Noncanonical autophagy at ER exit sites regulates procollagen turnover.

Cell Lines and Primary Cell Culture. MC3T3-E1 Subclone 4 osteoblast cell lines were acquired from ATCC (ATCC CRL-2593). Cells were cultured in αMEM + Glutamax (32571-036; Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% Pen/Strep (Corning). To stimulate procollagen synthesis and secretion, ascorbic acid 2-phospate (Sigma-Aldrich) was supplemented 18–24 h before imaging experiments. Primary osteoblasts were extracted from mice harboring the G610C mutation and their wild-type littermates (B6.129(FVB)-Col1a2tm1Mcbr/J; Jackson Laboratories), which were maintained on the C57BL/6J background ( 4 , 23 ). Osteoblasts were extracted from parietal bones of 3- to 8-d-old mice as previously described ( 4 ). All care and procedures were performed in accordance with a Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal Care and Use Committee-approved protocol. Constructs. Two FP-proα2(I) constructs were generated. In one construct, FP cDNA was placed between the signal sequence and exon 6 Col1a2 cDNA (Origene), replacing exons 1–5 that encode the N-propeptide and its cleavage site, following the cloning protocol generously provided by Sarah Dallas ( 21 , 54 ). In another construct, FP cDNA replaced exons 2–3, retaining the cleavage site and the minor triple helix of the N-propeptide. Both constructs demonstrated identical trafficking and secretion patterns. FP-proα1(I) construct was similar to the second FP-proα2(I) construct. Other FP-tagged proteins were based on the following constructs: GM130-Cherry modified from GM130-CFP ( 34 ), Ii33-Cerulean ( 55 ), and IL33-mRFP ( 55 ). FP-LC3 constructs were modified from CFP-LC3 ( 56 ); LAMP1-FP from LAMP1-Cherry ( 57 ); FP-Sec23 from YFP-Sec23A, a gift from David Stephens, University of Bristol, Bristol, United Kingdom (Addgene 66611) ( 10 ); FP-Sec31 modified from pECFP-Sec31A, a gift from David Stephens (Addgene 66612) ( 10 ); FP-Ub modified from GFP-Ub, a gift from Nico Dantuma, Karolinska Institutet, Stockholm (Addgene 11928) ( 58 ); FP-p62 modified from pMXs-puro GFP-p62, a gift from Noboru Mizushima, University of Tokyo, Tokyo (Addgene 38277) ( 59 ); ssHalo-KDEL modified from mEmerald-ER-3, a gift from Michael Davidson, Florida State University, Tallahassee, FL (Addgene 54082); Halo-Sec61 modified from mApple-Sec61-C-18, a gift from Michael Davidson (Addgene 54946); Halo-KLHL12 modified from XE250 pCDNA3.1 + (zeo)- VSV-KLHL12-Q405X, a gift from Randall Moon, University of Washington, Seattle (Addgene 16759); and FP-Halo modified from pENTR4-HaloTag (w876-1), a gift from Eric Campeau, University of Massachusetts, Worcester, MA (Addgene 29644). The following FPs were utilized: eGFP-N1 (GFP), mCherry-N1 (Cherry), HaloTag-N1 (Halo), mApple-N1 (Apple), mVenus-N1 (Venus), mCerulean-N1 (Cerulean), and mTagBFP2-N1 (TagBFP2). Janelia Fluor Dye 646 ( 60 , 61 ) was utilized for marking Halo-tagged molecules in live cells. FP-proα2 (I) and FP-proα1 (I) with GFP and Venus FP had low transfection efficiencies but were otherwise well-expressed and appeared to have minimal or no effect on procollagen synthesis and trafficking as well as cell function, unless dramatically overexpressed. Only the cells with low or moderate expression levels of FP-procollagen were utilized for imaging experiments. Cells that accumulated large aggregates within the ER lumen, although physiologically relevant and recapitulating in vivo observations of ER stress ( 4 ), were not utilized for studies of trafficking or degradation. TagBFP2, Cerulean, or Apple FP-procollagen constructs had even lower transfection efficiencies than GFP and Venus constructs but displayed no abnormal distributions. FP-procollagen with Cherry had an abnormal localization pattern inside the cell, which appeared to be caused by Cherry dimerization. Transfection and Treatments. MC3T3 cells were transfected with Fugene 6 (Promega) and primary cells were transfected with TransIT-LT1 (Mirus Bio), using the manufacturer’s protocols. Cells were imaged 18–24 h after transfection and subsequent incubation in αMEM + Glutamax media supplemented with 100 µM ascorbic acid 2-phoshate (Sigma-Aldrich) and 10% FBS from Valley Biomedical (lot no. 2C0550 tested for supporting osteoblast differentiation). Fifty micromolar H89 (Sigma-Aldrich), 5 µg/mL BFA (Cell Signaling), 100 nM bafilomycin A1 (Sigma-Aldrich), and 100 µM leupeptin (Sigma-Aldrich) were added to the cell culture media as needed at the time points indicated in the text. Immunofluorescence. Cells were fixed in freshly prepared methanol-free 2% formaldehyde (Thermo Fisher Scientific) solution in PBS, pH 7.4, for 10–15 min, washed in PBS, permeabilized in 0.4% Triton X in PBS for 10 min, and returned to PBS. After 30-min blocking in 3% BSA in PBS for 30 min, cells were incubated overnight at 4 °C with primary antibody diluted in the same blocking buffer then washed and incubated for 30–60 min with secondary Alexa Fluor-labeled antibodies (Thermo Fisher Scientific) diluted with 1.5% BSA in PBS. After the final PBS wash, cells were either imaged immediately or mounted with Prolong Diamond Antifade with DAPI (Thermo Fisher Scientific) for subsequent imaging. The following primary antibodies were utilized: anti-procollagen (AB765P; Millipore), anti-LC3 (3868; Cell Signaling), anti-SEC31 (13483; Cell Signaling), anti-GM130 (610822; BD Biosciences), and anti-PDI (1D3; Enzo Life Sciences). Imaging. Live- or fixed-cell imaging was performed on an LSM 880 microscope (Zeiss) with a 63× oil objective at standard confocal resolution or enhanced Airyscan resolution. Live-cell imaging was performed with line scanning whereas fixed-cell imaging was performed with frame scanning. For CLEM imaging, MC3T3 cells were grown on fibronectin-coated gridded cover glass (72265-50; EMS) and transfected as described above. After transfection for 18 h, cells were fixed with 2% formaldehyde and 0.1% glutaraldehyde (Sigma-Aldrich) in PBS for 10 min and imaged in PBS with Airyscan resolution. After imaging, the cells were additionally fixed in 2.5% glutaraldehyde, 2% formaldehyde, and 2 mM CaCl 2 in 0.1 M sodium cacodylate, pH 7.4, for 15 min at room temperature followed by 45 min on ice. The coverslips were washed for 5 min four times, postfixed with 2% OsO 4 for 2 h in the same buffer at 4 °C, extensively washed with water, stained with 2% uranyl acetate in water, dehydrated through series of increasing ethanol concentrations (30, 50, 70, and 90%, three changes of 100%) and embedded in EMBed 812 epoxy resin (EMS). After resin polymerization, the coverslip was removed with hydrofluoric acid. Cells previously imaged by light microscopy were identified by their position on the grid. A 1- × 1-mm area containing the cell(s) of interest was cut out using a jeweler’s saw, mounted on an aluminum holder, and trimmed to 300 µm × 300 µm. Serial 70- to 80-nm sections were cut parallel to the plane of the coverslip and mounted on formvar/carbon-coated slot (0.5 × 2 mm) EM grids. Sections were stained with 2% uranyl acetate in 50% ethanol and imaged in an FEI Tecnai 20 transmission electron microscope operated at 120 kV. Images were recorded on AMT XR81 widefield CCD camera. Light and electron microscopy images were manually aligned based on well-defined organelles.

Photobleach Corrections for Time-Lapse

Videos of Fluorescence Recovery Experiments. Time-lapse sequences and videos were corrected for photobleaching associated with acquisition of multiple images from the same area by using a bleach correction plugin based on histogram matching within the FIJI image processing package ( 62 ). In FP-Sec23 fluorescence recovery after photobleaching experiments ( Fig. 9 and SI Appendix , Fig. S8 ), intensity of FP-Sec23 puncta within the bleached fluorescence recovery area was normalized to the intensity of adjacent FP- Sec23 puncta in the same image outside this area. Biochemical Assays. Procollagen folding, secretion, and degradation were measured by Western blotting and pulse-chase experiments with azidohomoalanine as described in SI Appendix , Fig. S3 C – E ( 4 , 63 – 65 ). Quantitation and Statistical Analysis. Images were quantitatively analyzed using custom generated macros for FIJI image processing package as illustrated in SI Appendix , Fig. S6 . Two-way ANOVA with a Holm–Sidak post hoc test (SigmaPlot 13.0; SYSTAT) was performed for analysis of transfected and treated primary osteoblasts ( SI Appendix , Figs. S2 and S3 E ). One-way repeated-measures ANOVA with a Holm–Sidak post hoc test was performed on time-series data with BFA or H89 treatment ( Fig. 2 C and D ). Heteroscedastic, two tailed t tests were performed for all other data.

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