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.2020 Jan 6;219(1):e201904054.
doi: 10.1083/jcb.201904054.

Regulated resurfacing of a somatostatin receptor storage compartment fine-tunes pituitary secretion

Affiliations

Regulated resurfacing of a somatostatin receptor storage compartment fine-tunes pituitary secretion

Walaa Alshafie et al. J Cell Biol..

Abstract

The surfacing of the glucose transporter GLUT4 driven by insulin receptor activation provides the prototypic example of a homeostasis response dependent on mobilization of an intracellular storage compartment. Here, we generalize this concept to a G protein-coupled receptor, somatostatin receptor subtype 2 (SSTR2), in pituitary cells. Following internalization in corticotropes, SSTR2 moves to a juxtanuclear syntaxin-6-positive compartment, where it remains until the corticotropes are stimulated with corticotropin releasing factor (CRF), whereupon SSTR2 exits the compartment on syntaxin-6-positive vesicular/tubular carriers that depend on Rab10 for their fusion with the plasma membrane. As SSTR2 activation antagonizes CRF-mediated hormone release, this storage/resurfacing mechanism may allow for a physiological homeostatic feedback system. In fact, we find that SSTR2 moves from an intracellular compartment to the cell surface in pituitary gland somatotropes, concomitant with increasing levels of serum growth hormone (GH) during natural GH cycles. Our data thus provide a mechanism by which signaling-mediated plasma membrane resurfacing of SSTR2 can fine-tune pituitary hormone release.

© 2019 McGill University.

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Figures

Figure 1.
Figure 1.
Following internalization SSTR2 traffics to a syntaxin-6–positive compartment through an endosomal pathway. (A–C) AtT20 cells were serum starved for 2 h and then transferred to ice. The cells were incubated on ice for 1 h in the presence of 5 µg/ml Alexa Fluor 647–labeled Trf and 100 nM [D-Trp8]-SOM. Cells were than fixed (0 min) or transferred to 37°C for either 5 or 40 min as indicated before being fixed and processed for immunofluorescence with antibodies recognizing SSTR2 and syntaxin-6. Images were acquired using a LSM 710 confocal microscope. The images in B and C correspond to the boxed areas in A. For C, the intensity distributions for the lines passing through the images are indicated. Scale bars represent 10 µm in A and 5 µm in B and C.(D) AtT20 cells were washed extensively with Earle’s buffer and then incubated with 80 µM dynasore or vehicle control for 20 min before and during a 40-min incubation without (no stimulation) or with 100 nM [D-Trp8]-SOM at 37°C. Cells were than fixed and processed for immunofluorescence with antibodies recognizing SSTR2 and syntaxin-6.(E) Sucrose (0.45 M) was added to cells in Earle’s buffer and incubated for 15 min before and during a 40-min stimulation without (no stimulation) or with 100 nM of [D-Trp8]-SOM at 37°C. The stimulation was ended by washing in ice-cold Earle’s buffer and the cells were processed for immunofluorescence with antibodies recognizing SSTR2 and syntaxin-6. Images were acquired using the superresolution mode of the LSM 880 confocal microscope. Scale bars represent 5 µm.
Figure 2.
Figure 2.
SSTR2 displays slow recycling kinetics. (A) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6 (top two rows of panels). In parallel, cells treated with 100 nM [D-Trp8]-SOM for 40 min were subsequently transferred to 4°C, surface-bound agonist was stripped by a brief acid wash, and the cells were returned to 37°C for 1, 4, or 24 h before being processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6 (bottom three rows of panels). The boxes in the merged images are shown at higher magnification on the far right. Scale bars represent 5 µm and 1 µm for the low and high magnifications, respectively.(B) Quantification of SSTR2 cell surface immunofluorescence intensity from three successive experiments as shown in A and from three parallel experiments in the presence of Brefeldin A as shown in Fig. S1 with 10–15 cells quantified per condition per experiment. Data were analyzed by two-way ANOVA and Bonferroni comparison test (and procedures implemented in GraphPad statistical package). Data are presented as least-square means ± SEMs with treatment effects significant at 0.05. *, P ≤ 0.05; ***, P ≤ 0.001.
Figure S1.
Figure S1.
SSTR2 displays slow recycling dynamics in the presence of Brefeldin A and cycloheximide. (A) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM in the presence of 10 µM Brefeldin A and then fixed and processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6 (top two rows of panels). In parallel, cells treated with 100 nM [D-Trp8]-SOM for 40 min in the presence of Brefeldin A were subsequently transferred to 4°C, surface-bound agonist was stripped by brief acid wash, and the cells were returned to 37°C for 1 or 4 h before being processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6 (bottom two rows of panels). The boxes in the merged images are shown at higher magnification on the far right. Scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively. Dashed line indicates cell boundaries.(B) AtT20 cells were incubated with DMSO or 100 µg/ml cycloheximide 2 h before the start of experiment. Th cells were then incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM in the presence of vehicle control or cycloheximide (left 4 most panels). In parallel, cells treated with [D-Trp8]-SOM were subsequently transferred to 4°C, surface-bound agonist was stripped by a brief acid wash, and the cells were returned to 37°C for 1, 4, and 24 h (right six most panels). In all cases, at the end of the incubations cells were processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6. Scale bar represents 5 µm.
Figure 3.
Figure 3.
Confocal and STED microscopy reveals that internalized SSTR2 localizes to a cellular compartment distinct from the TGN. (A) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for each treatment condition. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively.(B) AtT20 cells were fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for each set of images. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Dotted lines indicate the plasma membrane.(C) AtT20 cells were incubated for 3 min with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SST2R. Images were collected using standard confocal microscopy, and the area under the magnifying glass was acquired by STED microscopy. Scale bars represent 5 µm and 1 µm for the confocal and STED images, respectively. Dashed areas in the STED image indicate individual SSTR2-labeled vesicles.(D and E) AtT20 cells were incubated for 40 min with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SSTR2 (D) or syntaxin-6 (E). Images were collected using standard confocal microscopy and the area under the magnifying glass was acquired by STED microscopy. Scale bars represent 5 µm and 1 µm for the confocal and STED images, respectively. Dashed lines in the confocal image indicate the cell boundary and dashed lines in the STED image indicate tubulovesicular structures.(F) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for the [D-Trp8]-SOM treatment condition. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Images were acquired using an Abberior STED microscope as a single optical section through the cells and cropped using ImageJ. Dashed lines indicate cell boundaries.(G) AtT20 cells were fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification in the bottom row for each image. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Images were acquired using an Abberior STED microscope as a single optical section through the cells and cropped using ImageJ. Dashed lines indicate cell boundaries. The arrowheads indicate the spatial segregation of immunoreactive signals of the indicated proteins.
Figure S2.
Figure S2.
SSTR2 and syntaxin-6 remain colocalized under nocodazole treatment. (A) AtT20 cells were treated with vehicle or 20 µM nocodazole for 2 h in DMEM and for the last 40 min of the nocodazole treatment with 100 nM [D-Trp8]-SOM before being fixed and processed for immunofluorescence with antibodies against the indicated proteins.(B–D) AtT20 cells were treated with vehicle or 20 µM nocodazole for 2 h in DMEM before being fixed and processed for immunofluorescence with antibodies against the indicated proteins. The arrowheads in A and B point to colocalizing structures, while the arrowheads in C and D point to noncolocalizing structures. Scale bars represent 1 µm. Dashed lines indicate cell boundaries.(E and F) Mander’s correlation coefficient measurements of at least 30–40 cells from two or three independent experiments for cells treated with vehicle (E) or nocodazole (F). Data were analyzed by one-factor ANOVA and Tukey comparison test (procedures implemented in the GraphPad Prism 5 statistical package). Data are presented as least-square means ± SEMs with treatment effects significant at ***, P < 0.001.
Figure 4.
Figure 4.
The SSTR2/syntaxin-6 compartment is resistant to Brefeldin A treatment. (A–C) AtT20 cells (A), cultures of primary cortical neurons (B), and C2C12 myoblasts (C) were incubated with vehicle control or Brefeldin A for 40 min before fixation and processing for immunofluorescence with antibodies recognizing the indicated antibodies. For B and C, the areas indicated by boxes in the merged images are shown at higher magnification on the right. Arrowheads represent nonoverlapping signals. For all images, scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively, and the dashed lines indicate cell boundaries. The outline of the nucleus (N) is also indicated by a dashed line in B.(D and E) AtT20 cells were incubated for 15 min with vehicle control or Brefeldin A before the 40-min stimulation of [D-Trp8]-SOM. The cells were then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification in the bottom sets of panels. The dashed lines indicate cell boundaries. Scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively.(F) Pearson coefficient measurement of SSTR2/syntaxin-6 or SSTR2/PIST colocalization in a juxtanuclear ROI. The quantification is based on 40–60 cells per condition from four successive, independent experiments. Data were analyzed by one1-factor ANOVA and Tukey comparison test (procedures implemented in JMP statistical package; SAS Institute). Data are presented as least-square means ± SEMs with treatment effects significant at *, P < 0.05.(G–I) HEK-293 cells (G), HeLa cells (H), and MCF10A cells (I) were incubated with vehicle control or Brefeldin A for 40 min before fixation and processing for immunofluorescence using antibodies recognizing the indicated antibodies. Scale bars represent 5 µm. The dashed lines indicate cell boundaries.
Figure 5.
Figure 5.
SSTR2 fractionates with a syntaxin-6 compartment distinct from the TGN.(A)Immunoisolation of syntaxin-6–positive vesicles. HEK-293 cells were transfected with T7-SSTR2 alone or in combination with HA-EGFP-syntaxin-6. Cell lysates were prepared in detergent free buffer, and HA-EGFP-syntaxin-6 structures were isolated using anti-HA magnetic beads. The proteins were separated by SDS-PAGE and probed by immunoblot using antibodies recognizing the indicated proteins. The migration of molecular weight markers in kilodaltons is indicated. IP, immunoprecipitate.(B) Sucrose gradient separation of the syntaxin-6 compartment. 2 ml of AtT20 postnuclear supernatant collected from three 10-cm dishes was fractionated on a 10-ml continuous 10–40% (wt/vol) sucrose density gradient. Fractions (500 µl) were harvested, beginning at the top of the gradient. Equal volumes from every other fraction were separated by SDS-PAGE and processed for immunoblot using antibodies against recognizing PIST, TGN38, GM130, and syntaxin-6.n = 4. The migration of molecular weight markers in kilodaltons is indicated.
Figure 6.
Figure 6.
STORM of SSTR2. (A and D) AtT20 cells were left at steady state (no stimulation) or incubated with 100 nM [D-Trp8]-SOM for 60 min.(A) Cells were immunostained for SSTR2 and syntaxin-6 and (B) for SSTR2 and PIST. Cross-nearest-neighbor measurements are shown between SSTR2 and syntaxin-6 versus the spatially unrelated proteins PIST and Giantin (B and C) or for SSTR2 and PIST versus PIST and Giantin (E and F). PM, plasma membrane. Note the increase in spatial association between SSTR2 and syntaxin-6 following [D-Trp8]-SOM stimulation in the juxtanuclear clump in B but the random distribution of SSTR2 and PIST in E. Images were acquired using a Vutara SR-350 Biplane point-localization superresolution microscope. Data volumes are displayed in point splatting view.(G) Representative segmentation of juxta-nuclear clump versus the rest of the cells as used for the STORM analysis. Note that the image used to indicate the segmentation in G is identical to the image used to represent the localization SSTR2 and syntaxin-6 in A.(H) The spatially unrelated Giantin and PIST single-molecule localizations are used as a negative control for the distance analysis. Scale bars represent 5 µm.
Figure 7.
Figure 7.
Dynamics of SSTR2 trafficking. (A) HeLa cells transfected with EGFP-SSTR2 were imaged for 5 min in the absence of stimulation (top two panels), for 42 min 6 min after applying [D-Trp8]-SOM (middle two panels), or for 22 min 8 min after agonist washout (bottom two panels). The images are the first and last frames from Videos 1, 2, and 3, respectively.(B) HeLa cells were transfected with EGFP-SSTR2 and Ds-Red-syntaxin-6. Cells were incubated for 60 min with 100 nM of [D-Trp8]-SOM followed by brief acid wash, and the cells were then imaged using a Zeiss LSM 880 microscope with airyscan at 10- to 20-s intervals. The images represent frames from Videos 4 and 5. The boxed areas in the top panel are shown at higher magnification below. The dashed lines in B indicate SSTR2/syntaxin-6–positive vesicles. Scale bars in A and B represent 5 µm and 1 µm for the low- and high-magnification images, respectively.(C) HeLa cells were transfected with EGFP-SSTR2 and imaged live following 60 min of [D-Trp8]-SOM incubation and a brief acid wash. Representative cropped frames from Video 6 are shown. The arrows indicate SSTR2 transport on a tubular carrier. Scale bar represents 5 µm.
Figure 8.
Figure 8.
Rab10 is involved in SSTR2 recycling. (A) HeLa cells expressing EGFP-SSTR2 along with mCherry-Rab8, mCherry-Rab10, or mCherry-Rab13 were imaged live, with the figure revealing cropped frames from Videos 7–9. The boxed area from the Rab10-transfected cells is shown at higher magnification below. Arrows indicate SSTR2/Rab10-positive vesicles moving toward and fusing with the plasma membrane, which is indicated by the dashed line. Scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively.(B) Representative immunoblots from wild-type (WT) AtT20 cells and two Rab10-knockout (KO) cell lines generated using CRISPR/Cas9. The migration of molecular weight markers in kilodaltons is indicated.(C) AtT20 cells, either wild-type, two Rab10 knockout lines (KO-1 and KO-2), and an isogenic control were left untreated (no stimulation) or were treated for 60 min with [D-Trp8]-SOM to induce maximal SSTR2 internalization, followed by a brief acid wash and incubation in ligand-free media for 1 h at 37°C, without or with 100 nM CRF. Following the treatments, cells were fixed and processed for immunofluorescence with antibody recognizing SSTR2. Scale bar represents 5 µm.(D) Quantification of SSTR2 cell surface immunofluorescence intensity of three successive experiments as in C. Data were analyzed by two-way ANOVA and Bonferroni comparison test (procedures implemented in GraphPad statistical package). Data are presented as least-square means ± SEMs with treatment effects significant at 0.05. **, P ≤ 0.01; ***, P ≤ 0.001.
Figure 9.
Figure 9.
SSTR2 recycling is enhanced in response to physiological signals. (A) AtT20 cells were treated for 60 min with [D-Trp8]-SOM to induce maximal SSTR2 internalization followed by brief acid wash and incubation in ligand-free media for 1 h at 37°C without or with 100 nM CRF, 50 nM Bay K8644, a Ca2+ channel agonist, or 10 µM forskolin/1 mM IBMX. Following the treatments, cells were fixed and processed for immunofluorescence with antibody recognizing SSTR2. Scale bar represents 10 µm.(B) Quantification of SSTR2 cell surface immunofluorescence intensity of three successive experiments as in A. Data were analyzed by one-factor ANOVA and Tukey comparison test (procedures implemented in GraphPad statistical package). Data are presented as least-square means ± SEMs with treatment effects significant at 0.05.(C) AtT20 cells were treated for 60 min with [D-Trp8]-SOM with or without 10 µM PKI followed by brief acid wash and incubation in ligand-free media for 1 h at 37°C without or with 100 nM CRF and PKI. Following the treatments, cells were fixed and processed for immunofluorescence with antibody recognizing SSTR2. Scale bar represents 10 µm.(D) Quantification of SSTR2 cell surface immunofluorescence intensity of two successive experiments as in C. Data were analyzed by one-factor ANOVA and Tukey comparison test (procedures implemented in GraphPad statistical package). **, P ≤ 0.01; ***, P ≤ 0.001.(E) Cartoon model for the induced SSTR2 recycling from the GLSV compartment. Following internalization after SOM binding, SSTR2 is sequestered in a GLUT4 like storage vesicles in endocrine cells. The recycling dynamics is very slow and can be stimulated by activation of CRF receptor (CRFR) or activation of its downstream signaling pathways.
Figure S3.
Figure S3.
SSTR2 is in a vesicle that requires Rab10 for recycling and is positive for transfected GLUT4. (A) Control AtT20 cells or the two Rab10 knockout lines (KO-1 and KO-2) were electroporated with 5 µg EGFP-Rab10Q68L. At 72 h after transfection, cells were treated for 60 min with [D-Trp8]-SOM to induce maximal SSTR2 internalization followed by brief acid wash and incubation in ligand-free media for 1 h at 37°C without or with 100 nM CRF. After the treatments, cells were fixed and processed for immunofluorescence with antibody recognizing SSTR2. Scale bar represents 5 µm.(B) GLUT4 is sorted to GLSVs in AtT20 cells. AtT20 cells were electroporated with 5 µg GLUT4-GFP or GLUT4-mCherry. 24 h after transfection, cells were treated for 40 min with [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibody recognizing SSTR2 and syntaxin-6. Scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively.
Figure 10.
Figure 10.
Coronal sections through the mice anterior pituitary gland immunostained for SSTR2.(A) Coronal sections through the anterior pituitary of mouse 5, which was at a GH blood nadir at time of sacrifice, and mouse 11, which was at a GH blood apex at time of sacrifice. The sections are stained with antibody recognizing SSTR2 and with DAPI to reveal nuclei. Arrowheads point to predominantly intracellular staining in mouse 5 and cell surface staining in mouse 11. Scale bar represents 25 μm.(B) Average SSTR2 immunoreactive signal presented as ratio of plasma membrane to cytoplasmic signals from mice grouped depending on the GH status. Data were analyzed by Student'st test in GraphPad prism 5. Data are presented as least-square means ± SEMs with treatment effects significant at P < 0.05. ***, P ≤ 0.001.(C) Blood samples (2 µl) were collected from the tip of the tails at least 2 h before the sacrifice time at 15- to 20-min intervals. The level of GH in blood was measured using a sensitive sandwich ELISA.
Figure S4.
Figure S4.
Analysis of the SSTR2 surface to intracellular ratio in mouse pituitary. (A) SSTR2 staining in sections of pituitary with blood GH levels at the nadir, declining or at the apex as indicated. The mouse number is stated on each panel and corresponds to the GH profiles shown in Fig. 10 C. Scale bars represent 5 µm.(B and C) The cell surface to intracellular ratio of SSTR2 immunofluorescent signal from individual cells from individual animals (B) or grouped depending on the GH status (C). PM, plasma membrane. (D) Sections of pituitary glands from selected mice as indicated were immunostained with antibodies recognizing SSTR2 and GH. The mice were selected from the animals that were sacrificed at the apex or nadir of blood GH levels. Scale bars represent 5 µm.
Figure S5.
Figure S5.
Antibody validation. Validation of syntaxin-6 (A) and PIST (B) antibodies was done using predesigned siRNA directed against mouse syntaxin-6 (FlexiTube siRNA, NM-021433, #SI02674231, and #S102717729; Qiagen) or PIST (FlexiTube siRNA, NM-001199272, #SI01054900; Qiagen). AtT20 cells were transfected with 50 nM of siRNA 48 h before immunostaining for the indicating proteins. Note the disappearance of the immunofluorescent signal in the siRNA-transfected cells, but not in the cells transfected with a scrambled siRNA.(C) GH antibody was tested on a pituitary section showing selective staining of a subset of pituitary cells.
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