Golgi Apparatus

Cells were cultured on glass coverslips (Matsunami) pre-coated with 10 µg/ml fibronectin (Sigma) and fixed with 4% (w/v) paraformaldehyde in PBS or BRB80 [80 mM Pipes (pH 6.8), 1 mM MgCl₂, and 1 mM EGTA] for 10 min at room temperature. Fixed cells were stained with the respective antibodies, phalloidin conjugated with either Alexa Fluor 488 or rhodamine (Invitrogen), along with DAPI (Sigma) as described previously^{2 (link), 54 (link)}. In situ proximity ligation assay (PLA) was performed using Duolink kit (Olink Bioscience) according to the manufacturer’s instructions. After completion of the PLA reaction, samples were refixed with 4% (w/v) paraformaldehyde and incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) to detect the individual proteins. Fluorescence images were obtained using a laser scanning confocal imaging system (LSM700, Carl Zeiss) and processed using the ImageJ software. Number of Golgi fragments was quantified by using the ImageJ particle analysis tool. Colocalization was examined using the ImageJ JACoP plugin^{64 (link)} or Metamorph (Molecular Devices).

SaOS2 cells were transfected with the respective siRNAs and then with the respective expression plasmids for VSVG-GFP, VSVG-Myc, VSVG-MT1-MMP, or VSVG-KDELR-Myc with or without expression plasmid for sr-IFT20 or IFT20. To examine the ER-to-cell surface transport, VSVG-GFP-transfected SaOS2 cells were incubated overnight at 40 °C in DMEM containing 1% FBS to accumulate VSVG-GFP at the ER, and then shifted to DMEM containing 1% FBS and 0.1 mg/ml cycloheximide pre-warmed at 32 °C to allow transport through the Golgi. After 30 or 60 min in culture, cell surface proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-ss-biotin (Thermo) in Dulbecco’s PBS (DPBS) for 30 min at 4 °C and quenched with 50 mM NH₄Cl in DPBS for 10 min at 4 °C. After washing with ice-cold DPBS, cells were solubilized with Triton X-100 lysis buffer [25 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.4% (w/v) sodium deoxycholate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF]. Biotinylated proteins were affinity-purified with streptavidin-Sepharose beads and subjected to SDS-PAGE followed Western blot analyses. ER-to-cis-Golgi and intra-Golgi transport of VSVG and VSVG-MT1-MMP and retrograde transport of VSVG-KDELR have been described previously^{48 (link)}.

Transfected cells were treated with 3 µg/ml nocodazole (NZ) in culture medium for 2 hr. Cells were washed with ice-cold DMEM 5 times on ice to remove NZ and then incubated for 0∼8 min in CO₂-independent medium (Life Technologies) containing 1% (v/v) FBS at 25 °C. Cells were fixed with 4% (w/v) paraformaldehyde in BRB80 for 5 min at 25 °C, followed by 10 min fixation with methanol at −20 °C. Fixed cells were stained with antibodies against GM130 and tyrosinated (Tyr)-tubulin to visualize the cis-Golgi and newly nucleated MTs, respectively. Serial optical confocal z sections spanning the entire cell were obtained and stacked using a maximal intensity projection. Number of Golgi-MTs (non-centrosomal MT that has one end attached to a Golgi fragment) and non-centrosomal, non-Golgi-MT was quantified from the z sections and their stacked images.

For surface staining, single cell suspensions were stained with anti-CD4, anti-CD3, (from Invitrogen and eBioscience, Germany). For intracellular staining of Foxp3, cells were fixed and permeabilized with a FOXP3 staining buffer set (eBioscience, Germany) following the manufacturer’s instructions and stained with respective antibodies (Invitrogen) for 30 min. Intracellular cytokines were stained with anti- IFN-γ APC (Invitrogen) and anti-IL-17-Alexa 488 (BD), after PMA (30 nM) and Ionomycin (1.5 µM) (both Sigma-Aldrich, USA) restimulation in the presence of Golgi Plug for 5 h (antibodies see Table 1). Representative gating strategies and dot plots are shown in Supp Fig. 2.Table 1

Antibodies used in the experiments.

Antibody	Lot Number	Company	Clone
CD11b Pacific Blue	4329941	Invitrogen	M1/70
Gr1 FITC	2233545	BD Biosciences	1 A8
CD3 APC	2293305	Invitrogen	145-2C11
CD4 PE	E01009-1635	eBioscience	GK1.5
CD4 Pacific Blue	2261418	Invitrogen	RM4-5
IFN-γ APC	2011141	Invitrogen	XMG1.2
IL-17 Alexa 488	7096813	BD Biosciences	TC11-18H10
CD25 Pacific Blue	2055195	Invitrogen	PC61.5
Foxp3 FITC	2152040	Invitrogen	FJK-16 s

Mice were sacrificed and transcardially perfused with 4% paraformaldehyde (PFA) in cold phosphate-buffered saline (PBS). Mouse brains, superior cervical ganglions, and adrenal glands were collected, post-fixed in 4% PFA/PBS solution overnight, submerged in 30% sucrose in PBS for at least 72 h, and sectioned at 40 μm thickness using CM1950 cryostat (Leica)^{22 (link)}. Frozen sections were stained with antibodies specific to p150^Glued (amino acid 3–202 at the N-terminus of p150^Glued, BD Biosciences, #610474, 1:200, recognizing p150^Glued but not p135+), p150^Glued & p135+ (amino acid 1266–1278 at the C-terminus of p150^Glued, Abcam, #ab11806, 1:500, recognizing both p150^Glued and p135+), tyrosine hydroxylase (TH, Pel-Freez, #P40101-150, 1:2500; ImmunoStar, #22941, 1:500; Synaptic Systems, #213104, 1:500), dopamine transporter (DAT, Millipore, #MAB369, 1:500), vesicular monoamine transporter 2 (VMAT2, Synaptic Systems, #138302, 1:1000), glial fibrillary acidic protein (GFAP, Abcam, #ab7260, 1:1000), TAR DNA-binding protein 43 (TDP-43, Proteintech, #10782-2-AP, 1:500), α-synuclein (Santa Cruz, #sc-7011-R, 1:500; Santa Cruz, #sc-69977, 1:500), phosphorylated α-synuclein (Ser129) [p-α-synuclein (Ser129), Abcam, #ab51253, 1:500], neuronal nuclei (NeuN, Millipore, #ABN91, 1:500), synaptophysin (Millipore, #AB9272, 1:500), binding immunoglobulin protein (BiP, also referred to as GRP78, Abcam, #ab21685, 1:500), reticulon 3 (RTN3, Proteintech, #12055-2-AP, 1:500), 63 kDa cytoskeleton-linking membrane protein (CLIMP63, Proteintech, #16686-1-AP, 1:500), calnexin (Abcam, #ab22595, 1:500), protein disulfide isomerase (PDI, Proteintech, #11245-1-AP, 1:500), receptor binding cancer antigen expressed on SiSo cells (RCAS1, Cell Signaling Technology, #12290, 1:500), early endosome antigen 1 (EEA1, Cell Signaling Technology, #3288, 1:500), sequestosome 1 (SQSTM1, MBL, #PM066, 1:500), cathepsin D (R&D Systems, #AF1029, 1:500), ER-Golgi intermediate compartment 53 kDa protein (ERGIC53, Sigma-Aldrich, #E1031, 1:500), 130 kDa cis-Golgi matrix protein (GM130, BD Biosciences, #610822, 1:500), phosphorylated eukaryotic translation initiation factor 2α (Ser51) [p-eIF2α (Ser51), Abcam, #ab32157, 1:500], and phosphorylated inositol-requiring enzyme 1α (Ser724) [p-IRE1α (Ser724), Abcam, #ab48187, 1:500] as suggested by manufacturers. Alexa Fluor 488-, 546-, or 647-conjugated secondary antibody (Invitrogen, 1:500) was used to visualize the staining. Fluorescent images were captured using LSM 880 laser-scanning confocal microscope with Zen software (Zeiss) in conventional or Airyscan mode. As a high-resolution imaging modality, the Airyscan technology is reported to improve resolution 2-fold and signal-to-noise ratio 8-fold relative to the conventional confocal microscopy^{61 (link)}. The paired images in all the figures were collected at the same gain and offset settings. Post-collection processing was applied uniformly to all paired images. The images were presented as a single optic layer after acquisition in z-series stack scans at 1.0 μm intervals from individual fields or displayed as maximum-intensity projection or three-dimensional (3D) reconstruction to represent confocal stacks.

Tissue preparation started 24 h after the end of extinction test in matured mice. For each genotype, 6 mice were used. Mice were anesthetized with 3% isoflurane and decapitated. Brains were quickly removed from the skull and washed with ddH₂O to remove blood from the surface. Golgi–Cox impregnation of neurons was performed using the FD Rapid GolgiStain™ kit (FD NeuroTechnologies, # PK401)[68 ]. After a 3-week incubation, dye-impregnated brains were rapidly frozen in isopentane at -50 °C and then stored at -80 °C. For cryosection, brains were embedded in TissueTek O.C.T. compound (Sakura Finetek, # 4583) and coronally cryosectioned in 100-µm thickness and directly mounted on gelatin-coated slides (FD NeuroTechnologies, # PO101) with the help of solution C provided in the kit. Sections were stained according to the manufacturer’s protocol and mounted using the ROTI®Histokitt embedding medium (Carl Roth, # 6638)[68 ].