A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells.

Materials

Rhodamine-Phalloidin was from Life Technologies (Grand Island, NY). Latrunculin B was from Calbiochem (Billerica, MA). Rabbit anti-phospho-ERK1/2, mouse anti-ERK1/2, rabbit anti-phospho-MEK1/2, rabbit anti-MEK1/2, and rabbit anti-PAK1 antibodies were from Cell Signaling Technology (Danvers, MA). Diazoxide, IPA3, and rabbit anti-phospho-PAK1 (Thr423) antibody was purchased from Santa Cruz (Santa Cruz, CA). Mouse anti-SNAP-25, anti-VAMP2 and rabbit anti-actin antibodies were purchased from BD Biosciences (Mountain View, CA), Synaptic Systems (Germany), and Sigma (St. Louis, MO), respectively. Goat anti-mouse horseradish peroxidase secondary antibody and PD0325901 were obtained from Thermo Fisher Scientific (Rockford, IL). Goat anti-rabbit horseradish peroxidase secondary antibody and TransFectin™ lipid reagent were acquired from Bio-Rad (Hercules, CA). ECL reagent and SuperSignal™ Femto were purchased from GE Healthcare (Piscataway, NJ) and Pierce (Rockford, IL), respectively. The rat insulin radioimmunoassay kits were obtained from Millipore (Billerica, MA). The Lifeact-GFP plasmid was kindly provided by Dr. Louis Philipson (University of Chicago, ( 30 )). The ML-141 inhibitor was kindly provided by the KU Specialized Chemistry Center (University of Kansas, Lawrence, KS). The F/G Actin ratio kit was obtained from Cytoskeleton (Denver, CO). All other chemicals were obtained through Thermo Fisher Scientific or Sigma. Cell culture, transient transfection, and secretion assays MIN6 β cells (gift from Dr. John Hutton, University of Colorado Health Sciences Center, Denver, CO) were cultured in Dulbecco's modified Eagle's medium (25 mM glucose), supplemented with 15% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml L-glutamine, and 50 μM β-mercaptoethanol as described previously ( 31 ). MIN6 cells were transfected with TransFectin™ (Bio-Rad) according to the manufacturer's instructions and cultured 48 h before use in experiments. For experiments where cells were starved and stimulated, MIN6 cells were washed twice with and incubated for 2 h in freshly prepared modified Krebs-Ringer bicarbonate buffer (MKRBB: 5 mM KCl, 120 mM NaCl, 15 mM HEPES, pH 7.4, 24 mM NaHCO 3 , 1 mM MgCl2, 2 mM CaCl 2 , and 1 mg/ml radioimmunoassay-grade BSA). Cells were stimulated with 20 mM glucose as indicated and insulin secreted into the buffer was quantified using a rat insulin radioimmunoassay kit. Cells were lysed in 1% Nonidet P-40 (NP-40) lysis buffer (25 mM HEPES, pH 7.4, 1% Nonidet P-40, 10% glycerol, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 137 mM NaCl, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml leupeptin) and cleared of insoluble material by centrifugation for 10 min at 4°C for subsequent use in co-immunoprecipitation experiments.

Subcellular fractionation

As described previously ( 18 ), all fractionation steps were performed at 4°C. Briefly, MIN6 cells at 70-80% confluence were harvested into 1 ml of homogenization buffer (20 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 100 μM phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, 2 μg/ml pepstatin, and 10 μg/ml leupeptin). Cells were homogenized by 10 strokes through a 27-gauge needle, and then centrifuged at 900 × g for 10 min. Post-nuclear supernatants were centrifuged for 5500 × g for 15 min. The resulting supernatant was centrifuged at 25,000 × g for 20 min to obtain the secretory granule fraction in the pellet. The plasma membrane (PM) fraction was prepared by mixing the post-nuclear pellet with 1 volume of Buffer A (0.25 M sucrose, 1 mM MgCl 2 , and 10 mM Tris-HCl, pH 7.4) and 2 volumes of Buffer B (2 M sucrose, 1 mM MgCl 2 , 10 mM Tris-HCl, pH 7.4). This mixture was overlaid with Buffer A and centrifuged at 113,000 × g for 1 h to obtain an interface containing the PM. The interface was collected, diluted to 1.5 ml with homogenization buffer, and centrifuged at 6000 × g for 10 min. The resulting pellet contained the PM fraction. All collected pellets were resuspended in 1% NP-40 lysis buffer.

Co-immunoprecipitation and immunoblotting

For immunoprecipitation, 2-3 mg of cleared detergent lysate protein was combined with 1 μg of antibody per mg protein and the reaction rotated for 2 h at 4°C. Protein G Plus agarose beads (Santa Cruz) were added and reactions rotated at 4°C for an additional 2 h. Beads were pelleted and washed three times with lysis buffer and resulting immunoprecipitates were resolved on 10-12% SDS-PAGE and transfer to PVDF membranes for immunoblotting. Immunoreactive bands were visualized with ECL, ECL Prime (GE Healthcare, Piscataway, NJ), or Supersignal Femto (Pierce) reagents and imaged using a Chemi-Doc gel documentation system (Bio-Rad, Hercules, CA). Phosphorylated and total ERK1/2 blots were visualized using goat anti-mouse 680 and goat anti-rabbit 800 simultaneously and imaged on a Licor imaging system.

Confocal microscopy

MIN6 cells plated onto glass coverslips at 30% confluence were transiently transfected with 4 μg of Life-Act-GFP plasmid DNA/35 mm well. After 48 h incubation, cells were placed in MKRBB for 2 h, followed by stimulation with 20 mM glucose for 5 min and then immediately fixed and permeabilized in fixation/permeabilization buffer (4% paraformaldehyde, 0.1% Triton X-100 at 4°C) for 10 min in the dark. Fixed and permeabilized cells were blocked (1% BSA plus 5% donkey serum in PBS) for 1 h at room temperature, followed by incubation with 0.17 μM Rhodamine-Phalloidin for 1 h. Cells were washed three times with PBS and during the final wash, DAPI was added to stain nuclei. All cells were washed again with PBS, and mounted (using Vectashield) for confocal microscopy. Fluorescent cells were imaged using single-channel scanning with a 60X objective (2X zoom) using an Olympus FV1000-MPE confocal microscope (Olympus, Center Valley, PA). To score F-actin remodeling, only cells on the outside of clusters were counted. Cells with discontinuous cortical F-actin after 5 min of glucose stimulation were scored as exhibiting F-actin remodeling. At least 100 cells were counted for each condition in the experiments for Figure 5.

Live-cell imaging

MIN6 cells were plated on 35 mm glass-bottom MatTek culture dishes (MatTek), grown to 30-50% confluency and transfected with Lifeact-GFP. Forty-eight h later cells were incubated in MKRBB containing either 1 μl DMSO (vehicle) or inhibitors as stated in figure legends. For confocal live-cell imaging, cells were constantly perfused with MKRBB containing DMSO or IPA3 and imaged on an Olympus FV1000-MPE. For each condition, 11 cells were analyzed for F-actin remodeling across three independent experiments. For Apotome™ live-cell imaging data was collected using a Zeiss Axio Observer™ with a Plan-Apochromat 63X objective equipped with a Hamamastu Orca-ER digital camera and an Apotome™ and analyzed using ImageJ software (NIH).

Human islet culture

Pancreatic human islets were obtained through the Integrated Islet Distribution Program, IIDP. Criteria for human donor islet acceptance: receipt within 36 h of isolation, and of at least 70% purity and 75% viability. Upon receipt, human islets were first allowed to recover in CMRL medium for 2 h, and then were handpicked using a green gelatin filter to eliminate residual non-islet material. Human islets were cultured overnight in CMRL containing either DMSO or IPA3 (7.5 μM) prior to glucose time course experiments: 100-200 islets were preincubated for 1 h in KRBH buffer plus DMSO or IPA3 (10 mM HEPES pH 7.4, 134 mM NaCl, 5 mM NaHCO 3 , 4.8 mM KCl, 1 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 containing 0.5 mg/ml BSA) supplemented with 2.8 mM glucose. Islets were then stimulated for the times stated in figure legends with 16.7 mM glucose, washed in cold PBS, and lysed and boiled in Laemmli sample buffer for SDS-PAGE protein resolution and immunoblot analysis.

Statistical analysis

All data were evaluated for statistical significance using Student's t test. Data are expressed as the average ± SE. One-way ANOVA was performed using GraphPad Prism™ software (La Jolla, CA).

Materials

Rhodamine-Phalloidin was from Life Technologies (Grand Island, NY). Latrunculin B was from Calbiochem (Billerica, MA). Rabbit anti-phospho-ERK1/2, mouse anti-ERK1/2, rabbit anti-phospho-MEK1/2, rabbit anti-MEK1/2, and rabbit anti-PAK1 antibodies were from Cell Signaling Technology (Danvers, MA). Diazoxide, IPA3, and rabbit anti-phospho-PAK1 (Thr423) antibody was purchased from Santa Cruz (Santa Cruz, CA). Mouse anti-SNAP-25, anti-VAMP2 and rabbit anti-actin antibodies were purchased from BD Biosciences (Mountain View, CA), Synaptic Systems (Germany), and Sigma (St. Louis, MO), respectively. Goat anti-mouse horseradish peroxidase secondary antibody and PD0325901 were obtained from Thermo Fisher Scientific (Rockford, IL). Goat anti-rabbit horseradish peroxidase secondary antibody and TransFectin™ lipid reagent were acquired from Bio-Rad (Hercules, CA). ECL reagent and SuperSignal™ Femto were purchased from GE Healthcare (Piscataway, NJ) and Pierce (Rockford, IL), respectively. The rat insulin radioimmunoassay kits were obtained from Millipore (Billerica, MA). The Lifeact-GFP plasmid was kindly provided by Dr. Louis Philipson (University of Chicago, ( 30 )). The ML-141 inhibitor was kindly provided by the KU Specialized Chemistry Center (University of Kansas, Lawrence, KS). The F/G Actin ratio kit was obtained from Cytoskeleton (Denver, CO). All other chemicals were obtained through Thermo Fisher Scientific or Sigma.