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Mapping the circadian clock-dependent regulation of the secretory pathway


Project Description

The protein secretory pathway in cells comprises a series of membranous compartments and transport vesicles that the cell uses to synthesise, post-translationally modify, and move proteins for delivery to the plasma membrane or for secretion to the extracellular matrix. The conventional pathway describes protein synthesis on the luminal face of the endoplasmic reticulum, transport of proteins in 60 nm-diameter COPII vesicles to the Golgi apparatus, and subsequent transport to the plasma membrane in Golgi-to-plasma membrane carriers (GPCs). Ground-breaking studies on the secretory pathway (which led to the Nobel Prize in Physiology and Medicine 2013) have been carried out using fibroblasts in which small viral proteins have been used as the cargo. However, the most abundant cargo in fibroblasts is procollagen (which is ~450,000 kDa and 350 nm in length), which is not expected to fit into 60 nm-diameter transport vesicles.

Using time-series microarray analysis of gene transcription, proteomics, electron microscopy, and immunofluorescence for proteins in the secretory pathway during 24 hours, we have shown that the secretory pathway is under the control of the circadian clock (manuscript in preparation). Our data show that the circadian clock exercises transcriptional and translational control of key proteins that are chaperones of protein folding, interaction partners for COPII-vesicle formation, and regulators of retrograde Golgi-to-ER transport.

The project is to use state-of-the-art fluorescence and super resolution imaging using a variety of fusion proteins, electron microscopy, cells from specific knockout mice, and targeted knockdown and knockout of novel gene targets including the use of CRISPR/Cas9. We will: generate a complete three-dimensional map of the protein secretory pathway in fibroblasts at 20 nm resolution, understand how the arrangement and size of compartments changes during 24 hours, locate the positions of key proteins in the pathway, and identify mechanisms of transporting large cargo through the cell. To obtain a deeper understanding of the function of the circadian clock in maintaining tissue homeostasis, we will use mass spectrometry approaches to determine which extracellular matrix proteins are present in the extracellular matrix and in the secretory pathway at 4-hour intervals during 24 hours, in mice that have had specific genes edited by CRISPR/Cas9.

Bioinformatics is a powerful tool that we are using to analyse our proteomics datasets. The PhD student will have the opportunity to work alongside our bioinformaticians.

The project will shed new light on how fibroblasts transport large cargo, such as procollagen, and how this process is perturbed in fibrosis and ageing.

Training/techniques to be provided:

Molecular biology (cloning, DNA sequencing, PCR, Q-PCR, RNA isolation)
Cell biology (cell culture, immunofluorescence, expression of fluorescently-tagged proteins)
Circadian biology (bioluminescence, timed microarrays and timed proteomics and phoshoproteomics)
Mass spectrometry
Biochemistry
Bioinformatics

Funding Notes

Candidates are expected to hold (or are about to obtain) a First Class or Upper Second Class honours degree (or equivalent) in biochemistry, molecular biology, genetics or related degree.

This project has a Band 3 fee. Details of our different fee bands can be found on our website (View Website). For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (View Website).

Informal enquiries may be made directly to the primary supervisor.

References

1. Canty, E. G., Lu, Y., Meadows, R. S., Shaw, M. K., Holmes, D. F. and Kadler, K. E. (2004) Co-alignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon. Journal of Cell Biology 165: 553-563
2. Yeung, C-Y C, Gossan N, Lu Y, Hughes A, Hensman J, Bayer M, Kjær M, Kadler KE and Meng QJ (2014). Gremlin-2 is a BMP antagonist that is regulated by the circadian clock. Scientific Reports. 4, 5183; DOI:10.1038/srep05183.
3. Dudek M, Gossan N, Nan Yang N, Im H-J, Ruckshanthi JPD, Yoshitane H, Li X, Jin D, Wang P, Boudiffa M, Bellantuono I, Fukada Y, Boot-Handford RP, and Meng QJ (2016). The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J Clin. Invest. 126 (1), 365-376.
4. Canty, E. G. and Kadler, K. E. (2005) Procollagen trafficking, processing and fibrillogenesis. Journal of Cell Science 118: 1341-1353
5. Taylor, S. H., Yeung, C-Y., C., Kalson, N. S., Lu, Y., Zigrino, P., Starborg, T., Warwood, S., Holmes, D. F., Canty-Laird, E. G., Mauch, C. and Kadler, K. E. (2015) MMP14 is required for fibrous tissue expansion. eLIFE doi: 10.7554/eLife.09345.
6. Starborg, T., Kalson, N. S., Lu, Y., Mironov, A., Cootes, T. F., Holmes, D. F. and Kadler, K. E. (2013) Using transmission electron microscopy and 3View® to determine collagen fibril size and three-dimensional organization. Nature Protocols 8: 1433-1448
7. Kalson, N. S., Starborg, T., Lu, Y., Mironov, A., Humphries, S. M., Holmes, D. F. and Kadler, K. E. (2013) Non-muscle myosin II powered transport of newly-formed collagen fibrils at the plasma membrane. Proceedings of the National Academy of Sciences U. S. A. 110: E4743-E4752. doi: 10.1073/pnas.1314348110

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