Clockagen: Understanding and quantifying the link between collagen and the circadian clock through mathematical modelling

  • Ben Calverley

Student thesis: Phd


Without collagen, our bodies would have no structural integrity. As the most abundant protein in metazoan bodies it makes up the majority of the mass of tissues including bone, muscle, tendon, ligament, and cornea. The ability of vertebrate cells to assemble complex tissues capable of withstanding a lifetime of cyclical loading is directly due to an extracellular matrix (ECM) where collagen is assembled in roughly cylindrical fibrils. The fibrils can be centimetres (perhaps metres in large animals such as whales and giraffes) in length and provide a scaffold that both protects cells from environmental forces and maintains the shape and form of organs. A more complete understanding of how these scaffolds are maintained would have important ramifications in diseases where the collagen network is disrupted or abnormal, including osteoarthritis, fibrosis, and cancer. It was long thought that collagen is permanent in mammalian ECM. However recent studies have shown that cells retain the ability for regular (and sometimes rapid) production of collagen throughout their lifetime. This creates a conundrum: how can fibrils that are never renewed withstand millions of cycles of stress without suffering fatigue failure, and, in the absence of turnover, why do cells retain the ability to synthesise new collagen? This thesis has contributed to the discovery that there exists two pools of collagen in tendon, in which one pool (˜90-95%) is permanent and the other is under the control of the circadian clock, turned over on a 24-hour rhythmic basis. However, this discovery has opened up new questions. Why does such an important and potentially long-lived protein need to be circadianly regulated? How is that regulation achieved? In this thesis I show how the cell achieves circadian regulation of collagen levels and discuss potential reasons and applications. In the first chapters, I built a mathematical model of the circadian clock regulation of the collagen secretory pathway. I then designed a biological tool to quantify collagen dynamics and test the model (among other potential applications). I created a predictive mathematical model for collagen regulation at a cellular level, allowing for better understanding and exploration of the ways this vital protein is maintained. The novel experimental tool I developed helps to refine this model and quantify protein levels across multiple scales and in real time using CRISPR-Cas9 and NanoLuciferase, with other potential uses in therapeutic drug screening. We measured accurately the number of collagen molecules in a cell consistently across platforms and scales, as well as quantifying at sub-cellular vesicle level. In the final part of the work described in this thesis, I developed a mathematical mechanical model for the stress response of tendon starting from the microstructure of the tendon as seen in electron microscopy images, incorporating the effects of the fluid in the extrafibrillar matrix. Using this I investigated the circadian differences observed in both the distribution of fibrils and mechanical response of the tissue. This tissue mechanics modelling illustrates the importance of including the extrafibrillar matrix in mechanical tissue modelling, as well as accounting for the specific topology of the tendon itself. This thesis is a step forward in our understanding in a number of areas. A predictive model of tendon mechanics based on tendon EM images and incorporating the contributions of the extrafibrillar matrix will more accurately explain the behaviour of this important tissue and its response to stresses. Further refinement of the collagen pathway model will lead to a better understanding of how the cells maintain such an important homeostatic mechanism. The NLuc-based, easy-to-use, reliable tool for protein quantification will have wide ranging benefits and uses, from sub-cellular dynamic resolution of protein trafficking to live quantitation of tissue level changes to protein amou
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorKarl Kadler (Supervisor) & Tom Shearer (Supervisor)


  • Quantitation
  • Circadian
  • Mechanics
  • Biology
  • Mathematics
  • Collagen

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