Abstract
Collagen is the most abundant secreted protein in vertebrates that persists throughout life without renewal. The unchanging nature of collagen contrasts with observed continued collagen synthesis throughout adulthood and with conventional transcriptional and translational homeostatic mechanisms that replace damaged proteins with new copies. Here we show circadian clock regulation of procollagen transport from ER-to-Golgi and Golgi-to-plasma membrane by sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis, assembly and degradation affects only a pool of newly-synthesized collagen whilst maintaining the persistent collagen network. Disabling the circadian clock causes collagen accumulation and abnormal fibrils in vivo. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis in which a sacrificial pool of collagen is synthesized and removed to maintain tissue function.
One-third of the eukaryote proteome enters the secretory pathway 1, including the collagens that assemble into centimeter-long fibrils in the extracellular matrix (ECM) 2. The fibrils account for one-third of the mass of vertebrates 3 and are sites of attachment for a wide range of macromolecules including integrins, making them essential for metazoan development 4,5. The importance of tight cellular control of collagen fibril homeostasis is exemplified in fibrosis, which is characterized by accumulation of collagen fibrils and is a feature of 45% of all deaths 6 including those caused by cancer 7, and in musculoskeletal disease in which loss of collagen leads to tissue frailty. A remarkable feature of collagen fibrils is that they are formed during embryogenesis 8 and remain without turnover for the life of the animal as evidenced by studies showing that the half-life of collagen in tendon and cartilage exceeds 200 years 9–13. This has led to the notion that collagen fibrils are static and unchanging.
However, the difficulty with the zero-turnover description of collagen is that it ignores the observation that the fibrils do not suffer from fatigue failure, which would be expected in the face of lifelong cyclic loading. In contrast to the evidence of zero replacement, fibroblasts synthesize new collagen 14,15 in response to mechanical and hormonal stimuli 16–18, and microdialysis of human Achilles tendon shows elevated levels of the C-propeptides of procollagen-I (PC-I, the precursor of collagen-I) after moderate exercise 19,20.
These opposing observations led to the alternative hypothesis presented in this study, in which zero turnover and continued synthesis can co-exist. We hypothesized that a pool of ‘persistent’ collagen co-exists with a pool of ‘sacrificial’ collagen, in which the latter is synthesized and removed on a daily basis under the control of the circadian clock. Support for this alternative hypothesis comes from observations of a circadian oscillation in the serum concentrations of the N- and C-propeptides of PC-I 21,22 and of collagen degradation products in bone 23. However, despite physiological and clinical observations, direct mechanistic support for these observations was lacking. Whilst the suprachiasmatic nucleus of the hypothalamus is the master circadian pacemaker, almost all tissues have self-sustaining circadian pacemakers that synchronize rhythmic tissue-specific gene expression in anticipation of environmental light and dark cycles 24,25. Disruption of the circadian clock leads to musculoskeletal abnormalities involving the ECM e.g. chondrocyte-specific disruption of the circadian clock resulted in progressive degeneration of the ECM in articular cartilage 26 and fibrosis in intervertebral disc 27, and mice with a global knockout (KO) of Bmal1 28 or the ClockΔ19 mutation 29 develop thickened and calcified tendons with associated immobilization. These observations were indicative of circadian control of ECM homeostasis.
Here, we performed time series electron microscopy, transcriptomics and proteomics over day/night cycles, which showed that the synthesis and transport of procollagen by the protein secretory pathway in fibroblasts is regulated by the circadian clock. We showed that SEC61, TANGO1, PDE4D and VPS33B regulate collagen secretion, are 24-hour rhythmic, and are located at the entry and exit points of the endoplasmic reticulum (ER), Golgi and post-Golgi compartments, respectively. CTSK is a collagen-degrading proteinase that is rhythmic in phase with collagen degradation to maintain collagen homeostasis. The result is nocturnal PC-I synthesis and a daily wave of collagen-I with no net change in total collagen content of the tissue. Importantly, the levels of SEC61, TANGO1, PDE4D and VPS33B do not fall to zero; therefore, there is a low-level persistent secretion of collagen which has been previously detected using radioactive amino acid biosynthetic labeling of PC-I (e.g. 30). Crucially, we discovered that arrhythmic ClockΔ19 and Scleraxis-Cre-dependent Bmal1 deletion mutant mice accumulate collagen and have a disorganized and structurally abnormal collagen matrix that is mechanically abnormal, which demonstrates the importance of secretory pathway rhythmicity in maintaining collagen homeostasis.
Results
Collagen fibril network in tendon follows a rhythmic pattern during 24 hours
Tendon is a peripheral circadian clock tissue with collagen fibrils arranged parallel to the long axis of the tissue. Time-series transmission electron microscopy (TEM) of wild-type C57/BL6 mice identified a trimodal distribution of diameters (D1, <75 nm; D2, ∼75-150 nm; D3 >150 nm) that varied with time of day (Figure 1A-C and Figure S1A). The relative proportions of D1 and D2 fibrils were antiphase to that of D3 fibrils during 24 hours (Figure 1D). This was the first evidence, to our knowledge, that the distribution of collagen fibril diameters is rhythmic during 24 hours. In further experiments, we mechanically dissociated whole tendons and performed time-series mass-mapping scanning transmission electron microscopy on the dispersed fibrils (Figure S1B, C), using previously described methods 31. Analysis of the mass per unit length data showed an abundance of D1 fibrils from tendons sampled at early night (Figure 1E), which corresponded to the time of day when levels of D3 fibrils were building up in undisrupted tendons. Thus, D3 fibrils can be separated into D1 fibrils by mechanical disruption. Next, we examined tendon tissue by serial block face-scanning electron microscopy (SBF-SEM) (Figure 1F). Stepping through the image stacks showed bundles of collagen fibrils in near parallel alignment with the long axis of the tendon (Video 1). Automated segmentation of the fibrils in three-dimensional reconstructions showed that D2 and D3 fibrils maintained relative positions to each other (Figure 1G, Video 2). In contrast, the narrow D1 fibrils exchanged relative positions with each other and with D2 and D3 fibrils, and made contacts with different D2 and D3 fibrils (Figure 1H, Video 3). The tips of D1 fibrils were attached to the shafts of D3 fibrils, and D3 fibrils appeared to become loose and give rise to new D1 fibrils (Video 4). We also showed that fibrils sampled at ZT15 (zeitgeber time, 3 hours into darkness) had more circular outlines than those sampled at ZT3 (3 hours into light, Figure 1I). In further experiments we showed that the percentage energy loss during dynamic cyclic loading at various strain rates varied between day and night; tendons at ZT15 exhibited more energy loss than at ZT3 independent of strain rate (Figure S1D-G). Three distinct time constants were calculated from stress-relaxation curves, all of which were higher on average in ZT3 tendons than in ZT15 tendons (T2, in particular, was statistically significantly higher) (Figure S1F), suggesting that the tendons relax more quickly at night.
Rhythmic protein secretory pathway
We considered that the diurnal changes in collagen fibril diameters, fibril circularity, and tissue mechanical properties could be due to rhythmic changes in transcription, translation and secretion leading to rhythmic changes in ECM composition. To identify a molecular mechanism, we first examined data from time-series transcriptomic profiling of mouse tendon 29. The data showed that the expression of Col1a1 and Col1a2 mRNAs is not rhythmic in mouse tendon during 24 hours (Figure S2). Therefore, the diurnal behavior of collagen fibrils could not be explained by rhythmicity of collagen gene transcription. However, previous studies in liver had revealed circadian clock regulation of ribosome binding of the Col1a1 transcripts 32. Here, we showed by electron microscopy that the packing of ribosomes along the ER was time-of-day dependent in mouse tendon fibroblasts in vivo and maximal at ZT15 (Figure 3SA, B). This was an indicator that circadian clock control of collagen homeostasis begins early in the secretory pathway by temporally regulating protein translation.
Intriguingly, transcripts encoding several key secretory pathway proteins were conspicuously rhythmic (Figure 2A). These included: Sec61a2 (a core member of the translocon 33), Mia3 (which encodes TANGO1 with a proposed role in facilitating cargo loading at ER exit sites 34), Pde4d (functionally related to another phosphodiesterase, Pde7a1, which has been proposed to be important in regulating PC-I transport in COS7 cells in culture 35), and Vps33b (a post-Golgi protein reported to be important in PC-IV post-translational modification 36). We also noted that the transcript encoding Ctsk (a collagenase important in collagen degradation 37,38) is 24-hour rhythmic. Western blot analysis showed that proteins encoded by these transcripts were rhythmic in adult mouse tendon (Figure 2B).
Circadian clock-synchronized cells in culture exhibit a rhythmic collagen matrix
Collagen secreting fibroblasts have been widely used as a model of peripheral clocks 39. Therefore, we used clock-synchronized cultured fibroblasts as an in vitro model system to evaluate the importance of Sec61a2, Tango1, Pde4d, Vps33b and Ctsk in collagen secretion and homeostasis. Using collagen I- and PDI-(ER-resident protein disulfide isomerase) specific antibodies, we showed that PC-I localizes to the ER early after synchronization (3 hours) and collagen fibrils begin to appear 7-15 hours later and are maximal at the end of the 24 hours post-synchronization (Figure 3A). The rhythmicity of collagen fibril assembly and disassembly continues for at least 3 days in culture (Figure S4). In further experiments, co-immunofluorescence studies using collagen I- and GM130-(an ERGIC/cis-Golgi resident protein) specific antibodies, showed a time-dependent localization of PC-I in ERGIC/cis-Golgi compartments (Figure S5). These experiments showed that a rhythmic secretory pathway was cell-intrinsic. Disappearance of some extracellular collagen fibrils at the end of the circadian cycle was indicative of a clock-regulated degradation step. Cathepsins are a family of cysteine proteases of which cathepsin K (CTSK) deficiency in pycnodysostosis results in accumulation of non-digested phagocytosed collagen in fibroblasts 40. Ctsk is transcriptionally and translationally rhythmic in tendon fibroblasts (Figure 2A, B and Figure 3B). Incubation of ex vivo tendons with odanacatib to inhibit CTSK resulted in collagen accumulation, as shown by quantitative hydroxyproline analysis (Figure 3C). Quantitative-PCR showed that odanacatib also led to increased expression of Col1a1 mRNA (Figure 3D) suggestive of a feedback mechanism between collagen turnover and transcription.
SEC61, TANGO1, PDE4D, and VPS33B are required for collagen fiber assembly
SEC61, TANGO1, PDE4D, and VPS33B are sequentially expressed during a circadian period and serially located within the secretory pathway in the direction of travel of secreted proteins. Thus, we postulated that they may have an essential function in regulating procollagen trafficking and collagen fiber assembly. To test this notion, we used knockdown and knockout strategies combined with immunolocalization fluorescence confocal microscopy.
SEC61 The mRNAs of Col1a1 and Col1a2 (which encode α1 and α2 of type I collagen, respectively) have a 5’ UTR consisting of 2 stems flanked by a bulge (termed the collagen 5’ stem-loop) that binds LARP6 (La ribonucleoprotein domain family member 6) and are specifically targeted to the SEC61 translocon 41. The protein-conduction pore of the translocon is formed from SEC61α which associates with a β and a γ subunit to form a heterotrimeric complex SEC61αβγ in eukaryotes 42–44,45. There are two Sec61a genes (Sec61a1 and Sec61a2), which encode SEC61α. CRISPR/Cas9-mediated knockout of Sec61a1 and Sec61a2 in MEFs (Figure 3E, Figure S3C) resulted in markedly fewer collagen fibers (Figure 3F). In the absence of the SEC61 translocon, transcription from Col1a1 and Col1a2 would be expected to continue, which raised the question about the fate of the proα1(I) and proα2(I) polypeptide chains. Co-immunofluorescence using anti-collagen-I- and anti-calnexin-specific antibodies showed that collagen was translated into the cytosol in the absence of the SEC61 translocon (Figure S3D).
TANGO1 TANGO1 has been suggested to corral COPII coats to accommodate PC-I 46. We showed that siRNA-mediated knockdown of Mia3/TANGO1 in MEFs (Figure 3G and Figure S3E) resulted in reduced numbers of collagen fibers (Figure 3H). Knockdown of Mia3/TANGO1 resulted in co-localization of PDI (ER-resident protein disulfide isomerase) and collagen-I in the absence of extracellular collagen fibrils (Figure 3H).
PDE4D Previous studies in COS7 cells had shown that PDE7A1 regulates PKA-dependent retrograde transport of the KDELR in COS7 cells 35. HSP47 is an ER-resident molecular chaperone that contains a C-terminal KDEL signal and is required for procollagen secretion 47. Thus, we investigated if PDE4D regulated HSP47 transport. The results showed that HSP47 co-localized with GM130 (a marker of cis-Golgi/ERGIC) between 15- and 23-hours post-synchronization (Figure 4A). RNAi was highly effective at reducing the levels of Pde4d mRNA in MEFs (Figure S3F, G). Immunofluorescence using a collagen-I-specific antibody showed that Pde4d knockdown severely reduced the ability of cells to assemble collagen fibers and caused intracellular retention of PC-I, when compared to control cells (Figure 4B). In Pde4d KD cells, HSP47 showed poor co-localization with GM130 (Figure 4C), indicative of a blockage of HSP47 transport.
VPS33B Post-Golgi trafficking of PC-I is poorly understood but recent studies suggest a role for VIPAR in the regulation of lysyl hydroxylation of collagen IV by PLOD3 (procollagen-lysine,2-oxoglutarate 5-dioxygenase 3) 36. The transcript and protein for Vps33b, a binding partner of VIPAR peak at the end of the circadian cycle (Figure 2A, B). Thus, we used CRISPR-Cas9 to knockout Vps33b in MEFs (Figure S3H) and immortalized tendon fibroblasts (iTTFs) (Figure S3H-J), and used collagen-I-, PDI- and GM130-specific antibodies to observe transport of PC-I through the secretory pathway during the circadian cycle. In non-CRISPR treated cells, PC-I co-localized with PDI 3 hours after synchronization (Figure 4D), with GM130 7-15 hours after synchronization (Figure 4E), and assembled collagen fibrils between 15- and 23-hours post-synchronization (Figure 4D, E). In contrast, PC-I co-localized with PDI throughout the circadian cycle, Also, whereas PC-I had transited through the Golgi by 23-hours post-synchronization in control cells, PC-I co-localized with GM130 at 23-hours post-synchronization in VPS33B knockout cells. Furthermore, numbers of collagen fibrils were severely reduced in VPS33B knockout cells (Figure 4D, E).
A predictive mathematical model of the collagen secretory pathway
We used the above protein data, together with published data on mRNA transcriptional rates 48, to generate a predictive model of the transport of PC-I through the secretory pathway. We built a deterministic ordinary differential equation system based on the principles of mass-action enzyme kinetics, with variables representing cellular concentrations of collagen and relevant proteins in ER (translation via SEC61 with phase of CT23-7), ER exit sites (pathway progression controlled by TANGO1 and HSP47, CT7-11), Golgi (PDE4D directed transport of HSP47 at CT15-23), post-Golgi (pathway progression controlled by VPS33B, CT19-23), and plasma membrane (release step at CT0-3). Collagen degradation is modeled based on cathepsin K (CTSK, CT15-27). Simulations and subsequent analysis were carried out using the differential equation solver NDSolve in Wolfram Mathematica 11.3 (see STAR★METHODS). The simulation predicted that the relative amounts of PC-I in ER, ER exit sites, Golgi, post-Golgi compartments and the extracellular matrix varied depending on the circadian cycle (Figure 5A and Video 5). The model predicted low levels of PC-I throughout the circadian cycle in ER exit sites (compared to other transport compartments) (Supplementary Video 5), which was due to the narrow window of time when TANGO1 was expressed in the circadian period and the rapidity of microtubule transport of vesicles. The model predicted an inverse relationship (to a close approximation) between intracellular PC-I and extracellular collagen concentration (Figure 5B), assuming that PC-I is converted to collagen-I during transport or in the ECM, which agreed with previous observations 49,50. However, the shape of the phase curve (Figure 5B) predicted a 6-hour time delay between the peak in PC-I and the peak in collagen-I. Importantly, the model predicted that extracellular collagen-I was rhythmic within a circadian period, with the peak of collagen occurring in the presumptive day (Figure 5C).
Rhythmic proteome revealed daily surge of collagen production in tendons in vivo
To explore the prediction that a rhythmic secretory pathway generates a wave of collagen-I in the day, we performed a time-series proteomic analysis of mouse tendon using an extraction buffer that solubilized non-covalently bound collagen whilst leaving the permanent, cross-linked collagen intact (see STAR★METHODS). This allowed for separate analysis of newly-synthesized collagen from the permanent collagen. Electron microscopy showed the persistence of D2 and D3 fibrils, and depletion of D1 fibrils, in the non-solubilized (pellet) fraction (Figure S6A-C). Hydroxyproline analysis showed that the solubilized collagen accounted for 18% of total collagen (Figure S6D). The soluble proteins were treated with trypsin and the resultant peptides analyzed by liquid chromatography-tandem mass spectrometry 51. We detected ∼10,000 peptides and identified 1,474 proteins with ≥ 2 peptides per protein (Figure 6A) (Supplementary Data 1), with good reproducibility between biological replicates as indicated by Pearson analysis across two consecutive 24-hour periods (Figure 6B). Rhythmic profiles of >100 proteins were detected during 48 hours (Figure 6C), with prominent changes occurring at the night-day and day-night transitions for rhythmic and non-rhythmic proteins (Figure 6D, E and Figure S7A). Protein ontology analysis showed that actin-binding proteins were most abundant in night samples (Figure S7B). This supports a recent proteomics study in fibroblasts, which indicated regulation of actin dynamics is under circadian control 52. Approximately 10% of detected proteins (141) were robustly rhythmic (with periods of 20-27.5 h) (Figure 6F). The α1(I) and α2(I) chains of collagen-I were conspicuously rhythmic and peaked during daylight hours, equivalent to 1.6-fold difference in extractable collagen between day and night sampling (Figure 6G). It was possible to identify peptides specific for the C-propeptide region of the proα1(I) proα1(I) chain of PC-I, which peaked ∼6 hours prior to the α1(I) and α2(I) peptides and was consistent with the slow cleavage of PC-I to collagen-I 53,54 (Figure 6H and Supplementary Data 2). To investigate the rate of collagen-I production between the day and night, tendon tissues dissected 12 hours apart were labeled with 14C-proline ex vivo 30. Both Achilles and tail tendon tissues had significantly greater PC-I production at night (Figure 6I and Figure S7C, D), corroborating the LC-tandem mass spectrometry data. 14C-amino acid labeling of newly-synthesized proteins every 4 hours during 24 hours confirmed that PC-I production was ramped up throughout the night (Figure S7E, F).
Mistiming of the secretory pathway results in collagen accumulation and abnormal fibril structure
If a 24-hour rhythmic secretory pathway generates a daily wave of collagen and rhythmic assembly of collagen fibers (as shown in Figure 3A), an arrhythmic pathway may be expected to result in arrhythmic or non-24-hour cyclical synthesis of collagen fibers. To test this prediction we cultured fibroblasts from wild type and ClockΔ19 mice bred on a PER2::LUC background, and treated the cells with the clock synchronizing agent dexamethasone. ClockΔ19 mice have disrupted circadian rhythms 55 and the in-frame fusion of luciferase at the Per2 locus enabled direct measurement of the endogenous circadian rhythm. Wild type cells responded well to the synchronizing effect of dexamethasone and showed a robust bioluminescence rhythmicity that persisted for 3 days (Figure 7A). In contrast, ClockΔ19 fibroblasts did not sustain a rhythm (Figure 7A). Time-series quantitative-PCR analysis of Nr1dr1 confirmed that dexamethasone was effective in synchronizing the clock in wild type cells only but not in ClockΔ19 cells (Figure 7B). Levels of Col1a1 mRNA were not rhythmic in synchronized wild type ClockΔ19 cells (data not shown), which agreed with in vivo data (see Figure S2). However, the overall levels of Col1a1 mRNA were elevated (Figure 7C) and the overall levels of Ctsk mRNA were decreased (Figure 7D) in ClockΔ19 compared to wild type cells.
Next, we synchronized wild type and ClockΔ19 fibroblasts and performed a time-series immunofluorescence study to quantitate the number of collagen fibers formed (Figure 7E). The results showed that wild type and ClockΔ19 fibroblasts synthesized collagen fibers during 55 hours in culture; therefore, a rhythmic secretory pathway is not necessary for collagen fiber formation. However, ClockΔ19 fibroblast cultures accumulated more fibers than wild-type cells (Figure 7F). Metacycle analysis of the numbers of fibers indicated that the increase and decrease in fiber number during days in culture in wild type and ClockΔ19 cultures was 24 and 28 hours, respectively (Figure 7G).
the number of collagen fibers synthesized by wild-type cells increased and decreased to reach a steady-state level after 55 hours (Figure 7F). In contrast, the number of collagen fibrils synthesized by ClockΔ19 fibroblasts increased and decreased with a periodicity of 28 hours (Figure 7G) and increased during 55 hours without reaching a plateau (Figure 7F). As a consequence, the number of collagen fibers per cell was greater in ClockΔ19 than in wild-type fibroblasts (Figure 7H).
The ClockΔ19 is a global mutant mouse that shows systemic loss of rhythmicity, with potential confounding factors. To selectively disrupt circadian rhythm in tendon-lineage cells we generated a new line of Scx-Cre::Bmal1 lox/lox knockout mice that lacked circadian rhythm in tail and Achilles tendons (Figure S8A, Videos 6 (wild-type) and 7 (Scx-Cre::Bmal1 lox/lox)). Using TEM, we showed that the tendon fibrils in ClockΔ19 and Scx-Cre::Bmal1 lox/lox mice were irregular in outline (Figure 7I and Figure S8B), had disrupted fibril diameter distributions (Figure 7J and Figure S8C) and decreased circularity values (Figure 7K and Figure S8D). Tendons in clock-disrupted mice were significantly thicker and had reduced elastic modulus and maximum load compared to wild type littermates (Figure S8E and F). There were no differences in fibril volume fraction between arrhythmic and wild type tendons (Figure 8SE); hence, the increased tendon width in the mutant mice was due to an increased volume of collagen. These results showed that disrupting the circadian clock results in increased collagen deposition, decreased elastic modulus, decreased maximum load, and irregular fibril structure, which demonstrate disrupted matrix homeostasis.
Discussion
In our study we have shown a rhythmic pool of collagen in the extracellular that results from circadian clock regulation of the protein secretory pathway. Mistiming of the pathway in cells and mice lacking a functional circadian clock resulted in dysregulated collagen homeostasis as seen in fibrosis. The synthesis and removal of a distinct pool of collagen whilst leaving the bulk protein unchanged illustrates a surprisingly dynamic matrix and a selective mechanism of protein homeostasis that maintains tissue function and organization.
Intracellular proteins are constantly being synthesized and degraded in a cyclical process arising from transcription, translation and protein folding to replace old and damaged polypeptides with newly-synthesized copies 56. However, the extreme longevity of collagen in tissues would suggest that these conventional transcriptional and translational homeostatic mechanisms do not apply to collagen. Presumably, the energy- and time-dependent developmental processes that are required to establish extensive networks of collagen fibrils in tissues (which in humans equates to ∼17 years) rule out a protein homeostatic mechanism in which all of the collagen is turned over. Tissues that are rich in collagen are some of the highest mechanically-loaded tissue in vertebrates and are exposed to repeated cycles of stress and strain throughout the life of the animal. Therefore, collagen molecules that are never replaced would be expected to undergo mechanical damage leading to fatigue failure. A mechanism of protein homeostasis must therefore exist to protect, rather than replace, the collagen that was assembled during skeletal growth. Such a mechanism must be able to account for the observed continued synthesis of collagen in the face of zero turnover of the bulk of the tissue collagen. The circadian clock mechanism described here would appear to fulfill these requirements.
Our study showed that a population of thin (D1) fibrils are interspersed between thick (D2 and D3) collagen fibrils, and make frequent contact with the surfaces of the thick fibrils. It has been suggested that the thin fibrils are part of a sheer stress loading mechanism in tendon 57. Our data would agree with this suggestion, particularly as the D1 fibrils do not appear to be under tensile strain. However, the fact that the proportion of D1 fibrils varies with time of day, and that the collagen-I in these fibrils is circadian clock rhythmic, may suggest an additional function to maintain matrix homeostasis in the face of bouts of activity and long-term wear and tear on the tissue. Collagen fibrils are ‘molecular alloys’ that comprise a variety of collagen types 58 and associated molecules including small leucine rich proteoglycans (SLRPs) (for a review of SLRPs, see 59). We noted that collagen α2(V) was rhythmic in-phase with collagen α1(I) and collagen α2(I) (Supplementary Data 1). Collagen-V controls the initiation of collagen fibril assembly, and haplo-insufficient mice have fewer, large and irregular collagen fibrils 60. In addition, decorin (an abundant SLRP found in a wide range of tissues) was rhythmic, peaking at CT21 (Supplementary Data 1). Mice deficient in decorin have weakened tissues and abnormal collagen fibrils with irregular profiles 61. This raises the possibility that the rhythmic collagen-I-rich D1 fibrils are part of a multicomponent chronomatrix.
Early work using electron microscopy, histochemistry and autoradiography demonstrated that procollagen molecules could be present in the Golgi apparatus and post-Golgi secretory vesicles within 20-30 mins of synthesis in the ER 62. Later studies using 14C-labeled amino acids showed that PC-I could be synthesized and secreted within 20 mins 30,50. The need to use radiolabel is these studies to detect the procollagen suggests that the concentrations of procollagen were low, and perhaps below the critical concentration for collagen fibril assembly 63. Taken together, cells have the ability to secrete low levels of procollagen rapidly (∼minutes) and to increase the concentration of procollagen being delivered to the plasma membrane by circadian clock regulation of SEC61, TANGO1, PDE4D and VPS33B. Presumably, damming procollagen flow from one secretory compartment to the next is a mechanism of increasing procollagen concentration to facilitate D1 collagen fibril assembly. The existence of two temporal mechanisms of procollagen secretion might have implications for day time protection of the collagenous matrix following bouts of nocturnal activity in mice. Furthermore, the absence of rhythmic collagen homeostasis, or a shift to continuous collagen secretion, might be a disease mechanism in poor wound healing, fibrosis, and musculoskeletal degeneration.
METHODS
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author Karl Kadler (karl.kadler{at}manchester.ac.uk).
A separate resource document is uploaded.
EXPERIMENTAL MODELS
Mice
The care and use of all mice in this study was carried out in accordance with the UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under the Home Office Licence (#70/8858 or I045CA465). The permission included the generation of conditional knock out animals.
ClockΔ19 mice (C57BL/6 on a PER2::Luciferase reporter background) were as described previously 55. To generate mice in which Bmal1 is ablated in tendon-lineage cells, we crossed mice expressing Cre recombinase under the control of the Scleraxis promoter (Scx-Cre; C57BL/6) 64 with C57BL/6 mice carrying loxP sites flanking the exon encoding the BMAL1 basic helix-loop-helix domain 65 bred on a background with PER2::Luciferase reporter 66 to produce Scx-Cre::Bmal1 lox/lox mice. Six-week old wild type C57BL/6 mice housed in 12-h dark/12-h dark cycle (DD) were used for circadian time (CT) time-series microarray and time-series protein extraction for western blotting as previously described (Yeung et al., 2014). Wild type C57BL/6 mice housed in 12-h light/12-h dark cycle (LD) were used for all remainder zeitgeber time (ZT) studies, including time-dependent electron microscopy experiments, cyclic loading mechanical testing, ex vivo 14C-proline labelling experiments, proteomics analyses, and ex vivo odanacatib treatment.
METHOD DETAILS
Preparation of Cells
Tail tendon and lung fibroblasts from wild type and ClockΔ19 were released from 6-8-week mouse tail tendons by 1000 U/ml bacterial collagenase type 4 (Worthington Biochemical Corporation) in trypsin (2.5 g/L) as previously described 67. Wild type tail tendon fibroblasts were immortalized by retroviral expression of mTERT (iTTFs) 68. Mouse embryonic fibroblasts (MEFs) were isolated from E14 wild type mouse embryos. Cells were cultured in complete media (Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 containing 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, supplemented with 10% fetal calf serum, 200 μM L-ascorbic acid 2-phosphate, 10,000 U/ml penicillin/Streptomycin) at 37°C, in 5% CO2.
To synchronize the circadian clocks of a population of cells, cells were incubated with either 100 nM dexamethasone (Merck) in complete media for 30 min at 37°C, in 5% CO2 or 50% horse serum (Thermo Fisher Scientific) in DMEM F-12 for 2 h at 37°C, in 5% CO2 and then for 24 h in complete medium at 37°C, in 5% CO2. After (at 0 h after synchronization), cells were fixed for immunofluorescence or used for protein or RNA isolation, at 3-h or 4-h intervals.
CRISPR-Cas9-Mediated Knockout
Cells were treated with CRISPR-Cas9 to delete Sec61a1, Sec61a2 or Vps33b genes. Ribonucleoprotein complexes of crRNA, tracrRNA and recombinant 3xNLS Cas9 were formed according to manufacturer’s protocol (Integrated DNA Technologies). Two different guides were used in combination to target Sec61a1 (g1: TCC GGC AAG ATG ACA CAG AA, g2: TCT GCA AAA AGG GTA CGG CT), Sec61a2 (g1: AGT TTA GAG AGA AGG TAC TA, g2: TTT ATG ATC GCT CAC ATT TC) or Vps33b (g1: CGT TAG CAA TTC GAT CCA AA, g2 AGT CAT CCT AAG CCC TCA GA). 60 pmol complexes (2 pmol Cas9) were nucleofected into 4 × 105 MEFs using P1 Nucleofection Buffer (Lonza). Gene knockout was confirmed by western blotting and qPCR. For iTTF studies, 5 × 104 cells were plated out in a 24-well plate, and the aforementioned ribonucleoprotein complexes were introduced into the culture using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific), according to manufacturer’s instructions. Clones were then generated using serial dilution in a 96-well plate. Gene knockdown was confirmed by western blotting and qPCR.
RNA Interference
Cells were treated with siRNAs to deplete Mia3 or Pde4d transcripts in mouse cells. siRNAs targeting Pde4d (SMARTpool ON-TARGETplus Pde4d siRNA; Dharmacon) or Mia3 (GeneSolution Mia3 siRNA; Qiagen) were transfected into MEFs using MEFs Nucleofector Kit (Lonza) according to the manufacturer’s instructions. Briefly, 4 × 106 cells (in a well of a 6-well plate) were transfected with a 0.8 nmol of siRNA in a final volume of 3 mL (yielding a final concentration of 250 nM).
RNA isolation and Quantitative Real-Time PCR
RNA was isolated from ex vivo tendon and cells as described previously 67. In brief, tissues and cells were disrupted in TRIzol Reagent (Thermo Fisher Scientific) and RNA was isolated according to manufacturer’s protocol. DNase treatment of RNA was performed using RQ1 RNase-Free DNase (Promega Corporation) according to manufacturer’s protocol. RNA integrity was assessed by gel electrophoresis and RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific). Complementary DNA was synthesized from 2 µg RNA using TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific).
SensiFAST SYBR kit reagents were used in qPCR reactions. Primer sequences used were Col1a1 GCC TGC TTC GTG TAA ACT CC and TTG GTT TTT GGT CAC GTT CA, Ctsk GGA CCC ATC TCT GTG TCC AT and CCG AGC CAA GAG AGC ATA TC, Gapdh AAC TTT GGC ATT GTG GAA GG and ACA CAT TGG GGG TAG GAA CA, Mia3 AGC CAC GGA CGG CGT TTC TC and CCC CTG CCA GTT TGT AGT A, Nr1d1 CTT CCG TGA CCT TTC TCA GC and GGT AAG TGC CAG GGA GTT GA, Pde4d CCA ACT AGC CTC CCC TTG AA and GCT GTC AGA TCG GTA CAG GA, Sec61a1 CAG GTT CTT CTG CCA AAG ATG and TCA TGC ACC ATG GAG GTC T, Sec61a2 TGT ATG TCA TGA CGG GGA TG and CAA ACC AGC AAC AAA CAA CTG, and Vps33b GCA TTC ACA GAC ACG GCT AAG and ACA CCA CCA AGA TGA GGC G. Primers were validated by DNA sequencing of PCR products and verified using BLAST. For qPCR analysis, the 2-ΔΔCt method 69 was used to analyze relative fold changes in gene expression compared to the first time point or to wild type or control.
Protein Extraction and Western Blotting
Protein was extracted and analyzed by western blotting as previously described 29. Briefly, tendons were snap frozen and homogenized with ice-cold cell lysis buffer (20 mM Tris/HCl pH 7.6, 150 mM NaCl, 1 mM ethylene diamine tetra-acetic acid (EDTA), 1% (v/v) Igepal CA-630, 50 mM sodium fluoride) containing complete, mini EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche). Lysates were cleared by centrifugation at 10000 × g, at 4°C. Pierce BCA Protein Concentration Assay (Thermo Fisher Scientific) was used to determine protein concentration.
Proteins were separated on NuPAGE Novex 10% polyacrylamide Bis-Tris gels with 1x NuPAGE MOPS SDS buffer (both from Thermo Fisher Scientific) and transferred onto PVDF membranes (GE Healthcare). Membranes were blocked in 5% skimmed milk powder in 1x PBS containing 0.05% Tween 20. Antibodies were diluted in 2.5% skimmed milk powder in 1x PBS containing 0.05% Tween 20. Primary antibodies used were goat pAb to collagen α1 type I (1:1000; Santa Cruz), mouse mAb to GAPDH (1:10000; Merck), rabbit pAb to PDE4D (1:500; Santa Cruz), rabbit pAb to Sec61A (1:500; Santa Cruz), rabbit pAb to Tango1 (1:500; Santa Cruz), mouse mAb to vinculin (1:200; Oncogene), and mouse mAb to VPS33B (1:500; Proteintech). HRP-conjugated antibodies and Pierce ECL Western Blotting Substrate (both from Thermo Fisher Scientific) were used and reactivity was detected on Hyperfilm (GE Healthcare).
Immunofluorescence and Laser Confocal Imaging
Cells were fixed with 100% methanol at −20°C and permeabilized with 0.5% Triton-X/PBS. Collagen-I was detected using a rabbit pAb (1:500; Gentaur), HSP47 was detected using mouse mAb (1:100; Santa Cruz), ER was detected using rabbit pAb to calnexin (1:200; Abcam) or mouse mAb protein disulfide isomerase (PDI) (1:100; Abcam), cis-Golgi was detected using rabbit mAb to GM130 (1:250; Abcam) or mouse pAb to GM130 (1:500; 610822, BD Biosciences). Secondary antibodies conjugated to FITC (Abcam) and Cy5 (Thermo Fisher Scientific) were used. Stained cells were mounted using VECTASHIELD Hardset antifade mounting medium with DAPI (Vector Labs).
Images were collected on a Leica TCS SP5 AOBS inverted confocal using an [63x / 0.50 Plan Fluotar] objective [and 3x confocal zoom]. The confocal settings were as follows, pinhole [1 airy unit], scan speed [1000 Hz unidirectional], format 1024 × 1024. Images were collected using [PMT] detectors with the following detection mirror settings; [FITC 494-530 nm; Cy5 640-690 nm] using the [488 nm (20%), 594 nm (100%) and 633 nm (100%)] laser lines respectively. To eliminate crosstalk between channels, the images were collected sequentially. When acquiring 3D optical stacks the confocal software was used to determine the optimal number of Z sections. Only the maximum intensity projections of these 3D stacks are shown in the results.
Ex Vivo Tendon Culture
Following euthanization hind legs were amputated just below the knee. The skin was removed to expose the Achilles tendon. The whole bone-muscle-tendon unit (tibia, calcanium, gastrocnemius and soleus muscles with the Achilles tendon) was placed in complete media containing 1 µM odanacatib (Cambridge Bioscience Ltd.) or 0.1% DMSO vehicle control. Ex vivo tendons were cultured for 72 h at 37 °C, in 5% CO2.
14C-Proline Labelling of Ex Vivo Tendons
Achilles tendons were dissected from mice, rinsed in PBS, and incubated at 37°C in complete media containing 2.5 Ci/ml of 14C-proline (Perkin Elmer) for one hour, with the tubes agitated every 20 min. After incubation, tissues were washed, spun down and transferred to fresh Eppendorf tubes with 100 μl of salt extraction buffer (1 M NaCl, 25 mM EDTA, 50 mM Tris-HCl, pH7.4) containing protease inhibitors and phosphatase inhibitors. Tendons were extracted in 2 changes of salt extraction buffer – 12 h (S1), 6 h (S2) – these steps extracted collagen that was present in the extracellular fraction of the tissue. This was then followed by 12 hours in NP40 buffer (salt extraction buffer supplemented with 1% NP40 (Merck)) which allowed extraction of intracellular fractions. NP40 extracts were then analyzed on 6% Tris-Glycine gels (Thermo Fisher Scientific) under reducing conditions, followed by transfer onto nitrocellulose membranes. Membranes were then dried and exposed to a phosphoimaging plate (Fuji BAS-III or BAS-MS). After 14 days of exposure, the plates were processed using a phosphoimager (Fuji BAS 2000 or 1800), and images analyzed using ImageJ.
Hydroxyproline Assay
Samples were incubated in 6 M hydrochloric acid (diluted in ddH2O, (Fluka); approximately 1 mL per 20 mg of sample) in screw top tubes (StarLab) overnight in a sand-filled heating block at 100°C, covered with aluminium foil. Tubes were then allowed to cool down and centrifuged at 12000 g for 3 min. Hydroxyproline standards were prepared (starting at 0.25 mg/mL, diluted in ddH2O), and serially diluted with 6 M hydrochloric acid. Each sample and standard (50 µL) were transferred into fresh Eppendorf tubes, and 450 μL of chloramine T reagent (0.127g of chloramine T in 50% N-propanol diluted with ddH2O; bring up to 10 mL with acetate citrate buffer – 120 g sodium acetate trihydrate, 46 g citric acid, 12 mL glacial acetic acid, 34 g sodium hydroxide, pH to 6.5 then make to 1 L with dH2O. All reagents from Sigma) was added to each tube and incubated at room temperature for 25 min. After, 500 μL of Ehrlich’s reagent (1.5 g of 4-dimethylamino-benzaldehyde diluted in 10 mL of N-propanol/perchloric acid at 2:1 ratio) was added to each reaction tube and incubated at 65°C for 10 minutes and then the absorbance reading at 558 nm of 100 μL of each sample was measured in a 96-well plate format.
Mass Spectrometry
All reagents were of mass spectrometry grade. Tail tendons were homogenized with a bullet blender (with 1.6 mm steel beads; Next Advance) at maximum speed at 4°C for 5 min in 200 μL of SL-DOC buffer (1.1% SL (sodium laurate), 0.3% DOC (sodium deoxycholate), 0.5 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate) (all from Merck), supplemented with protease and phosphatase inhibitor cocktails (Roche). Samples were incubated at 4°C for 5 min, alkylated with 12 μL of 30 mM of iodoacetamide for 20 min at RT, followed by quenching with 12 μL of 30 mM DTT. Samples were centrifuged at maximum speed for 10 min. Supernatants were transferred into LoBind Protein tubes (Eppendorf) and protein concentrations measured using a Millipore Direct Detect spectrometer.
A total of 2 μg protein per sample was digested using trypsin beads (SMART digestion kit, Thermo Fisher Scientific) with rapid agitation (1400 rpm) at 37°C overnight. Peptide/bead mixtures were centrifuged at 12000 g for 5 min and supernatants transferred into new tubes. The pH was adjusted to <3 by addition of 10% formic acid (Merck) and 400 μL of EA (ethylacetate; Merck). Samples were then spun for 5 min at 16000 g, and the upper layer of solvent (EA) was discarded. EA extraction was repeated twice and samples were lyophilized in a speed-vac centrifuge (Heto).
Samples were then mixed with 100 μL of 5% acetonitrile with 0.1% formic acid (Merck), and proceeded to R3 sample desalting clean-up. Poros R3 beads (1 mg, Applied Biosystems) were added to each well of a 96-well plate with 0.2 µM PVDF membrane (Corning), and the plate was centrifuged at 200 × g for 1 min. Centrifugation was repeated to wash the beads with 50% acetonitrile (wet solution), with the flow-through discarded each time. Beads were then washed twice with wash solution (0.1% formic acid) and flow-through discarded. Samples were added into the corresponding wells and mixed with beads on a plate mixer for 5 min at 800 rpm. Samples were washed twice with wash solution, and the flow-through was discarded after each centrifugation step. A fresh sample collection plate was then placed under the PVDF membrane plate, and samples were eluted and collected using 50 μL of elution buffer (40% acetonitrile, 0.1% formic acid). This elution step was repeated to give a total of 100 μL eluted samples. Eluted samples were transferred from the plate into individual chromatography sample vials, and dried in a speed-vac centrifuge for 60 min or until complete dryness was achieved. The resulting peptides were dissolved in 15 μL of 5% acetonitrile with 0.1% formic acid.
Digested samples were analyzed using an UltiMate® 3000 Rapid Separation liquid chromatography system (RSLC, Dionex Corporation) coupled to either an Orbitrap Elite (for quality control assessment; Thermo Fisher Scientific) or Q Exactive HF (Thermo Fisher Scientific) mass spectrometer. Briefly, for the Orbitrap Elite, peptides were separated using a 75 mm × 250 μm inner diameter 1.7 μM CSH C18, analytical column (Waters) with a gradient from 92% A (0.1% formic acid, FA, Sigma, in deionized water) and 8% B (0.1% FA in acetonitrile) to 33% B, in 104 min at 300 nL/min. For the Q Exactive HF, peptides were separated in a gradient of 95% A and 5% B to 7% B at 1 min, 18% B at 58 min, 27% B in 72 min and 60% B at 74 min at 300 nL/min using a 75 mm × 250 μm inner diameter 1.7 μM CSH C18, analytical column (Waters). Peptides were selected for fragmentation automatically by data dependent analysis.
Electron Microscopy
Tendons from 5- to 6-week old mice were prepared for transmission electron microscopy (TEM) and serial block face-scanning electron microscopy (SBF-SEM) as described previously 70. In brief, tendons were fixed in 1% osmium and 1.5% potassium ferrocyanide in 0.2 M sodium cacodylate buffer for 1 h, washed with distilled water, then incubated with 1% tannic acid in 0.1M cacodylate buffer for 1 h, washed with distilled water, then incubated with 1% osmium in water for 30 min. Samples were then washed with distilled water and stained with 1% uranyl acetate in water for 1 h, then dehydrated in acetone and embedded into resin.
For scanning transmission electron microscopy (STEM), mouse Achilles tendons were dissected from 6-week-old mice every 3 hours over a 24-hour period. Four mice were sacrificed at every time point. Each entire mouse Achilles was diced into ∼ 0.5 mm pieces and crushed between liquid nitrogen-cooled stainless-steel blocks. The resulting powder was then mechanically dispersed in a TRIS-EDTA buffer, pH 7.4, with added proteinase inhibitors in a hand-held Dounce homogenizer. Fibrils were adsorbed from solution on a carbon-filmed 400-mesh copper grid and washed with ultra-pure water. STEM was implemented on a Tecnai Twin (FEI, Eindhoven) electron microscope fitted with an annular dark-field detector using a camera length of 350 cm. Images were recorded at 120 kV with a sufficiently low electron dose (< 103 e/nm2) to give a negligible beam-induced mass loss. Tobacco mosaic virus was used as a standard of mass per unit length.
Mechanical Testing of Tendons
Mechanical properties of arrhythmic tendons were measured as described previously 71. In brief, tail tendons were dissected from 6-week-old mice and mounted onto a grade 100 sandpaper frame with a 1 cm length window using superglue. The sandpaper frame was clamped in an Instron 5943 fitted with a 10 N load cell (Instron Inc, UK). Only samples that failed in the mid portion of the tendon were included. Tendons were tested to failure with a displacement rate of 5 mm/min. The length and width of the samples tested were measured from a digital photograph using ImageJ software, which were used to derive corrected elastic modulus and calculate maximum load/cross sectional area. At least 10 tendons from each biological replicate were tested.
The dissected mouse Achilles tendons sampled at ZT3 and ZT15 were subjected to a cyclic test regime on an Instron 5943 fitted with a BioPuls specimen bath. Each sample was clamped at the bone and over the myotendinous junction using superglue and a sandpaper sandwich in the pneumatic clamps. The sample bath was filled with PBS and maintained at 22°C. The tendon was straightened with a tare load of 0.02 N and then subjected to successive strain cycles up to a 1 N load. A series of 30 cycles was used for each tendon sample and consisted of 6 sets of 5 cycles with a displacement rate for each set of 1, 2, 3, 4, 5 and 1 mm/min, respectively. The tendon was finally loaded to 1 N at 1 mm/min after the last strain cycle and the stress-relaxation load curve was recorded over 15 min. Energy loss measurements were obtained from the stress-strain hysteresis loop of the final cycle in each set. The elastic modulus was calculated from the linear part of the stress-strain curve on the load increasing part of the strain cycle. The length of the test portion of the tendon and the lateral dimensions were measured from calibrated images. The stress-relaxation curve was fitted to a sum of three exponential decay curves to yield 3 characteristic relaxation times (T1, T2, T3).
Bioluminescence Imaging and Recordings
Tendon explants from 6-week old mice were cultured on 0.4 µm cell culture inserts (Millipore) in recording media containing 0.1 mM luciferin (Merck) in the presence of 100 nM dexamethasone (Merck) and visualized using a self-contained Olympus Luminoview LV200 microscope (Olympus) and recorded using a cooled Hamamatsu ImageEM C9100-13 EM-CCD camera. Images were taken every hour for 3 days, and combined in ImageJ to generate movies. The recording parameters were the same for control wild type (Video 6) and Scx-Cre::Bmal1 lox/lox (Video 7) Achilles tendons but the brightness for the Scx-Cre::Bmal1 lox/lox recording was increased for the images and video to enable visualization.
LumiCycle apparatus (Actimetrics) was used for real-time quantitative bioluminescence recording of tendon explants and tendon fibroblasts as described above in the absence of dexamethasone for 3 days. Then 100 nM dexamethasone was added to re-initiate the circadian rhythms. Baseline subtraction was carried out using a 24-hr moving average. Period after addition of dexamethasone was calculated using LumiCycle analysis software (Actimetrics) or using the RAP algorithm. A minimum of three peaks were used to determine period. Amplitude was calculated as peak-trough difference in bioluminescence of the second peak using base-line subtracted data.
QUANTIFICATION AND STATISTICAL ANALYSIS
Data are presented as mean ± SEM unless otherwise indicated in figure legends. Sample number (n) indicates the number of independent biological samples in each experiment. Sample numbers and experimental repeats are indicated in figures and figure legends or methods section above. Data were analyzed using One-Way ANOVA or unpaired t-test. Data analysis was not blinded. Differences in means were considered statistically significant at p < 0.05. Significance levels are: *p<0.05; **p<0.01; ***p< 0.001; ****p< 0.0001; NS, non-significant. Analyses were performed using the Graphpad Prism 7 software.
Morphometric Analyses of EM images
Fibril diameter, perimeter and area were measured using ImageJ software to calculate circularity and fibril volume fraction as described previously (Starborg et al., 2013). Fibril diameter distributions were plotted and fitted to 3-Gaussian distribution using Sigma Plot 12.0 software (Systat Software) as previously described (Starborg et al., 2013).
For the STEM experiments, tobacco mosaic virus (TMV) was used as standard of mass per unit length (131 kDa/nm). Mass per unit length measurements were averages of 5D-periods for each fibril. Mass per unit length measurements were made from STEM images on samples of dispersed collagen fibrils as described 31. The mass per unit length frequencies were weighted according to the individual fibril length in each image.
Proteomics Data Analysis and Protein Quantification
Spectra from multiple samples were automatically aligned using Progenesis QI (Nonlinear Dynamics) with manual placement of vectors where necessary. Peak-picking sensitivity was set to 4/5 and all other parameters were left as defaults. Only peptides with charge between +1 to +4, with 2 or more isotopes were taken for further analysis. Filtered peptides were identified using Mascot (Matrix Science UK), by searching against the SwissProt and TREMBL mouse databases. The peptide database was modified to search for alkylated cysteine residues (monoisotopic mass change, 57.021 Da), oxidized methionine (15.995 Da), hydroxylation of asparagine, aspartic acid, proline or lysine (15.995 Da) and phosphorylation of serine, tyrosine, threonine, histidine or aspartate (79.966 Da). A maximum of 2 missed cleavages was allowed. Peptide detection intensities were exported from Progenesis QI as Excel (Microsoft) spread sheets for further processing. Peptide identifications were filtered via Mascot scores so that only those with a Benjamini-Hochberg false discovery rate (FDR)-adjusted p-value < 0.05 remained. FDR filtering was performed as described 72. Raw ion intensities from peptides belonging to proteins with fewer than 2 unique peptides (by sequence) per protein in the dataset were excluded from quantification. Remaining intensities were logged and normalized per MS run by the median logged peptide intensity. Peptides assigned to different isoforms were grouped into a single “protein group” by gene name. Only peptides observed in at least 2 samples were used in quantification. Missing values were assumed as missing due to low abundance, an assumption others have shown is justified 73. Imputation was performed at the peptide level following normalization using a method similar to that employed by Perseus 74 whereby missing values were imputed randomly from a normal distribution centered on the apparent limit of detection for this experiment. The limit of detection in this instance was determined by taking the mean of all minimum logged peptide intensities and down-shifting it by 1.6s, where s is the standard deviation of minimum logged peptide intensities. The width of this normal distribution was set to 0.3s as described 74. Fold-change differences in the quantity of proteins detected in different time-points were calculated by fitting a mixed-effects linear regression model for each protein with Huber weighting of residuals, as described in 73 using the fitglme Matlab (The MathWorks, USA) function with the formula: y_iptb= β_t+ β_p+ β_b; where y_iptb represents the log2(intensity) of peptide p belonging to protein i, at time-point t, in batch b. βs represents effect sizes for the indicated coefficients. Batch and peptide were entered as random effects whereas time-point was entered as a fixed effect. Standard error estimates were adjusted with Empirical Bayes variance correction as previously described 75,76. Conditions were compared with Student’s t-tests with Benjamini-Hochberg correction for false positives 77.
Periodicity Analysis
Periodicity analysis was performed using the MetaCycle package 78 in the R computing environment 79 using the default parameters. A Benjamini-Hochberg FDR<0.2 was used to detect rhythmic proteins.
Predictive Model of Collagen Homeostasis
Reactions and Equations
Variables
All variables are protein concentrations in units of μM relative to typical fibroblast cell size.
Parameters
Parameter values for the model were derived where possible from existing results in the literature (such as 48). This paper lists protein copy numbers in molecules per cell, translation rates in molecules per mRNA per hour, mRNA copy numbers in molecules per cell, and protein half-life in hours, among others. These, along with a typical fibroblast cell size of 2.04 pL were combined to give translation rates in micro-molar per hour, and concentrations in micro-molar, or time scale rates in per hour. For example, k1 was calculated by dividing protein copy number by the cell size and Avogadro’s number to give concentration. Where values for a particular protein were not available, translation rates were taken as the median values from 48, as detailed below. The values for procollagen transport rates from one intracellular compartment to the next were guided by knowledge of the speed of vesicular transport in the eukaryotic cell.
Collagen Fiber Quantification
Collagen fibers were independently and manually scored by two independent researchers using a Zeiss fluorescence microscope. An average of at least 12 fields of views were quantified for each time point. Number of fibers were counted based on collagen-I fluorescence staining; where collagen fibers appear branched it was counted as separate fibers. For each field of view, the number of fibers were corrected to the number of cells as defined by DAPI staining. For each time point, the number of fibers per cell was corrected to the time point with the maximum value, expressing all other quantifications as a percentage of the maximum. Representative images from selected time points were presented.
DATA AND SOFTWARE AVAILABILITY
The mouse tail tendon time-series microarray data 29 have been deposited to Array Express repository with the dataset identifier (ArrayExpress accession number E-MTAB-7743). All raw mass spectrometric data files from this study are available for download in Supplementary Information.
Author contributions
C-YCY and RG designed and performed experiments, interpreted data and drafted the manuscript. AP, JC, DFH, YL and BC designed and performed experiments and interpreted data. VM, JS and AA contributed materials and analysis tools. HR-H, OJ and TS supervised the mathematical aspects of the research. Q-JM and KEK conceived the project, supervised the experiments and wrote the manuscript.
Supplementary Data
MetaCycle output (columns A-X) for timed series LC-tandem mass spectrometry analysis of proteins extracted from 6-week-old mouse male tail tendon (columns Y-AJ).
Video 1
Step-through movie of serial block face-scanning electron microscopy of mouse Achilles tendon at 09:00 hrs. Bar, 7 µm. Z-depth, 486 µm, images recorded at 9.1 µm intervals to reduce file size.
Video 2
Step through movie displaying individual colourised fibrils in small region of mouse tail tendon, showing merging of D1 fibrils onto larger D2 and D3 fibrils. Data gathered from serial block face-scanning EM stack of images. Area shown is 4 × 4 µm, each frame is a longitudinal step of 100 nm through the tendon.
Video 3
Step-through movie of serial block face-scanning EM of mouse Achilles tendon at 09:00 hrs. Bar, 500 nm. D3 fibril highlighted with a blue line. Filled circles indicate D1 fibrils.
Video 4
Step-through movie of serial block face-scanning electron microscopy of mouse Achilles tendon showing diameter variability along the length of fibrils and tortuosity of D1 fibrils. Bar, 700 nm. Z-depth, 12.5 µm.
Video 5
Step-through animation showing the relative proportion of PC-I in the ER, ER exit sites, Golgi, post-Golgi, plasma membrane, and extracellular matrix, starting at CT0.
Video 6
Real-time bioluminescence microscopy of dissected Achilles tendon from wild type PER2::Luciferase mice during 5 days.
Video 7
Real-time bioluminescence microscopy of dissected Achilles tendon from ScxCre::Bmal1 lox/lox PER2::Luciferase mice during 5 days. The brightness for the Scx-Cre::Bmal1 lox/lox tendon recording was increased to enable visualization.
KEY RESOURCES TABLE
Acknowledgements
The research was funded by Wellcome (110126/Z/15/Z and 203128/Z/16/Z), MRC (MR/P010709/1) and Arthritis Research UK (20875). VM was supported by a studentship from the Sir Richard Stapley Educational Trust. JS was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB/L024551/1). BC is supported by a Wellcome 4-year PhD studentship (210062/Z/17/Z). The authors would like to thank Ronen Schweitzer (Shriners Hospital for Children, Portland, OR, USA) for Scx-Cre mice, Alison Hallworth and Raymond Hodgkiss and the University of Manchester Biological Support Facility for assistance in animal welfare and husbandry, and Michal Dudek and Nan Yang (University of Manchester) for assistance with bioluminescence imaging. The proteomics was performed at the Biological Mass Spectrometry Facility in the Faculty of Biology, Medicine and Health (University of Manchester) with the assistance of Stacey Warwood and Ronan O’Cualain, and electron microscopy was performed in the Electron Microscopy Facility, Faculty of Biology, Medicine and Health (University of Manchester).
Footnotes
↵$ Lead contact: KEK email karl.kadler{at}manchester.ac.uk