Collagen-I is endocytosed and reassembled into fibrils

A. Fluorescent images of tail tendon incubated with Cy3-colI for 5 days, showing presence of collagen-I within the cells, and fibril-like fluorescence signals outside of cells. Hoechst stain was used to locate cells within the tendon. Area surrounded by yellow box expanded on the right, and cells with Cy3-colI present intracellularly pointed out by yellow triangles. Area surrounded by grey box expanded on the right, and fibril-like fluorescence signals indicated with white arrows. Scale bar = 50 µm. Representative of N = 3.

B. Bar chart showing an increase of percentage of fluorescent cells incubated with 1.5 µg/mL Cy3-colI over time (top), and an increase of percentage fluorescent cells incubated with increasing concentration of Cy3-colI for one hour (bottom), suggesting a non-linear time-dependent and dose-dependent uptake pattern. N = 3.

C. Flow cytometry imaging of fibroblasts incubated with 5 µg/mL Cy3-labeled collagen-I for one hour, showing that collagen-I is taken up by cells and held in vesicular structures. Images acquired using ImageStream at 40x magnification. Scale bar = 10 μm. Cy3 – Cy3 channel, BF/Cy3 – merged image of BF and Cy3. Representative of >500 cells images collected per condition.

D. Fluorescent image series of fibroblasts incubated with Cy3-labeled collagen-I at 0 min (t = 0min) 69 min (t = 69 min) and 107 min (t = 107 min) after addition (rows) and viewed from different angles (columns), with cell mask (cyan) to distinguish the cell volume. White arrows point to intracellular collagen in vesicular like structures. Representative of N = 3. Scale bar = 20 µm.

E. Fluorescent image series of fibroblasts incubated with 5 µg/mL Cy3-colI for one hour, trypsinized and replated in fresh media, and cultured for 72 h. Top left labels denotes the fluorescence channel corresponding to proteins detected. Representative of N>3. Scale bar = 20 µm.

F. Fluorescent image series of Cy3-colI incubated at different concentrations for 72 h, either cell-free (right panel), or with fibroblasts (left panel). Representative of N = 3. Scale bar = 20 µm. Red box - zoomed out to the left, and separated according to fluorescence channel. White arrows highlighting Cy3-positive fibrils assembled by fibroblasts when incubated with 0.1 µg/mL Cy3-colI.

Inhibition of endocytosis leads to changes in collagen-I homeostasis, and endocytosis is a rhythmic event

A. Left: fluorescent images of collagen-I (red) counterstained with DAPI (blue) in fibroblasts treated with DMSO (top) or Dyng (bottom) for 72 h. Scale bar = 20 µm. Right: quantification of area occupied by collagen-I fibrils, corrected to number of nuclei.

B. Western blot analysis of conditioned media taken from fibroblasts treated with DMSO (ctrl) or Dyng (Dyng) for 72 h, showing a decrease in collagen-I secretion. Top: probed with collagen-I antibody (Col-I), bottom: counterstained with Ponceau (Pon) as control. Representative of N = 3.

C. Left: fluorescent image series of collagen-I (green), fibronectin (magenta) counterstained with DAPI (blue) in fibroblasts treated with DMSO (top) or Dyng (bottom) for 72 h. Scale bar = 20 µm. Right: quantification of area occupied by fibronectin fibrils, corrected to number of nuclei.

D. Percentage Cy3-colI taken up by synchronized fibroblasts over 48 h. Meta2d analysis indicates a circadian rhythm of periodicity of 23.8 h. Bars show mean ± s.e.m. of N = 3 per time point.

E. Percentage of Cy3-colI taken up by synchronized cells, corrected to the maximum percentage uptake of the time course (pink, bars show mean ± s.e.m. of N=3 per time point), compared to the percentage collagen fibril count over time, corrected to the maximum percentage fibril count of the time course (black, fibrils scored by two independent investigators. Bars show mean± s.e.m. of N=2 with n=6 repeats at each time point).

Collagen-I recycling can generate fibrils

A. Fluorescent image series of fibroblasts treated with scrambled control (top panel, scr), and siRNA against col1a1 (bottom panel, siCol1a1). Labels on top denotes the fluorescence channel corresponding to proteins detected. Representative of N = 4. Scale bar = 25 µm.

B. Fluorescent image series of scr (left column) and siCol1a1 (right column) fibroblasts incubated with Cy3-colI. Labels on left denotes the fluorescence channel(s) corresponding to proteins detected. Cy3-colI fibrils highlighted by red arrows, and collagen-I fibrils highlighted by white arrows. Both scr cells and siCol1a1 cells can take up exogenous collagen-I and recycle to form collagen-I fibril. Representative of N>3. Scale bar = 10 μm.

C. Fluorescent image series of siCol1a1 cells treated with DMSO control (left) or Dyng (right) during Cy3-colI uptake, followed by further culture for 72 h. Labels on left denotes the fluorescence channel corresponding to proteins detected. Dyngo treatment led to a reduction of Cy3-colI fibrils. Representative of N>3. Scale bar = 25 µm.

VPS33B controls collagen fibril formation at the plasma membrane in a rhythmic manner

A. Electron microcopy images of fibroblasts plated on Aclar and grown for a week before fixation and imaging. Ctrl culture has numerous collagen-I fibrils, as pointed out by arrows. Yellow arrow points to a fibripositor, and green box is expanded to the left bottom corner, showing the distinct D-banding pattern of collagen-I fibril when observed with electron microscopy. VPSko clones all have fewer and thinner fibrils present in the culture (pointed out by red arrows). Representative of N = 3. Scale bar = 0.5 µm.

B. Fluorescence images of collagen-I (red) and DAPI counterstain in ctrl and VPSko fibroblasts. Yellow arrows indicating collagen fibrils, and white arrows pointing to collagen-I presence in intracellular vesicles. Representative of N>6. Scale bar = 25 µm.

C. Matrix deposition by ctrl or VPSko cells, after one week of culture. Left: decellularized matrix mass. N = 4, * p = 0.0299. Right: hydroxyproline content presented as a ratio between ctrl and VPSko cells. N = 4, *p = 0.0254. Ratio-paired t-test used.

D. Fluorescence images of collagen-I (red) and DAPI counterstain in ctrl and VPSoe fibroblasts. Representative of N>6. Scale bar = 20 µm.

E. Matrix deposition by ctrl or VPSoe cells, after one week of culture. Left: decellularized matrix mass, N = 4. Right: hydroxyproline content presented as a ratio between ctrl and VPSoe cells, N = 4. Ratio-paired t-test used.

F. Relative collagen fibril count in synchronized ctrl (black) and VPSoe (pink) fibroblasts, corrected to the number of fibrils in ctrl cultures at start of time course. Fibrils scored by two independent investigators. Bars show mean± s.e.m. of N = 2 with n=6 at each time point.

G. Western blot analysis of conditioned media taken from ctrl and VPSoe after 72 h in culture. Top: probed with collage-I antibody (ColI), bottom: counterstained with Ponceau (Pon) as control. Representative of N = 3.

Procollagen-I and VPS33B localize to the same compartments

A. Schematic depicting the proposed membrane topologies of VPS33b.

B. Fibroblasts expressing BFP-tagged VPS33B. Left: BFP tagged on the N-terminal end of VPS33B (VPSnBFP), Right: BFP tagged on the C-terminal end (VPScBFP). Images taken in Airy mode. Representative of N>4. Scale bar = 10 µm.

C. Schematic of the split-GFP system. GFP1-10 barrel is introduced into VPS33B (VPS-barrel), and GFP11 to alpha-1 chain of Collagen-I (GFP11-pro⍺1(I)). If the two tagged proteins co-localize (e.g. in a vesicle), a GFP signal will be emitted.

D. Brightfield (top) and fluorescence (middle) images of fibroblasts expressing both VPS-barrel and GFP11-pro⍺1(I) constructs. Representative of N=5. Green box is expanded to the bottom, to highlight the punctate fluorescence signals within intracellular vesicular structures, as well as fibril-like structures suggestive of fibril assembly sites. Scale bar = 20 µm.

E. Fluorescence images of VIPAS (green), collagen-I (red) and DAPI counterstain in fibroblasts. Representative of N=3. Green box is expanded to the right (flipped 90°) to show strong VIPAS signal encasing collagen-I. Scale bar = 25 µm.

F. Quantification of average number of fibrils per cell (left) and average fibril length (right) in control Dendra-colI expressing 3T3 cells (ctrl) and Dendra-colI expressing 3T3 overexpressing VPScBFP (VPScBFP). >500 cells quantified per condition. N=12. *P=0.048.

G. Bar charts comparing the percentage of cells that have taken up 5 µg/mL Cy3-colI (left) and Cy5-colI (right) after one hour incubation between control (ctrl), VPS33B-knockout (VPSko) and VPS33B- overexpressing (VPSoe) cells, corrected to control. RM one-way ANOVA analysis was performed. N = 4.

H. Fluorescence images of cells of different levels of VPS33B expression, fed with Cy5-colI and further cultured for 72 h. Cultures were counterstained with DAPI. Bottom panel are zoomed-in images of the fibrils produced by the fibroblasts. Representative of N = 2.

Integrin α11 subunit mediates VPS33B-effects and is required for collagen-I fibrillogenesis

A. Top 25 Functional Annotation of proteins detected in biotin-enriched samples when compared to non-enriched samples based on p-values. Y-axis denotes the GO term, X-axis denotes –log (P value).

B. Heatmap representation of spectral counting of collagens detected in biotin-enriched surface proteins from control (ctrl), VPS33B-knockout (VPSko), and VPS33B-overexpressing (VPSoe) fibroblasts. Scale denotes quantitative value as normalized to total spectra, as determined by Proteome Discoverer.

C. Heatmap representation of spectral counting of integrins detected in biotin-enriched surface proteins from control (ctrl), VPS33B-knockout (VPSko), and VPS33B-overexpressing (VPSoe) fibroblasts. Scale denotes quantitative value as normalized to total spectra, as determined by Proteome Discoverer.

D. Heatmap representation of spectral counting of Plod3 and VPS33B detected in biotin-enriched surface proteins from control (ctrl), VPS33B-knockout (VPSko), and VPS33B-overexpressing (VPSoe) fibroblasts. Scale denotes quantitative value as normalized to total spectra, as determined by Proteome Discoverer.

E. Western blot analysis of integrin α11 subunit levels in control (ctrl), VPS33B-overexpressing (VPSoe), VPS33B-knockout (VPSko) cells. Top: probed with integrin α11 antibody, bottom: reprobed with GAPDH antibody. Representative of N = 3.

F. qPCR analysis of Itga11 transcript levels in ctrl compared to VPSko fibroblasts (left), and ctrl compared to VPSoe fibroblasts (right). N>3, ****P<0.0001, *P = 0.0226.

G. qPCR analysis of Itga11 mRNA expression in ctrl (left) or VPSoe (right) fibroblasts treated either with scrambled control (scr) or siRNA against Itga11 (siItga11), collected after 96 h. N=3, **P=0.0091, ****P<0.0001.

H. IF images of ctrl and VPSoe fibroblasts treated either with control siRNA (scr) or siRNA again Itga11 (siItga11), after 72 h incubation; collagen-I (red) and DAPI (blue) counterstained. Representative of N = 3. Scale bar = 25 µm.

I. Bar chart comparing the percentage of cells that have taken up 5 µg/mL Cy5-colI after one hour incubation between fibroblasts treated with scrambled control (ctrl) or siRNA against Itga11 (siItga11), corrected to scr. N=3. **P=0.0062.

The IPF fibrotic focus is positive for integrin α11 subunit and VPS33B.

A. qPCR analysis of patient-derived fibroblasts isolated from control (ctrl) or IPF lungs. Bars showing mean± s.e.m, 5 patients in each group from 2 independent experiments (technical repeats not shown here). Itga11, *p = 0.0259; VPS33B, *p = 0.0183.

B. Fold change of percentage Cy5-colI (left) or Cy3-colI (right) taken up by ctrl or IPF lung fibroblasts, corrected to average of control fibroblasts. Bars showing mean± s.e.m., 5 patients in each group from 2 independent experiments (technical repeats not shown here). **p = 0.003.

C. Fluorescent image series of ctrl or IPF lung fibroblasts that have taken up Cy3-colI (magenta), followed by further culture for 48 h, before subjected to collagen-I staining (cyan). Labels on top denotes the fluorescence channel corresponding to proteins detected. IPF fibroblasts produced more Cy3-labelled fibrillar structures. Representative of N = 4. Scale bar = 20 µm.

D. Immunohistochemistry of IPF patient (patient 1) with red dotted line outlining the fibroblastic focus, the hallmark lesion of IPF. Sections were stained with hematoxylin and eosin (H&E), collagen-I (ColI), integrin-α11, VPS33B. Scale bar = 50 µm.

E. Immunohistochemistry of 5 µm thick sequential lung sections taken from lungs classified as control (Control 1). Sections were stained with hematoxylin and eosin (H&E), collagen-I (ColI), integrin-α11, VPS33B. Scale bar = 100 µm.

Proteins responsible for collagen fibrillogenesis are also co-localized to diseased areas of chronic skin wounds.

A. Immunohistochemistry of 5 µm thick sequential skin sections taken from normal skin regions of patients with chronic skin wounds (Patient 1, Patient 2, Patient 3, Patient 4). Sections were stained with pentachrome, integrin-α11, VPS33B. Scale bars positioned in top left corner: black (unzoomed pentachrome) = 100 µm, white (zoomed sections) = 50 µm.

B. Immunohistochemistry of 5 µm thick sequential skin sections taken from the chronic wound areas from patients with chronic skin wounds (Patient 1, Patient 2, Patient 3, Patient 4). Sections were stained with pentachrome, integrin-α11, VPS33B. Scale bars positioned in top left corner: black (unzoomed pentachrome) = 100 µm, white (zoomed sections) = 50 µm.

Proposed working model of collagen homeostasis in fibroblasts.

Endogenous collagen is either secreted as protomers (soluble secretion route, not circadian rhythmic) or made into fibrils (fibril assembly route, circadian rhythmic). Secreted collagen protomers can be captured by cells through endocytosis (circadian rhythmic) and recycled to make new fibrils. Integrin α11 and VPS33b directs collagen to fibril formation.

Reagents

Primer list for cDNAs generated by site-directed mutagenesis.

Primer list for PCR reactions used to create transcription templates.

Primer list for qPCR reactions.