. (47) Shevchenko, A., and Simons, K. (2010) Lipidomics: coming to grips with lipid diversity. Nat. Rev. Mol. Cell Biol. 11, 593−598. (48) Leitner, M. G., Halaszovich, C. R., Ivanova, O., and Oliver, D. (2015) Phosphoinositide dynamics in the postsynaptic membrane compartment: Mechanisms and experimental approach. Eur. J. Cell Biol. 94, 401−414. (49) Hammond, G. R. V., Schiavo, G., and Irvine, R. F. (2009) Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P-2. Biochem. J. 422, 23−35. (50) Kosaka, N., Iguchi, H., Yoshioka, Y., Hagiwara, K., Takeshita, F., and Ochiya, T. (2012) Competitive interactions of cancer cells and normal cells via secretory microRNAs. J. Biol. Chem. 287, 1397−1405.

1843

DOI: 10.1021/acschembio.6b00067 ACS Chem. Biol. 2016, 11, 1834−1843