hp47  (Santa Cruz Biotechnology)

Journal: Wellcome Open Research

Article Title: Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

doi: 10.12688/wellcomeopenres.23776.1

Figure Lengend Snippet: A ) Dendra2::Col1a2 NIH3T3 cells were imaged using Airyscan microscopy after 18 h of culture, 1000x magnification, and 2.5 × digital zoom; a maximum intensity projection is shown. Vesicles containing Dendra2 signals were observed at the sites of fibril assembly. The Dendra2 signal was observed at the periphery of the vesicles; the scale bar represents 1 µm. The area outlined by the box is enlarged in the right-hand panel. Small panels show looped structures containing Dendra2-positive collagen. B ) Individual frames taken from Extended data 14 demonstrate that a single fibripositor (highlighted in the red box) is in contact numerous times by both the endoplasmic reticulum and electron-dense vesicles. The Golgi apparatus did not contact fibripositor (Extended data 15). C ) Immunofluorescence imaging of type I collagen in mouse embryonic fibroblasts using the ER marker PDI. Scale bar represents 20 µm. D ) Fractionation of NIH3T3 cells using a sucrose density gradient, type I collagen is co-resident with the ER protein calreticulin (Calr), but also with the lysosomal protein Lamp1 and the collagen chaperone Hp47. E ) Proteomic analysis of lysosomal fractions 6 and 7 revealed significant enrichment of proteins identified by proteomic analysis of fractions 6 and 7 based on GO Cellular component terms. F ) Col1a1 (Red and Magenta) and Col1a2 (Blue and Green)-derived procollagen peptides were present in fractions 6 and 7, suggesting that the newly synthesized collagen transitioned to these compartments. G ) Dendra2::Col1a2 NIH3T3 cells were imaged by Airyscan microscopy 48 h after transfection with LAMP1-YFP. Images were recorded at 1000x magnification using a 2.5 × digital zoom. The maximum intensity projection of the 31 images is shown. LAMP1-YFP vesicles (green) containing photoswitched Dendra2 signals were observed. Three LAMP1-positive areas were also observed. H ) Live super-resolution microscopy of NIH3T3 cells stably transduced with Hsp47-BFP-RDEL lentivirus and transfected with LAMP1-YFP. Scale bar, 20 µm.

Article Snippet: Antibodies used in this study were collagen (Gentaur, OARA02579, dilution 1:2000 (WB), 1:500 (IF)), Dendra2 (Origene, TA180094, dilution 1:500), GAPDH (Sigma, G8795, dilution 1:10,000), Calreticulin (Stressgen; SPA-601, dilution 1:1000), Lamp1 (Santa Cruz, sc-20011, 1:500), vinculin (Chemicon; CBL233, 1:2000), Hp47 (Santa Cruz, sc-398579, 1:1000 (WB), 1:500 (IF)), and syntaxin 5 (Santa Cruz, sc-365124, 1:500(WB)) PDI (Abcam, ab180993, dilution 1:500 (IF)).

Techniques: Microscopy, Immunofluorescence, Imaging, Marker, Fractionation, Derivative Assay, Synthesized, Transfection, Super-Resolution Microscopy, Stable Transfection, Transduction

Journal: bioRxiv

Article Title: Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

doi: 10.1101/2024.05.09.593302

Figure Lengend Snippet: A. Dendra2::Col1a2 NIH3T3 imaged using Airyscan microscopy after 18 h after culture, 1000x magnification, and 2.5x digital zoom, a maximum intensity projection is shown. Vesicles containing Dendra2 signal are observed at sites of fibril assembly. Dendra2 signal was observed arranged at the periphery of vesicles, scale bar represents 1 µm. Area outlined by the box is enlarged in the right hand panel. Small panels show looped structures containing Dendra2-positive collagen. B. Individual frames taken from Video 6 demonstrating that a single fibripositor (highlighted in the red box) is contacted numerous times by both endoplasmic reticulum and electron dense vesicles. C. Immunofluorescence imaging of type I collagen in mouse embryonic fibroblasts with the ER marker PDI. Scale bar represents 20 µm. D. Fractionation of NIH3T3 using a sucrose density gradient, type I collagen is co-resident with the ER protein Calreticulin (Calr) but also with the lysosomal protein Lamp1 and the collagen chaperone Hp47. E. Proteomic analysis of lysosomal fractions 6 and 7 identified significant enrichment of proteins identified by proteomic analysis of fractions 6 and 7 based on GO Cellular component terms. F. Col1a1 (Red and Magenta) and Col1a2 (Blue and Green) derived procollagen peptides were present within both fractions 6 and 7 suggesting that newly synthesized collagen transitions these compartments. G. Dendra2::Col1a2 NIH3T3 imaged using Airyscan microscopy 48 h after transfection with LAMP1-YFP. Images were recorded at 1000x magnification with 2.5x digital zoom. A maximum intensity projection of 31 images is shown. LAMP1-YFP vesicles (shown in green) containing photoswitched Dendra2 signals are observed. Three LAMP1-positive areas are enlarged. H. Live cell super resolution microscopy of NIH3T3 stably transduced with Hsp47-BFP-RDEL lentivirus and transfected with LAMP1-YFP. Scale bar, 20 µm.

Article Snippet: HP47-BFP-RDEL was generated using PCR products of HP47 (PCR amplified from plasmid MC202692 (Origene)), and BFP-RDEL (PCR amplified from BFP-KDEL plasmid) using primers detailed In Supplementary table 2 .

Techniques: Microscopy, Immunofluorescence, Imaging, Marker, Fractionation, Derivative Assay, Synthesized, Transfection, Super-Resolution Microscopy, Stable Transfection, Transduction