The Ragulator complex and lysosomal calcium release are crucial for cell migration

Mice

C57BL/6J mice were used for in vivo experiments. Mice were housed under specific pathogen-free conditions with a 12-h light/dark cycle, at a temperature of 22 ± 2°C, and a relative humidity of 50 ± 5%. The mice were fed a standard mouse chow diet, and three to five mice were housed in the same cage. 8–9-wk-old mice were used in the experiments. Both male and female mice were used; however, the sex was always matched in each experiment. Animal experiments were approved by the ethics board of the Graduate School of Medicine, Osaka University (28-008-034), and all animal experiments were performed according to Osaka University’s regulations.

Cells and cell culture

THP1 cells, a human acute monocyte leukemia cell line, were obtained from the ATCC (TIB-202) and cultured in RPMI 1640 (Nacalai Tesque) supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.05 mM 2-mercaptoethanol. THP1 cells were differentiated with 50 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 72 h. HEK293T cells were obtained from the ATCC (CRL-3216) and cultured in DMEM supplemented with 10% FCS and 1% penicillin and streptomycin. All cells were maintained at 37°C in 5% CO₂. BMDCs were generated by culturing bone marrow cells with 50 μg/ml GM-CSF (R&D Systems) for 6–8 d as described previously (Inaba et al, 1992).

Antibodies, reagents, and fluorescent dyes

Reagents were obtained from the suppliers listed in Table S1.

NanoBRET

Plasmids of MPRIP1-539 tagged with a Halo-Tag donor at the amino terminus (MPRIP1-539-HTC), Lamtor1 tagged with a NanoLuc acceptor at the amino terminus (Lamtor1-NLF-C), and Lamtor2 tagged with a NanoLuc acceptor at the amino terminus (Lamtor2-NLF-C) were generated using In-Fusion HD Cloning Kit (Takara Bio). Briefly, cDNA fragments of human Lamtor1, Lamtor2, and MPRIP1-539 were ligated to homologous sequences by overlapping PCR using the primers listed in Table S1. These fragments were then cloned into the EcoRI-SacI sites of the pNLF1-C [CMV/Hygro] vector (Accession Number KF811458) for Lamtor1 and Lamtor2 and into the EcoRI-SacI sites of the pHTC Halo-Tag CMV-neo-Vector (Accession Number JF920305) for MPRIP1-539, using In-Fusion HD Cloning Kit. Transfection of HEK293T cells for luciferase assays was performed using Lipofectamine 2000 (Invitrogen). After 48 h of transfection, luminescence was measured after the addition of Nano-Glo Luciferase Assay Substrate (Promega) using GloMax Discover Microplate Reader (Promega). For evaluating the effect of TRPML1, HEK293T cells transiently expressing MPRIP1-539-HTC and Lamtor1-NLF-C were treated overnight with MLSA-1 8 μM and MLSI-3 16 μM before the assay.

Chemical compound library screening

A phytochemical and chemical library was obtained from Prestwick Chemical. HEK293T cells transiently expressing MPRIP1-539-HTC and Lamtor2-NLF-C were treated overnight with chemical compounds diluted to a concentration of 10 μM with DMEM. MPRIP1-539–Lamtor1 interactions were examined using the NanoBRET assay, as described above. The result is listed as Supplemental Data 1 and Supplemental Data 2.

Western blotting

Cells were solubilized in buffer A (1% Nonidet P-40 [NP-40], 50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 5% glycerol, and 2% n-octyl-b-D-glucopyranoside) with proteinase inhibitor (Roche), vortexed for 10 min, and centrifuged at 20,000g for 15 min at 4°C. Supernatants were mixed with 2× Laemmli sample buffer containing 2-mercaptoethanol and denatured for 5 min at 95°C. The reduced samples were electrophoresed on 4–12% Bis-Tris gels (Life Technologies), transferred to nitrocellulose membranes, and blotted with the antibodies listed in Table S1. Protein concentrations in the SDS–PAGE gel bands were determined using ImageJ software (NIH).

Immunoprecipitation

HEK293T cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen) and incubated for 48 h. Cells were solubilized with buffer A and centrifuged at 20,000g for 15 min at 4°C. Immunoprecipitation was performed using Dynabeads Protein G Immunoprecipitation Kit (10007D; Invitrogen). Briefly, after binding of the antibody to magnetic beads (15 min at room temperature, with rotation), cell lysates and antibody-coated beads were mixed and incubated for 1 h at room temperature. For the pharmacological assay, HEK293T cells were treated with ouabain (0, 50, and 100 nM) and MLSA-1(0, 20, and 50 μM) at specified concentrations for 6 h before the immunoprecipitation assay. For the Ca²⁺ assay, 3 mM EDTA was added to the cell lysate and incubated for 6 h, followed by the addition of CaCl₂ (0, 50, and 100 μM) to the lysate at 4°C for 12 h. For the Ragulator complex formation assay, HEK293T cells were transfected with Lamtor1-G2A-Flag with DMSO pretreatment for 6 h, Lamtor1-Full-Flag with DMSO pretreatment for 6 h, or Lamtor1-Full-Flag with ouabain 100 nM pretreatment for 6 h. Immunoprecipitation was performed with DYKDDDK-coated beads overnight at 4°C.

All immunoprecipitated samples were washed three times, and proteins were eluted with 2× Laemmli sample buffer containing 2-mercaptoethanol and denatured for 5 min at 95°C. The samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with the indicated antibodies.

Lysosomal fractionation

THP1 cells were cultivated at a density of 1.0 × 10⁶ cells/ml, and lysosomes were isolated from 3 × 10⁶ cultured cells by density gradient separation (14,5000g, 2 h), using Lysosome Enrichment Kit for Tissue and Cultured Cells (Pierce Biotechnology) according to the manufacturer’s protocol. The lysosome fraction band in the top 2 ml of the gradient was centrifuged as a lysosome pellet, and the remaining fraction band was centrifuged as a remnant pellet. Both pellets were mixed with 2× Laemmli sample buffer containing 2-mercaptoethanol and denatured for 5 min at 95°C. Samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with the indicated antibodies. For the pharmacological assay, before the assay, THP1 cells were treated with 100 nM ouabain for 6 h.

In vitro cell migration assay

For the Transwell assay, inserts (pore size, 5.0 μm for THP1; Corning) were placed in 24-well plates filled with 0.6 ml of 0.1% (wt/vol) BSA in RPMI medium containing recombinant CCL2. A cell suspension of THP1 (2 × 10⁵ cells/100 μl) was added to the upper well, followed by incubation for 4 h at 37°C. The cells in the lower chamber were detached for 5 min using 5 mM PBS-EDTA and counted using the Guava PCA system. Migration ability (% of input cells) was evaluated by dividing the number of cells in the lower chamber by the number of cells in the upper chamber and multiplying it by 100. For the pharmacological assay, THP1 cells were treated with ouabain (0, 50, and 100 nM) for 2 h before the assay, and MLSI-3 (0, 8, and 16 μM) and MLSA-1 (0, 4, and 8 μM) were added at the start of the assay. These substances were present in both the upper and lower chambers during the assay. For DC migration in 3D collagen matrices, chemotaxis assays were performed using μ-Slide Chemotaxis (ibidi GmbH) according to the manufacturer’s protocol. Briefly, LPS (200 ng/ml)-treated BMDCs were suspended in 1.5 mg/ml type I collagen (BD Biosciences) containing 5% FCS and seeded to the observation channel, incubating for 10 min. Subsequently, RPMI medium containing 0.1% BSA was filled to both sides of the chamber, with one side containing CCL19 (5 μg/ml). After 30 min of incubation, DC locomotion was examined at 1-min intervals by confocal time-lapse video microscopy. The velocity of cells was measured using ImageJ. To visualize lysosomes, BMDCs were labeled with 100 nM LysoTracker Red DND-99 for 30 min before being subjected to chemotaxis assays in μ-Slide Chemotaxis. For the pharmacological assay, MLSA (8 μM), MLSI-3 (16 μM), and blebbistatin (50 μM) were treated with BMDC for 2 h before and during the assay.

Expression vector cloning

cDNAs encoding Lamtor1 (Met1–Pro161) and its G2A mutant variant, both with a C-terminal 3×FLAG tag, have been previously described (Nakatani et al, 2021; Tsujimoto et al, 2023). Lamtor2–5, each tagged at the C terminus with 3×FLAG, were synthesized by Eurofins Genomics and cloned into the NotI–BamHI sites of the CSII-EF-IRES2-Venus vector (provided by Dr. Hiroyuki Miyoshi, Keio University) using In-Fusion HD Cloning Kit. The previously characterized V5-tagged MPRIP (Met1–Asp2457)–IRES-RFP lentiviral vector (Nakatani et al, 2021) was subcloned into the same sites as the CSII-EF-IRES2-Venus vector. cDNAs encoding the truncated forms of MPRIP (MPRIP^1–150, MPRIP^1–384, MPRIP^1–545, and MPRIP^545–1024) with the V5 sequence at the C terminus were amplified by overlapping PCR using the completed MPRIP-V5 vector as a template and subsequently cloned into the same vector sites. The cloned primers are listed in Table S1. TRPML1 tagged at the C terminus with V5 was synthesized by Eurofins Genomics and cloned into the NotI–BamHI sites of the CSII-EF-IRES2-Venus vector. The MYPT1-Myc vector (VB190904-1066bwp, pLV[Exp]-EGFP-EF1A> hPPP1R12A [NM_002480.3]/Myc) was purchased commercially and cloned. These vectors and constructs were used to express the respective proteins in specific cell lines for subsequent functional assays.

Generation of KO or knockdown cell lines

Lamtor1-deficient THP1 cells were generated as described previously (Nakatani et al, 2021). MPRIP-deficient cell lines were generated by transfecting cells stably expressing Cas9 with a mixture of several gRNAs using the Avalanche-Omni transfection reagent (EZ Biosystems). The target sequences used for MPRIP-KO are listed in Table S1. ATP1A1 knockdown was performed using lentiviral plasmids expressing shRNAs specific for human ATP1A1; the target sequence is listed in Table S1 (TRCN0000043226; Sigma-Aldrich).

Establishment of stable transfectants

Lamtor1−/− THP1 transfectants stably expressing full-length or mutant Lamtor1 were established by lentivirally introducing FLAG-tagged full-length Lamtor1 (Lamtor1-KO-Full-THP1) or FLAG-tagged G2A-Lamtor1 (Lamtor1-KO-G2A-THP1) into Lamtor1−/− THP1 cells, and single clones were screened by Western blotting with anti-FLAG mAb (M2; Sigma-Aldrich).

Enzyme-linked immunosorbent assay

Serum cytokine levels (TNF-α) were measured with DuoSet ELISA Development System (R&D Systems) following the product manual.

Immunostaining and fluorescence microscopy

For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS before incubation with primary antibodies, followed by incubation with Alexa Fluor secondary antibodies. Nuclei were stained with DAPI in a mounting medium (ProLong Gold with DAPI; Thermo Fisher Scientific). For imaging with multiple channels, extensive control experiments were performed to ensure that there was no nonspecific staining or crosstalk between channels. These control experiments included the following: (a) cells that lacked one of the proteins of interest and (b) staining without one of the primary or secondary antibodies. Fluorescence images of the fixed cells were obtained using an FV3000 confocal laser scanning microscope (Olympus).

Pharmacological application in cells

HEK293T cells were treated with ouabain, MLSI-3, or MLSA-1 at specified concentrations for 6 h. Immunoprecipitation and lysosomal enrichment assays were then performed. THP1 cells were pretreated with ouabain for 6 h, and the targets included phosphorylation levels of MLC and Rac GTPase, cell cytotoxicity, TNF-α activity, and localization of the indicated targets, which were assayed as mentioned above. Regarding the migration assay, the indicated concentrations and durations have been explained in their respective sections.

Active Rac1 detection

WT-THP1 cells were pretreated with 100 nM ouabain or DMSO for 6 h and lysed. The active form of Rac1 was pulled down with GST-PAK1-PBD fusion protein and immunoprecipitated with glutathione resin (Active Rac1 Detection Kit; Cell Signaling Technologies). Active Rac1 levels were determined by Western blotting using a Rac1/Cdc42 antibody (Cell Signaling Technologies).

RhoA pull-down assay

WT-THP1 cells were pretreated with 100 nM of ouabain or DMSO for 6 h and lysed. Then, the active form of RhoA was pulled down with a GST-Rhotekin-RBD fusion protein and immunoprecipitated with glutathione resin (Active RhoA Detection Kit; Cell Signaling Technologies). Active RhoA levels were determined by Western blotting using an anti-rabbit RhoA antibody (Cell Signaling Technologies). As a negative control, WT-THP1 cells were loaded with GDP before pull-down.

Adhesion assay

For the THP1 cell adhesion assay, 96-well plates were coated with VCAM (10 μg/ml). THP1 cells were stained with calcein-AM (5 mM) and suspended in RPMI medium supplemented with 0.1% BSA at a concentration of 5 × 10⁵ cells/well. The cell suspension was added to VCAM-coated wells and simultaneously treated with either MLSI-3 (16 μM) or MLSA-1 (8 μM). After incubation at 37°C for 2 h, nonadherent cells were removed by washing with PBS, and adherent cells were lysed in 0.1% NP-40. For the total group, 5 × 10⁵ THP1 cells were added to uncoated wells and lysed in 0.1% NP-40. Fluorescence intensity of each well was measured using a microplate reader set to an excitation wavelength of 485 nm. The adhesion rate was calculated by dividing the fluorescence intensity of each sample by that of the total group.

Fluorescein-labeled DC-trafficking assay

For the fluorescein-labeled DC-trafficking assay, 3 × 10⁶ BMDCs were pretreated with ouabain 200 nM for 6 h and subsequently treated with LPS 200 ng/ml for 2 h. Next, CFSE was labeled with 5 μM CFSE and injected into the footpads of WT mice. After 48 h, popliteal LNs were collected, and the frequency of CFSE-positive cells was assessed by flow cytometry.

Crystal-induced ankle arthritis model

C57BL/6 WT mice were subcutaneously anesthetized with ketamine and xylazine. Monosodium urate crystals (0.5 mg) suspended in 20 μl endotoxin-free PBS were co-administered with ouabain (10 μM, 0.2 μl) or DMSO (0.2 μl), and injected intra-articularly into the right tibia–tarsal joints. After 24 h, the joint sizes were measured using an electronic caliper, and the mice were euthanized to collect the joints for hematoxylin and eosin staining.

LPS-induced lung injury model

C57BL/6 WT mice were intraperitoneally injected with LPS (10 mg/kg). To assess the effect of ouabain on sepsis-related lung infiltration, 1 μg/kg of ouabain was co-administered intraperitoneally with LPS. At 36 h post-administration, the mice were euthanized, and lung tissues were fixed by intratracheal instillation of 4% paraformaldehyde. The tissues were stained with hematoxylin and eosin (HE) and examined by optical microscopy. Lung injury scores were quantified by a staff member who was blinded to the treatment groups, following the guidelines provided by the American Thoracic Society (Matute-Bello et al, 2011).

Mouse peritonitis model

C57BL/6 WT mice were intraperitoneally injected with 600 μl alum solution (20 mg/ml). Then, 1 μg/kg of ouabain was co-administered intraperitoneally with alum. To screen for immune cell populations, peritoneal infiltrating cells were stained with the indicated antibodies and analyzed using a FACSCanto II (BD Biosciences).

Flow cytometry

To detect the expression of CD80/CD86 in BMDCs, the cells were first stimulated with LPS for 6 and 24 h and simultaneously treated with 100 nM ouabain or DMSO for 6 h, then stained with the indicated antibodies. For the DC-trafficking assay, CFSE-positive cells were assessed by flow cytometry. Data from both experiments were collected by a FACSCanto II (BD Biosciences) by FACSDiva (v5.0.3) software and analyzed by FlowJo (v10) software.

RNA-seq

Overall 2 × 10⁶ Lamtor1-KO-Full and Lamtor1-KO-G2A-THP1 cells were collected for RNA extraction. Sequencing and data analysis were performed by the Genome Information Research Center at the Research Institute for Microbial Diseases, Osaka University. Quality control of the raw reads was performed using Trimmomatic (v0.39), and that of the resulting clean reads was performed with HISAT2 (v2.1.0). Gene expression analysis was performed by featureCounts (v2.0.6) and Cuffdiff (v2.2.1). All analysis data were evaluated and visualized by the server of RNAseqChef (Etoh & Nakao, 2023).

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 8. Normally distributed data were compared using a t test and are presented as means ± SEM. For non-normally distributed data, the Mann–Whitney U test was performed. Results for non-normal distributions are presented as the median with 25th and 75th percentiles, and minimum and maximum values, excluding outliers. Statistical significance was set at P < 0.05 for all analyses.