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Circadian control of the secretory pathway maintains collagen homeostasis

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Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the 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 and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function. Here, Chang et al. show that the circadian clock regulates secretion resulting in nocturnal procollagen synthesis and daytime collagen fibril assembly in mice to maintain the homeostasis of the collagen network.
Electron microscopy and biomechanics a, Collagen fibril diameter distributions measured from transverse TEM images of tail tendons sampled through a circadian period. Mice were housed in 12-hour dark/12-hour light cycles. ZT, zeitgeber time (hours into light). Fibrils (n = 1400) were measured for each panel. Black lines show 3-Gaussian fit curves. b, Typical scanning transmission electron microscopy image of fibrils from mechanically disrupted tendon. A set of 50 similar STEM images were acquired for each of 30 tendon samples. Bar, 500 nm. c, Representative mass-per-unit-length distribution measured from scanning transmission electron microscopy images of mechanically disrupted whole tendons. Time point shown is ZT12. The data shown is for n- = 997 fibrils from a single Achilles tendon. All mass-per-unit-length distributions from 30 tendon samples showed a characteristic prominent D1 peak. Percentage of D1 fibrils at all time points over 24 h is shown in Fig. 1e. d, Elastic modulus of Achilles tendon is not time-of-day dependent. Bars show mean ± s.e.m., n = 4 biological replicates, unpaired t-test, p = 0.46. e, Representative hysteresis curves from cyclic loading show the energy loss is greater in tendons taken at ZT15 compared with ZT3, n = 4 biological replicates, n = 5 hysteresis cycles for each displacement rate for each tendon sample. f, Time constants derived from stress–relaxation from an initial 1 N load of Achilles tendon sampled at ZT3 compared to ZT15 (T1, T2 and T3). Bars show mean ± s.e.m., n = 4 biological replicates; the p-values were 0.114, 0.026 and 0.75 for the time constants T1, T2 and T3, respectively. g, Comparison of energy loss values of Achilles tendons at ZT3 versus ZT15 for a 5-fold range of strain rates showing a consistent ~ 40% greater energy loss at ZT15 compared with ZT3. Bars show mean ± s.e.m., n = 4 biological replicates, p-values using the unpaired t-test are 0.27, 0.26, 0.21, 0.004, 0.01 for displacement rates of 1, 2, 3, 4 and 5 mm/min, respectively. See also Statistical Source Extended Data Fig. 1. Source data
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Articles
https://doi.org/10.1038/s41556-019-0441-z
1Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science
Centre, Manchester, UK. 2School of Mathematics, Faculty of Science and Engineering, University of Manchester, Manchester, UK. 3Present address:
Institute of Sports Medicine Copenhagen, Bispebjerg Hospital, Copenhagen, Denmark. 4These authors contributed equally: Joan Chang, Richa Garva,
Adam Pickard, Ching-Yan Chloé Yeung. *e-mail: qing-jun.meng@manchester.ac.uk; karl.kadler@manchester.ac.uk
One-third of the eukaryote proteome enters the secretory
pathway1, including the collagens that assemble into centi-
metre-long fibrils in the extracellular matrix (ECM)2. These
fibrils account for one-third of the mass of vertebrates3 and are the
sites of attachment for a wide range of macromolecules, including
integrins, making them essential for metazoan development3. A
remarkable feature of collagen fibrils is that they are formed dur-
ing embryogenesis4 and remain without turnover for the life of the
animal58. This has led to the idea that collagen fibrils are static and
unchanging. However, the difficulty with zero turnover is that it does
not explain the absence of fatigue failure, which would be expected
in the face of life-long cyclic loading. In contrast to the evidence
of zero replacement, fibroblasts synthesize collagen in response to
mechanical loading9, 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 exercise10.
These opposing observations led to the alternative hypothesis
presented in this study, in which zero turnover and continued syn-
thesis can coexist. We hypothesized that a pool of ‘persistent’ col-
lagen coexists 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 C-propeptides of PC-I11 and of collagen degradation products
in bone12. However, despite physiological and clinical observations,
direct mechanistic support for these observations was lacking.
Although 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 cycles of light and
dark13. Disruption of the circadian clock leads to musculoskeletal
abnormalities—for example, chondrocyte-specific disruptions of
the circadian clock result in progressive degeneration of articular
cartilage14 and fibrosis in the intervertebral disc15, and mice with
a global knockout of Bmal1 (ref. 16) or the ClockΔ19 mutation17
develop thickened and calcified tendons with associated immobi-
lization. These observations are indicative of circadian control of
ECM homeostasis.
Here, we performed time-series electron microscopy, tran-
scriptomics and proteomics over day–night cycles, which showed
that the synthesis and transport of PC-I by the protein secretory
pathway in fibroblasts is regulated by the circadian clock. We
show that SEC61, TANGO1, PDE4D and VPS33B regulate col-
lagen secretion, are 24-h 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, which is rhythmic in-phase with colla-
gen degradation to maintain collagen homeostasis. The result is
nocturnal PC-I synthesis and a daily wave of collagen-I with no
net change in the total collagen content of the tissue. 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. Finally, we show that ClockΔ19
fibroblasts in vitro amass collagen fibres compared with con-
trol cells and treatment of ClockΔ19 fibroblasts with the NR1D1
agonist SR9009 (ref. 18) or the cryptochrome (CRY1/2) agonist
KL001 (ref. 19) reduces the number of collagen fibres. Wild-type
fibroblasts treated with KL001 lose their circadian rhythm and
generate more collagen fibres. Together, these results provide
insights into the importance of the circadian clock in maintaining
collagen homeostasis.
Circadian control of the secretory pathway
maintains collagen homeostasis
Joan Chang1,4, Richa Garva1,4, Adam Pickard1,4, Ching-Yan Chloé Yeung1,3,4, Venkatesh Mallikarjun 1,
Joe Swift 1, David F. Holmes1, Ben Calverley1,2, Yinhui Lu1, Antony Adamson 1,
Helena Raymond-Hayling1,2, Oliver Jensen 2, Tom Shearer2, Qing Jun Meng 1* and Karl E. Kadler 1*
Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency
of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional tran-
scriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian
clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the 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
and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling
the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced invitro by the NR1D1 and
CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of
protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.
NATURE CELL BIOLOGY | VOL 22 | JANUARY 2020 | 74–86 | www.nature.com/naturecellbiology
74
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... Fibrils exhibit a regular, wavy, longitudinal morphology called crimp, thought to undulate in either a two-dimensional (2D) planar or helical fashion [15][16][17] over an approximately 100 μm period for adult mouse tendon [18] . Their diameter remains constant along most of their length, with some exceptions [19] , but there is a distribution of diameters in any given tendon cross-section. Recent work has shown that the diameter distribution can be described well by a trimodal Gaussian distribution [19] . ...
... Their diameter remains constant along most of their length, with some exceptions [19] , but there is a distribution of diameters in any given tendon cross-section. Recent work has shown that the diameter distribution can be described well by a trimodal Gaussian distribution [19] . ...
... An increase of 50% in fibril diameter has previously been found to account for a five-fold increase in ultimate tensile strength (the maximum stress before failure) and Young's modulus in human fibroblast tendon constructs [20] . Collagen fibrils in tendon are structurally continuous through large longitudinal distances ( ∼ 10 −2 m) [21] , and can be as small as 50 nm in diameter, and as large as 500 nm [19] , giving them an aspect ratio of 10 5 -10 6 . ...
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Tendons are crucial connective tissues made almost entirely of bundles of long, near-parallel collagen fibrils, and are vitally important to skeletal stability and mechanical function. Tendon structure is typically quantified in 2D, whereas, in this work, we have used serial block face-scanning electron microscopy to image tendons in 3D. We present a custom fibre tracking algorithm (FTA), with which we have characterised the 3D microstructure of tendon. Currently available tools for fibre tracing were unsuitable for tracking large numbers of fibrils and handling imaging artefacts associated with EM. We have tracked fibrils through a representative tendon volume and measured their relative length, diameter, orientation, chirality, tortuosity and volume fraction, which are just some of the measurements it is possible to make with the FTA depending on the research question. This algorithm has been developed in a general way and can be applied to a range of biological research questions relating to tendon structure-function relationships, on topics such as ageing, disease, development and injury. The FTA is also applicable to other fibrous biological materials, as well as engineered materials and textiles; it is written using Python and is freely available to download. Statement of significance We have created an algorithm for tracking fibres in 3D image stacks and applied it to tendon tissue. Previous studies have examined tendon structure in 2D, whereas we have imaged tendons in 3D using volumetric electron microscopy. Currently available fibre tracing tools could not track the large numbers of fibres or tolerate the artefacts present in biological imaging data. Using our algorithm, we have reconstructed and characterised the geometrical properties of the collagen fibrils (length, width, alignment, area, location). This algorithm could help to answer questions in biology which relate tissue microstructure to function in areas such as ageing, disease, development and injury. It could also be used to study engineered materials and textiles and is available freely to download.
... Recently, it was found that fibrillar collagen molecules are in fact undergoing a remarkable remodelling that is controlled by the circadian clock [103]. In the Achilles tendon, while collagen The table lists the half-lives of fibrillar matrix components reported in literature, the origin of the samples (organism and tissue), the age or body weight of source animals, and the method used to infer the half-lives. ...
... However, these results seem to disagree with the previous reports that collagen does not turnover daily [97,[99][100][101]. To solve this problem, it was proposed that there exist a large amount of 'persistent' collagen and a small pool of 'sacrificial' collagen, and the latter is undergoing the circadian turnover [103]. It remains to be elucidated exactly how much of collagen molecules are 'persistent' and 'sacrificial' in vivo. ...
... Disruption of the circadian clock leads to a number of abnormalities in connective tissues, e. g. the fibrosis, calcification, and degeneration of cartilages and tendons [104][105][106][107]. Failure in the circadian turnover of fibrillar matrices may underlie these diverse pathologies [103]. It is unknown why fibrillar matrices should undergo a circadian turnover. ...
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Extracellular matrices (ECMs) are essential for the architecture and function of animal tissues. ECMs have been thought to be highly stable structures; however, too much stability of ECMs would hamper tissue remodelling required for organ development and maintenance. Regarding this conundrum, this article reviews multiple lines of evidence that ECMs are in fact rapidly moving and replacing components in diverse organisms including hydra, worms, flies, and vertebrates. Also discussed are how cells behave on/in such dynamic ECMs, how ECM dynamics contributes to embryogenesis and adult tissue homoeostasis, and what molecular mechanisms exist behind the dynamics. In addition, it is highlighted how cutting-edge technologies such as genome engineering, live imaging, and mathematical modelling have contributed to reveal the previously invisible dynamics of ECMs. The idea that ECMs are unchanging is to be changed, and ECM dynamics is emerging as a hitherto unrecognized critical factor for tissue development and maintenance.
... We have a poor understanding of how biomechanical overload timing and severity from sports training influences the in vivo proteolytic activity that may drive ACL microtrauma beyond the threshold for natural repair homeostasis [19,113]. High frequency, intensity, or total volume sport training may create situations where youth and adolescent athletes are at greater risk for compromising any of a number of developmental processes through chronic overtraining and hormonal dysregulation [37,58,61,68]. ...
... The nature and acuity of this healing response can prompt either an anabolic, homeostatic, or catabolic state, in which ECM production and structural properties are respectively either increased, maintained, or reduced. Increased ECM collagen production and incorporation occurs within hours of loading [53,86] with circadian regulation of collagen synthesis, cellular export, and collagen degradation attempting to maintain or restore tissue homeostasis [19]. Excessive sports training intensity, frequency, or total volume, however, may upset this balance. ...
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... 72,73 Fibroblasts in culture show a circadian expression pattern of clock components 74 and seem to function in a rhythmic manner to maintain collagen homeostasis in surrounding tissues. 75 Lung fibroblasts also showed robust circadian rhythms that are accentuated in fibrotic areas. 76 However, the circadian biology of cardiac fibroblast function remains to be elucidated. ...
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... Multi-tissues time-serie proteomic data come from: Mauvoisin et al. [17] for the liver, Noya et al. [45] for the forebrain, Chang et al. [46] for the tendon, and Dudek et al. [47] for the cartilage. Finally, single-cell data of thousand of cells in organs for which we had time-series datasets, i.e. liver, lung, kidney, muscle, aorta, and heart, were downloaded from figshare using R objects from FACS single-cell datasets [29]. ...
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