Circadian clock regulation of ECM organisation and secretion

Abstract

The extracellular matrix (ECM), provides structural and biochemical support to cells,
tissues and organs. Collagens comprise the main structural component of the ECM,
making up more than a quarter of human protein mass. Recent reports have identified
the circadian clock as a key regulator of collagen biosynthesis and fibril assembly.
Inappropriate secretion of ECM, and disruption of the circadian clock can both
contribute to numerous connective tissue disorders such as fibrosis, cancer and
osteoarthritis, all major causes of pathology and morbidity. In this study I sought to
identify how key secretory pathway mediators and the circadian clock affect secretion
and assembly of the ECM. To investigate ECM on the nano to micro scale, I used high-
speed atomic force microscopy (HS-AFM) and confocal microscopy to visualise the
effects of circadian synchronisation and perturbation on ECM ultrastructure. I showed
that deletion of the clock gene BMAL1 using CRISPR-cas9 alters ECM organisation. By
performing time-series proteomics of decellularised ECM samples from WT and BMAL1
KO cells, I identified a subset of proteins showing circadian rhythmicity, which was lost in
the BMAL1 knock outs. These proteins, which comprised 14% of total proteins, included
key regulators of the ECM, ECM-proteases, the cytoskeleton and focal adhesions.
Imaging of the cytoskeleton and focal adhesions confirmed dysregulation of these
structures in BMAL1 KO cells, likely contributing to the observed ECM disorganisation.
To determine whether secretory pathway disruption contributes to these changes in
ECM composition, I investigated Golgi structure. Compaction of cis-Golgi golgin-160
elements in BMAL1 KO cells was observed, implicating the clock in secretion regulation.
Further investigation also highlighted the importance of the circadian rhythmic Golgi
organising protein, golgin-160, in ECM secretion for the first time. Altogether these
findings are consistent with a role of the circadian clock in the regulation of ECM
secretion, assembly and remodelling.

Date of Award	9 Dec 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Chrissy L Hammond (Supervisor) & Nicola L Stevenson (Supervisor)