Medium, serum, and antibiotics for cell cultures are from Biological Industries (Kibbutz Beit Ha’Emek, Israel). GnRH and PMA were obtained from Sigma (St. Louis, MO, USA). EGF was purchased from Prospec (East Brunswick, NJ, USA). U0126, SB203580, 8-Br-cAMP, mouse monoclonal anti-doubly phosphorylated-ERK1/2 antibodies, and rabbit polyclonal antibodies to general ERK were obtained from Sigma-Aldrich (Rehovot, Israel). jetPRIME Transfection reagent was obtained from polyplus transfection (Illkirch, France). GnRH antagonist (cetrorelix acetate) was from Merck (NJ, USA). The ROCK inhibitor Y-27632 was from Cayman Chemical Company (Ann Arbor, MI, USA). Secondary horseradish peroxidase-conjugated goat anti mouse antibodies or goats anti rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). GFP-ERK2 and c-Src-GFP were kindly provided by Dr. Rony Seger, and Vinculin-GFP was kindly provided by Dr. Benny Geiger (Weizmann Institute of Science, Rehovot, Israel). GnRHR-mCherry construct was kindly provided by Dr. Colin Clay (Colorado State University, USA). Paxillin-GFP and FAK-GFP were kindly provided by Dr. Kenneth Yamada (NIH, USA). Actin-YFP was kindly provided by Dr. Ilan Tsarfaty, and EMTB-3XGFP was kindly provided by Dr. David Sprinzak (Tel-Aviv University, Israel).

LβT2 cells (kindly provided by Prof. P. Mellon UCSD, USA) were grown in DMEM supplemented with 10% FCS, streptomycin (100 µg/ml), penicillin (100 U/ml), and 5% glutamine. Cells were maintained in humidified atmosphere of 5% CO₂ and at 37°C. At 70–80% confluence, the cells were serum starved overnight in DMEM with 0.1% FCS, and stimulants were added in DMEM. Cells were washed twice with ice-cold PBS and overlaid with lysis buffer (20 mM Tris–HCl pH 7.5, 20 mM NaCl, 5 mM MgCl₂, 1 mM Na₃VO₄, 0.5% Tryton x-100, 50 mM β-glycerophosphate, 30% glycerol, 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF), followed by centrifugation (15,000 × g, 15 min, 4°C). The supernatants were collected, and aliquots were separated on 10% SDS-PAGE, followed by Western blotting.

LβT2 cells were plated on 35 mm glass-bottom plates and were transfected with 1 µg of GnRHR-mCherry along with 1 µg of one of the complex proteins constructs (ERK2–GFP, FAK–GFP, etc.) by using the jet PRIME™ transfection reagent. Approximately 30 h after transfection, the cells were serum starved (0.1% FCS) for 16 h and later stimulated with various ligands and inhibitors as indicated. Images were acquired at 10-s intervals using Leica TCS STED microscope (Leica, Wetzlar, Germany) using the 63 objective. Cells were kept in a microscope stage incubator at 37°C in a humidified atmosphere of 5% CO₂ throughout the experiment. Data and image analysis was performed using ImageJ (NIH, Bethesda, MD, USA).

LβT2 cells (1 × 10⁵) were trypsinized and resuspended in starvation medium (0.1% FCS) and plated in Matrigel (1:150 dilution)-coated transwell inserts with or without the MEK inhibitor, U0126 (25 µM). Lower chambers contained starvation medium with 10 nm GnRH. After 24 h, cells were fixed in 2.5% glutaraldehyde for 15 min and washed with DDW. Cells were stained with 0.1% methylene blue for 60 min. Cells that did not migrate to the underside of the membrane were scraped off using a cotton swab. Migrated cells were observed under a microscope and counted from ten random fields.

Post-pubertal female Sprague-Dawley rats obtained from Taconic Farms (Germantown, NY, USA) were euthanized by asphyxiation with CO₂, and the anterior pituitary glands were removed after decapitation. The procedure was approved by the NICHD Animal Care and Use Committee (#14-041). The methods were carried out in accordance with the approved guidelines. Pituitary tissue was cut into 1 mm³ pieces, treated with trypsin (20 µg/ml diluted in PBS + 0.3% BSA medium) for 15 min at 37°C, and followed by addition of a pinch of DNAase and 2.6 mg/ml trypsin inhibitor. Mechanical dispersion of cells was done in calcium-deficient PBS medium. Dispersed anterior pituitary cells were plated on poly-l-lysine coated 25 mm circular coverslips (Thomas Scientific, Swedesboro, NJ, USA) at 7 × 10⁵ cells/coverslip density and cultured overnight in medium-199 containing Earle’s salts and supplemented with 10% horse serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) (Life Technologies). At least an hour prior to experiments, the medium was changed to Krebs-Ringer containing 2.5 µM Fura-2 AM (Life Technologies). The coverslips were then washed in Krebs-Ringer medium and mounted on the stage of an inverted Observer-D1 microscope (Carl Zeiss, Oberkochen, Germany) with an attached ORCA-ER camera (Hamamatsu Photonics, Hamamatsu City, Japan) and a Lambda DG-4 wavelength switcher (Sutter, Novato, CA, USA). Hardware control and image analysis was performed using Metafluor software (Molecular Devices, Downingtown, PA, USA). Experiments were performed with a 63× oil-immersion objective while alternatively recording transmitted light image and the image at 380 nm excitation beam. There were approximately 20 mixed pituitary cells in the field, and the gonadotropes were identified by their intracellular Ca²⁺ response to GnRH, i.e., rapid decrease of 380 nm-induced fluorescence intensity, followed by a slower increase. After that, blebbing was analyzed. The images were further analyzed using ImageJ (NIH, Bethesda, MD, USA).

In order to confirm our findings from the gonadotrope-derived cell line, we have prepared primary gonadotropes from transgenic mice that carry a fluorescent signal in their gonadotropes, the GRIC/Ai9 mice. GRIC mice express Cre recombinase driven by the promoter of the GnRHR gene. Therefore, when Ai9 mice are crossed with GRIC mice the stop cassette is excised, which activates constitutive expression of dTomato in the gonadotropes. These mice thus allow identification and sorting of the gonadotrope cells_. Animals were held and handled after protocol approval by the Technion IACUC and in accordance with their guidelines and regulations. We prepared primary gonadotrope culture from heterozygous mice created by breeding GRIC females with Ai9 males. Sexually mature female heterozygous mice were sacrificed, their pituitaries removed, and pituitary cells were prepared as previously described (42, 43). The gonadotropes were collected from the total pituitary population based on their fluorescence, using a FACS Aria 2 sorter. Following sorting, the cells were plated on glass-bottom plates for 12 h in fresh medium (DMEM 10% FCS). At least an hour prior to experiments, cells were serum starved (0.1% FCS), later stimulated with various ligands and inhibitors. Images were acquired using Leica TCS STED microscope (Leica, Wetzlar, Germany) using the 63 objective. Cells were kept in a microscope stage incubator at 37°C in a humidified atmosphere of 5% CO₂ throughout the experiment. Data and image analysis was performed using ImageJ (NIH, Bethesda, MD, USA).

Results from three or more experiments were expressed as mean ± SEM. Where appropriate, data were subjected to statistical analysis by Student’s t-test, or by one- or two-way ANOVA, depending on the experimental design. Values of p < 0.05 were considered statistically significant.

Time-lapse confocal microscopy of GnRH-treated LβT2 cells showed that GnRH induces bleb formation (Figure 1A) (see also Video S1 in Supplementary Material). The blebs appear within ~2 min and last for at least 90 min at the apparent turnover rate of ~2–3 blebs/min. In order to further investigate the involvement of the GnRHR in bleb formation, LβT2 cells were transfected with GnRHR-mCherry and then treated with GnRH for 30 min. Under basal conditions, GnRHR was observed in the membrane (Figure 1B), while after GnRH treatment, GnRHR decorated the blebs membrane, with no difference between blebs expansion and retraction. Preincubation with the GnRH antagonist (cetrorelix acetate), abolished bleb formation by GnRH (Figures 1C,D), confirming that bleb formation is mediated by the GnRHR. In addition, the cells returned to pretreatment morphology after removal of GnRH indicating that the process is reversible. Retreatment with GnRH (30 min) 6 h later resulted in a “priming effect,” which is defined as an increase in cells response to the second exposure to GnRH compared with the first. Indeed, the second exposure to GnRH elevated the percentage of blebbing cells (Figure 2).

ERK1/2 activation by GnRH in LβT2 cells was reported to involve PKC, Ca²⁺ influx, dynamin, and c-Src (20, 44, 45). Previous studies in our laboratory have examined the kinetics of ERK1/2 activation in response to GnRH treatment in LβT2 cells. GnRH treatment resulted in a rapid and robust activation of ERK1/2 with a peak 5 min after stimulation and decline but still detectable after 90 min (44). It is thought that RTK and GPCR ligands induce a rapid translocation of ERK1/2 to the nucleus to phosphorylate and activate transcription factors (10, 11). In order to understand the involvement of ERK1/2 in bleb formation, we followed the cellular localization of ERK1/2 in response to GnRH treatment. LβT2 cells were transfected with GnRHR-mCherry and ERK2-GFP and then treated with GnRH for 15 min. Time-lapse confocal microscopy showed that GnRH-induced ERK1/2 accumulation in the blebs within 1 min (Figure 3A) (see also Video S2 in Supplementary Material). A line intensity profile across the cell was obtained (Figure 3B) and intensity profiles shown on the right demonstrate accumulation of ERK1/2 in the blebs. Quantitation of mean fluorescence intensity showed higher values of ERK1/2 in the blebs vs. intracellular area (without the blebs area) (Figure 3C). Moreover, preincubation with the MEK inhibitor U0126 strongly inhibits GnRH-induced bleb formation (Figure 3D). Also, preincubation with the MEK inhibitor U0126 strongly reduced GnRH-induced cell migration (data not shown) suggesting that the bleb formation may be involved in cell migration. Pretreatment with SB203580, a p38 inhibitor did not attenuate the bleb formation (Figure 3E). Quantitation confirmed that compared to GnRH treated cells, U0126 reduced cell blebbing and SB203580 had no significant effect (Figure 3E). In addition, fluorescence intensity was measured across the cells and the profiles shown in line graphs on the right indicates that ERK2 accumulates in the blebs even in the presence of the p38 inhibitor (Figure 3F).

Epidermal growth factor (EGF) plays important roles in proliferation, differentiation, and migration via stimulation of the ERK1/2 signaling pathway (46). Moreover, Bonfil et al. (20) reported that ERK1/2 activation by GnRH in LβT2 cells is mediated by PKC, Ca²⁺ influx, dynamin, and c-Src, and not via transactivation of the EGFR. PMA is a PKC activator, which mimics the action of the naturally occurring DAG by binding to the C1 region of PKC, thus activating the enzyme (47–49). PMA mimicked the activation of ERK1/2 by GnRH (44). In addition, GnRH stimulates cAMP production in LβT2 gonadotrope cells via PKCδ (50). We therefore examined the effect of the various ligands on ERK1/2 activation and bleb formation since we have shown above that active ERK1/2 is required for bleb formation (Figure 3). Addition of EGF resulted in rapid activation of ERK1/2 with a peak after 5 min, similar to the effect of GnRH (33) (Figure 4A). LβT2 cells were treated with EGF for 30 min, and time-lapse confocal microscopy revealed minimal bleb formation (Figure 4B). GnRH was then added to the EGF-pretreated cells and significant elevation of bleb formation was observed (Figures 4B,C). The ligands induced ERK1/2 activation in the rank order of: EGF > GnRH > PMA > cAMP (Figure 4D). Later, we examined the effect of the ligands on bleb formation. cAMP and PMA induced relatively small amount of blebs (Figure 4E). The rank order for bleb formation differs from that obtained for ERK1/2 activation and is: GnRH > PMA > cAMP > EGF (Figures 4C,E). Therefore, we propose that ERK1/2 activation is required, but not sufficient for bleb formation. Furthermore, the data suggest compartmentalization of the ERK1/2 signal to the blebs in a ligand-dependent manner, since GnRH-activated ERK1/2, was preferentially sorted also to the blebs.

The Rho family members RhoA, Rac1, and Cdc42 are implicated in actin cytoskeleton rearrangements. Godoy at el. (35) showed that GnRH activates Rho family members and increases cell motility. We therefore examined whether RhoA/ROCK is involved in GnRH-induced bleb formation. Preincubation with the ROCK inhibitor Y27632 abolished GnRH-induced bleb formation (Figures 4F–H). The findings indicate that RhoA–ROCK is involved in GnRH-induced bleb formation.

Our previous studies showed that GnRH activates ERK1/2 in LβT2 gonadotrope cells in a c-Src-dependent manner (20). The modular SH1, SH2, and SH3 and kinase domains of the Src family tyrosine kinases allow these domains to act as scaffolds for diverse signaling proteins (51). Since c-Src is a member of the signalosome (33), we followed its cellular localization in response to GnRH treatment. LβT2 cells were transfected with c-Src-GFP and GnRHR-mCherry and later treated with GnRH for 30 min (Figure 5A). Time-lapse confocal microscopy showed that c-Src is present in the blebs, with no difference between expansion and retraction of the blebs (Figure 5A). Furthermore, the data show the colocalization of c-Src and the GnRHR in the blebs (Figure 5B). Inhibition of c-Src activity by PP2, which we have shown previously (20), had no effect on bleb formation (Figure 5C), suggesting that active c-Src is not required for bleb formation by GnRH. Thereafter, we have chosen to follow other members of the signalosome known to interact with c-Src in relation to their presence in the blebs.

Vinculin is a scaffold protein, which binds to actin filament and is localized to FAs (52). Vinculin controls and regulates FA formation and cell migration. It is known that vinculin regulates survival and motility via ERK1/2 by controlling the accessibility of paxillin for FAK interaction (53). Since vinculin is a member of the signalosome and binds paxillin, we examined its involvement in bleb formation in response to GnRH treatment by live imaging microscopy. LβT2 cells were transfected with GnRHR-mCherry and vinculin-GFP and then treated with GnRH for 30 min. Time-lapse confocal microscopy showed that vinculin accumulates in the blebs (Figure 5D). However, unlike ERK1/2, c-Src, FAK, and paxillin (see below), vinculin was not present in the initial bleb expansion and was detected after the blebs were stabilized (Figure 5E). Therefore, we assume that vinculin is involved in bleb retraction.

Focal adhesion kinase is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of proliferation and migration of normal and tumor cells. FAK associates with integrin receptors and recruits a number of SH2- and SH3-domain-containing proteins to the site of this interaction, thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton (54). Since FAK binds c-Src and paxillin and is a member of the signalosome (33), we followed its presence in bleb formation. LβT2 cells were transfected with GnRHR-mCherry and FAK-GFP and then treated with GnRH for 30 min (Figure 6A). Time-lapse confocal microscopy showed that FAK is present in the blebs, while the GnRHR decorates the membrane but most of the receptors are retained in the cells as observed by others (55). Fluorescence intensity was measured across the cells and the profiles shown in line graphs on the right indicate that FAK accumulates in the blebs (Figure 6B). In addition, histograms show higher mean fluorescence intensity in the blebs vs. intracellular area (without the blebs area) (Figure 6C).

Paxillin, a multi-domain adapter protein, belongs to the FAs protein family and is known to interact with Ras, c-Src, tubulin, vinculin, and FAK (members of the signalosome) and is targeted to FAs via its LIM 1–4 domains (56, 57). It is thought that c-Src phosphorylation of Tyr118 of paxillin creates an ERK1/2-binding site. The activated paxillin then binds to Raf and MEK to activate ERK1/2. Moreover, the interaction with paxillin partially prevents ERK1/2 nuclear translocation, indicating that the task of restricting ERK1/2 in the cytosol is apparently carried out at least in part by paxillin. ERK1/2 phosphorylation of paxillin on Ser/Thr residues facilitates paxillin association with FAK (58, 59). Paxillin, together with FAK, is essential for cell spreading and migration (60, 61) as we have suggested for the signalosome (33). As indicated paxillin binds c-Src, FAK, and ERK1/2 and is a member of the signalosome, and therefore, we followed the cellular localization of paxillin in response to GnRH treatment. LβT2 cells were transfected with GnRHR-mCherry and paxillin-GFP and then treated with GnRH for 30 min (Figure 6D). Time-lapse confocal microscopy showed that paxillin accumulates in the blebs. Intensity profiles across the cells were obtained (Figure 6E), and graphs shown on the right demonstrate accumulation of paxillin in the blebs. Quantitation of mean fluorescence intensity (Figures 6F) shows higher values in the blebs vs. intracellular area (without the blebs area) as described above for FAK.

Microtubules are an important part of the cytoskeleton and play a vital role in many cellular processes, such as intracellular transport, mitosis, meiosis, and motility. Microtubules are composed of α–β-tubulin heterodimers (62). Since α-tubulin binds paxillin (57) and ERK1/2 (63) and is a member of the signalosome, we followed its presence in the process of bleb formation. LβT2 cells were transfected with GnRHR-mCherry and EMTB-3XGFP, which is the microtubule binding domain of ensconsin (EMTB) fused to 3GFP molecules, allowing microtubules visualization. Then, cells were treated with GnRH for 30 min. Time-lapse confocal microscopy showed the presence of microtubules fibers in the cells but not in the blebs, while α-tubulin was present in the blebs (Figures 7A,B).

The actin cytoskeleton is a central structure for various intracellular processes, such as vesicle transport, cell shape, cell division, motility, cell signaling, and morphogenesis (64). Furthermore, actin depolymerization and polymerization are involved in blebs life cycle (39, 40). Moreover, actin is involved in GnRH to ERK1/2 signaling (30, 34, 65). To examine the present of actin in GnRH-induced bleb formation, LβT2 cells were transfected with actin-YFP and then treated with GnRH for 30 min. Actin is not present in the blebs during blebs expansion. However, we could detect actin at the steady phase of the blebs and during the retraction (Figure 7C). The results are interesting since actin is not present in the signalosome (33), suggesting that blebs member’s proteins are not restricted to those present in the signalosome.

The data shown above confirmed that GnRH induces ERK1/2-dependent bleb formation in LβT2 cells. To determine whether this effect is also evident in primary rat pituitary cells in culture, dissociated rat pituitary cells were prepared and the gonadotropes were identified by their intracellular Ca²⁺ response to GnRH. Addition of GnRH resulted in bleb formation (Figure 8A). Quantitation of the percentage of blebbing cells showed that GnRH treatment resulted in bleb formation and ERK1/2 inhibition by U0126 significantly reduced bleb formation by GnRH (Figure 8B). We then isolated mouse pituitary gonadotropes by the use of FACS-sorted cells from adult GRIC/Ai9 mice and kept them in culture (42, 43, 66, 67). Time-lapse confocal microscopy revealed that GnRH-induced bleb formation (Figures 8C–E). In addition, preincubation of the cells with the MEK inhibitor, U0126, abolished bleb formation induced by GnRH (Figures 8D,E). Quantitation of the percentage of blebbing cells confirmed that GnRH induced ERK1/2-dependent bleb formation in primary gonadotropes in culture (Figure 8E).