Myosin 1b and F-actin are involved in the control of secretory granule biogenesis

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.


Results
Myosin 1b is associated with the trans-Golgi network and immature secretory granules in neuroendocrine PC12 cells. We first analyzed the expression and distribution of myosin 1b (Myo1b) in neuroendocrine PC12 cells. Western blot analysis of PC12 cell lysates and purified secretory granules revealed the cofractionation of Myo1b and VAMP2 (vesicle-associated membrane protein 2), a specific marker of secretory granule membrane (Fig. 1a). Analysis of Myo1b distribution in PC12 cells by confocal microscopy coupled to immunofluorescence (IF) revealed that this protein is associated with 47 + 18% of secretory granules labeled with chromogranin A (CgA), a marker of secretory granules (Fig. 1b). Using antibodies raised against TGN46, a marker of the trans-Golgi network, and against furin, a prohormone convertase mainly localized in immature secretory granules just after their budding from the TGN membrane, we observed that Myo1b is mainly located in the TGN area ( Fig. 1c) and in 89 + 8% of immature CgA-containing secretory granules (Fig. 1d). Together, these results show that Myo1b is associated with secretory granules at the level of the TGN, most likely to promote the budding of immature secretory granules.

Myosin 1b controls the biogenesis of secretory granules. To demonstrate the implication of Myo1b
in the biogenesis of secretory granules, we used the neuroendocrine PC12 cells as well as a simplified model consisting of COS7 cells expressing CgA which has been shown to induce secretory granule-like structures in these cells 8,13 . Myosin 1b gene silencing in these cells was achieved through a home-designed short interfering RNA (Myo1bH siRNA) validated in the study of Almeida et al. 6 , which affords a strong reduction in Myo1b expression. Indeed, transfection of this siRNA significantly reduced Myo1b expression in both COS7-CgA (Fig. 2a) and PC12 (Fig. 2b) cells. This decrease in Myo1b expression did not alter CgA expression in PC12 cells (Fig. 2c), thus ruling out the possibility of an effect of Myo1b through altered CgA expression. Under these conditions and using confocal microscopy coupled to IF, we observed a significant reduction in the number of CgA-containing granules in both PC12 (Fig. 2d) and COS7-CgA (Fig. 2e) cells. This result shows that Myo1b could play a key role in the formation of secretory granules. We then investigated whether Myo1b knockdown alters the formation of secretory granules at the TGN level by fast time-lapse imaging of COS7 cells expressing CgA-GFP, using a spinning disc-equipped confocal microscope. Five hours after cell transfection, we observed numerous CgA-GFP-containing granules around the Golgi area moving towards the cytoplasm in control siRNA-treated cells ( Fig. 3a; Movie 1), and we found that the number of CgA-GFP granules generated was significantly reduced in Myo1b siRNA-treated cells (Fig. 3b,c; Movie 2). Moreover, in the latter case, the granules generated moved faster than those observed in control cells treated with a scramble siRNA ( Fig. 3d; Movies 1, 2). Thus, the absence of Myo1b alters not only the number but also the dynamics of CgA-GFP containing vesicles, which suggests together with the distribution data described above that the motor protein is involved in the initiation of secretory granule formation from the Golgi complex.
Myosin 1b depletion increases basal secretion. Given the impact of Myo1b expression on the number of secretory granules, we analyzed the effect of this myosin on the secretory competence of PC12 and COS7-CgA cells using CgA release as an index of the regulated secretory pathway. This analysis showed that Myo1b knockdown results in increased basal release of CgA in both cell models (Fig. 4a,b). We also studied the distribution of CgA in the constitutive and the regulated secretory pathways in the absence of Myo1b. Using an antibody raised against collagen III, a protein of the extracellular matrix of fibroblasts which is addressed to the constitutive secretory pathway, we found that Myo1b down-regulation provokes a significant increase in the number of vesicular organelles containing both collagen III and CgA (Fig. 4c). These results indicate that CgA is misrouted to the constitutive secretory pathway in the absence of Myo1b, leading to its increased release in basal conditions. Together, these data show that Myo1b controls secretory protein sorting toward the regulated pathway.
Actin and actin-related proteins are also required for the biogenesis of secretory granules. In addition to those previously identified through proteomic characterization of whole secretory granules 8 , further analysis of purified secretory granule membranes revealed the occurrence of several other F-actin components, effectors and regulators with known functions at the level of the Golgi complex (Table 1). Among these proteins, Arp2/3 and actin cytoplasmic 1 and 2 have been reported to promote the formation of post-Golgi carriers by regulating membrane remodeling at the TGN in HeLa cells 6 , suggesting that actin could cooperate with Myo1b at the level of the TGN membrane to regulate secretory granule formation. To determine the role of F-actin in secretory granules biogenesis, we depolymerized F-actin using latrunculin B and inhibited actin nucleation mediated by the Arp2/3 complex using CK-666, in COS7-CgA and PC12 cells. F-actin depolymerization led to a significant decrease in the number of CgA granules (up to 50% and 10% in COS7-CgA and PC12 cells, respectively) ( Fig. 5a,b), thus revealing a key function of F-actin in the regulation of secretory granule formation. IF analysis of  Scientific RepoRts | 7: 5172 | DOI:10.1038/s41598-017-05617-1 the Arp2/3 p34 subunit distribution confirmed that CK-666 treatment inhibits the recruitment of Arp2/3 complex and F-actin to the Golgi region (Fig. 6a). Inhibition of Arp2/3 complex also provoked a significant reduction in the number of secretory granules (up to 50% in COS7-CgA and PC12 cells) (Fig. 6b,c). Taken together, these observations demonstrate that Arp2/3-dependent recruitment of F-actin in the Golgi area is required for the regulation of secretory granule biogenesis.  Myosin 1b promotes the recruitment of Arp2/3-dependent F-actin to the Golgi area. Myo1b has the ability to interact with actin via its motor domain and with the membrane via its pleckstrin homology motif 11 . Using Myo1b siRNA and the Arp2/3 inhibitor CK-666, we investigated the impact of Myo1b and Arp2/3 complex on F-actin distribution in the Golgi area of COS7-CgA cells. Fluorescent phalloidin and GM130 antibody allowed the analysis of actin distribution around the Golgi area. Analysis of line scans of fluorescence intensity taken through the cell showed that Myo1b depletion and Arp2/3 inhibition lead to F-actin decrease in the Golgi area (Fig. 7a). Quantification of actin and Golgi complex colocalization showed that both Myo1b knockdown and Arp2/3 complex inhibition lead to a 50% decrease in actin occurrence at the Golgi complex (Fig. 7b). These results show that blockade of Arp2/3 complex phenocopies Myo1b knockdown and that both Myo1b and Arp2/3 complex are involved in the recruitment of F-actin to the Golgi area in secretory cells.

Discussion
Secretory granules are key organelles allowing regulated hormone release in endocrine cells. Although considerable efforts have been devoted to understand the molecular events underlying secretory granule formation, the complex mechanisms that allow the specific sorting of hormones and the budding of secretory granules remain incompletely understood. It has been proposed that the sorting of peptide hormones through the regulated secretory pathway is controlled by chromogranins, which aggregate with peptides in the TGN lumen and function as the core around which budding of secretory granules is initiated from the TGN membrane 14 . In a previous study, we used a simplified model of CgA-induced vesicles in COS7 cells to identify potential effectors of granulogenesis through proteomic analysis of the newly formed granules. This analysis allowed the identification of a panel of cytosolic proteins interacting with actin, including myosins 8 , some of which have been associated with vesicular formation and exocytosis. Indeed, we identified myosin VI which is known to be involved in the regulated exocytosis of neuroendocrine cells 9,15,16 and in the maintenance of the Golgi morphology 17 . We also identified Myo1b, which has been shown to have an anchoring role that allows TGN membrane deformation through its attachment to the actin cytoskeleton, leading to the biogenesis of post-Golgi carriers in HeLa cells 6 . A more detailed analysis by mass spectrometry of proteins associated with the membrane of newly formed granules allowed us to identify Arp2/3 complex and actin cytoplasmic 1 and 2, which could act in concert with myosins to regulate the formation of secretory granules in endocrine cells. Although several studies reported the implication of myosins and F-actin in the trafficking and exocytosis of secretory granules 18 , the role of these multi-functional proteins in the formation of secretory granules has never been reported. We found that Myo1b is associated with the TGN and immature secretory granules in PC12 cells, indicating that Myo1b could affect the early stages of secretory granule biogenesis by inducing the budding of immature vesicles from the TGN membrane. When Myo1b expression is knocked down, the number of secretory granules is significantly reduced, thus pointing out a possible direct role of this protein in the formation of secretory granules. In addition, time-lapse imaging revealed that Myo1b depletion impacts the dynamics of the formed granules. Since our previous studies showed that constitutive vesicles move faster than CgA-induced granules in COS7 cells 8 , our present observations suggest that the absence of Myo1b leads to misrouting of CgA toward the constitutive secretory pathway. In support of this finding, we observed higher basal secretion and a higher number of CgA-containing constitutive vesicles when Myo1b was knocked down. Taken together, these data strongly support a role of Myo1b in the biogenesis of functional secretory granules underlying hormone regulated release in endocrine cells.
In addition to myosins, we found that Arp2/3 complex and F-actin are associated to secretory granule membranes. But more than controlling the integrity of the Golgi complex, F-actin, together with Arp2/3, has been shown to mediate the effect of Myo1b on TGN membrane deformation in HeLa cells, as Myo1b actively tethers and orients polymerizing F-actin to generate the required mechanical force 6 . In the present study, we demonstrate for the first time that F-actin controls the biogenesis of secretory granules, most likely via its recruitment to the Golgi region through Arp2/3 activation. Indeed, the Arp2/3 complex binds to actin and was shown here to exert an active role in secretory granule formation, potentially by providing de novo actin tracks for membrane curvature, as demonstrated during secretory granule exocytosis 19,20 . Therefore, Myo1b depletion or Arp2/3 inhibition could disrupt the interaction between the TGN membrane and F-actin, hampering secretory granule formation. Secretory carrier membrane protein 3 O14828 Golgi-derived transport carrier biogenesis and Golgi-to-ER protein transport 29 Table 1. F-actin components, effectors and regulators, with known functions at the Golgi complex, identified in the membrane fraction of purified granules from COS7-CgA cells. Identification was based on human sequences, as well as those of other mammalian species. Only proteins that were identified with one or more high scoring peptides from Mascot were considered to be true matches. "High scoring peptides" corresponded to peptides that were above the threshold in Mascot (P < 0.05) searches. The table shows a non-redundant list of the proteins and the reference to the Uniprot-access number used in database searches (http://uniprot.org).

Name Access number Function References
These results suggest that the actomyosin complex could contribute to secretory granule biogenesis by triggering membrane deformation, which could be stabilized in the nascent secretory granule by cytosolic BAR proteins, such as the protein interacting with C kinase 1 or arfaptin-1 21,22 , before the recruitment of scission proteins that will give rise to immature secretory granules. The present work represents the first evidence that Myo1b and F-actin are involved in secretory granule formation and in the establishment of the regulated secretory pathway. The action of the actomyosin complex could require its recruitment at the TGN to induce membrane remodeling, a mandatory process for the biogenesis of secretory granules. Additional studies will be needed to establish the molecular mechanisms promoting the recruitment of actomyosin complex to the TGN membrane in order to trigger secretory granule biogenesis.

Materials and Methods
Cell culture. African green monkey kidney fibroblast-derived COS7 cells (American Type Culture Collection; CRL 1651), wild type (COS7-WT) and stably expressing CgA (COS7-CgA) developed previously 8   Myosin 1b knockdown. For transient Myo1b knockdown, a home-designed Myo1b siRNA (Myo1bH siRNA, 5′-GCTTACCTGGAAATCAACAAG-3′) (Sigma Proligo) was used as previously described 6 . A non-targeting siRNA pool designed by Dharmacon-Thermo Scientific has been used as control siRNA. The non-targeting pool contains 2 siRNA which respectively reduces EGFR mRNA by ≈50% and targets firefly luciferase. COS7 cells were transfected with 10 nM siRNA and 1 µL of Lipofectamine RNAimax (Life Technologies) per well, in 24-well plate, according to the manufacturer's protocol. PC12 cells were transfected with 30 nM siRNA, following the same protocol. Four hours after the transfection, the medium was replaced by supplemented DMEM, and cells were cultured for 48 h. Transfected cells were then analyzed by SDS-PAGE and Western blotting for Myo1b or CgA levels or fixed for IF.  Immunofluorescence labelling. Cells cultured onto coated glass coverslips were transfected as described above and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were permeabilized and blocked for 30 min with 0.3% Triton X-100 in PBS containing normal donkey serum (1:50) and 1% BSA. Cells were then incubated for 2 h at room temperature with primary antibodies, and, after washing with PBS, for 1 h with secondary antibodies. Phalloidin-FITC (Invitrogen, 500 nM) was used to detect actin filaments. Nuclei were stained with DAPI (Molecular probes #D3571, 1 µg ml −1 ). To verify the specificity of the immunoreactions, the primary or secondary antibodies were substituted with PBS. Image acquisition. Confocal microscopy was carried out with a TCS-SP8 upright confocal laser scanning microscope equipped with 63× oil immersion objective (NA = 1.4; Leica, Microsystems). Alexa 488 and GFP were excited at 488 nm and observed in a 505-540 nm window. Alexa 594 was excited at 594 nm and observed in a 600-630 nm window. Alexa 647 was excited at 633 nm and observed in a 650-700 nm window. For dual color acquisition, images were sequentially acquired in line scan mode (average line = 2). Overlays were performed with post acquisition Leica Confocal Software functions to obtain the presented snapshots. Golgi exit of granules was monitored by time-lapse fluorescence microscopy using a spinning-disc confocal microscope. This microscopy was carried out with a Yokogawa CSU-22 spinning-disc head on a Nikon TE-2000 U microscope equipped with a 100× NA 1.4 oil immersion objective and a Coolsnap HQ2 camera, a NanoScanZ piezo focusing stage (Prior Scientific) and a motorized scanning stage (Marzhauser). This microscope was steered with Metamorph 7.1 (Universal Imaging Corporation). The fast scan mode at 512 × 512 pixel resolution was used. Video sequences were acquired at 5 frames s −1 (100-200 ms exposures).
Post-acquisition analysis. The extent of colocalization of two labels was measured using the "Colocalization" module of Imaris 7.6.5. 64-bit version (Bitplane AG, www.bitplane.com). This program analyzes plan of confocal sections acquired in two channels. Imaris colocalization analyzes the entire confocal plan by measuring the intensity of each label in each pixel. The program uses an iterative procedure to determine an intensity threshold (in the 0-255 scale of pixel intensity) for each of the two labels. Pixels with intensities above this threshold are considered to be above the background. A pixel is defined as having colocalization when the intensities of both labels are above their respective thresholds. The extent of colocalization was expressed by Pearson coefficient in pixels with colocalization. The Pearson coefficient is a number between + 1 and −1, with positive values indicating a direct correlation, negative values indicating an inverse correlation, and values near 0 indicating no correlation. In this case, the Pearson coefficient measures the correlation between the intensities of the two labels only in the pixels with colocalization. The following procedure was used to measure colocalization. A computer folder containing the stack of confocal sections for the two labels was generated by the Leica TCS-SP8 confocal microscope. This folder was opened with Imaris and converted into an Imaris file. A broad region of interest (ROI) was defined as all the pixels in which the intensity of one of the labels was above a pixel intensity defined in the 0-255 scale. Once the thresholds were set, the program outputs a canal of colocalization with channel statistics containing the Pearson coefficient. Then with the tool 'spots detection' on Imaris, the number of spots in each label (A, B and 'coloc' channel) was quantified to determine the percentage of colocalised granules. Spots statistics are automatically computed for each spot object. To each spot belongs a spatial position along the x-and y-axis, as well as the intensity of the point it represents. The spot object is available to model point-like structures in the data. It provides a procedure to automatically detect point-like structures, an editor to manually correct detection errors, a viewer to visualize the point-like structures as spheres, and statistics output. The rendering of the TGN surface was obtained using the Imaris 'surface' tool.
For the quantification of the number of CgA granules (Figs 2, 3, 5 and 6), the number of objects in the cytoplasm was counted automatically using the Imaris 'spots' function and a size filter (estimated xy diameter) of 0.5 µm. For the quantification of F-actin in the Golgi region, the Golgi region was first outlined on the plane showing more GM130 staining; then F-actin was thresholded and the overlapping pixels between the two structures was calculated with the Imaris 'coloc' function. The distributions of GM130 and F-actin were analysed using post acquisition 'line scan' mode of the Leica Confocal Software. For the analysis of live cell images, each granule in the Golgi area was identified after background correction by ND-Safir software 24 (Inria Rennes). The mean velocity of detected granules was determined using the ImageJ "manual tracking" tool. Subcellular fractionation. Cells were collected in PBS and sedimented by centrifugation at 400 g for 5 min at 4 °C. The cell pellet was disrupted by 5 pull/push through a 21-and then a 25-gauge needles attached to a syringe, in ice-cold buffer (0.32 M sucrose, 20 mM Tris-HCl, pH 8; 1 ml g −1 of cells). The resulting lysate was centrifuged at 800 g for 30 min at 4 °C. Post-nuclear supernatants were centrifuged at 20,000 g for 20 min at 4 °C. Pellets containing dense core granules were centrifuged on a multi-step gradient of 1 to 2.2 M sucrose (1, 1.2, 1.4, 1.6, 1.8, 2 and 2.2 M sucrose; 5 ml steps), at 100,000 g for 12 h at 4 °C. All gradient steps were collected from the top of the tube in 5 ml fractions, and analyzed by western blotting to check the granule-containing fractions and to verify their purity. The recovered granule fractions were used for liquid chromatography coupled to tandem mass spectrometry analysis.
Proteomic analysis. The fraction containing CgA granules purified from COS7-CgA cells was diluted in Buffer A (20 mM Tris pH 7.5, 150 mM NaCl and Complete Inhibitors (Roche)). The sample was submitted to two freeze-thaw cycles, sonicated for 1 min in a cold water bath and centrifuged at 50,000 g for 1.5 h at 4 °C to separate soluble from insoluble material. In order to precipitate soluble proteins, the soluble fraction was diluted in 4 volumes of cold (−20 °C) acetone and incubated for 4 h at −20 °C before centrifugation at 13,000 g for protein precipitation. On the other hand, the pellet containing the insoluble material was carefully washed twice with cold buffer A and incubated in 200 µl of Buffer A containing 1% n-dodecyl-beta-maltoside and 0.4% amidosulfobetaine-14 detergents in order to efficiently solubilize membrane proteins. After 2 h of incubation under soft agitation at 4 °C, the extract was centrifuged for 10 min at 13,000 g and the proteins contained in the supernatant were precipitated using standard chloroform/methanol method. Then, proteins were reduced, alkylated and separated by 1D-gel electrophoresis. Finally, proteins were cleaved in gel by trypsin and identified by liquid chromatography coupled to tandem mass spectrometry. Statistical analysis. Data were analyzed with the Prism program (GraphPad Software). All secretion experiments were repeated at least four times using the non-parametric Mann-Whitney U test. For the quantification of the number of CgA granules, statistical significance was determined by Student's t -test with Welch's correction or Mann-Whitney U test. Values are expressed as means ± s.e.m., and the level of significance is designated in the figure legend as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.