Publications

Recent publication highlights

New insight into the mechanisms of collagen secretion

Super-resolution microscopy of ERES

STED image of ERES

A new paper from our lab providing some new insight into the mechanism of collagen secretion has been published in Cell Reports.

McCaughey, J., Miller, V.J., Stevenson, N.L., Brown,  A.K., Budnik, A., Heesom,  K.J., Alibhai, D., and Stephens, D.J. (2016) TFG promotes organization of transitional ER and efficient collagen secretion. Cell Reports, Cell Reports, in press. doi: http://dx.doi.org/10.1016/j.celrep.2016.04.062

Collagen is the most abundant protein in the body. Fibril forming collagens, including collagen I, are made as large, 300 nm long precursors in the endoplasmic reticulum (ER), which and are then transported through the cell using the classical vesicular transport machinery. The first step in this pathway is the packaging into vesicles by the COPII coat, a complex of proteins that assemble at specific sites on the ER called ER exit sites. The COPII machinery is well described but classically generates 60-90 nm size vesicles that are too small to capture and transport large fibrillar collagens. A protein called TFG, known for its role at ER exit sites through interactions with COPII. The new work from the Stephens lab shows that TFG has a role in organizing ER exit sites into larger structures and that it has a key role in the secretion of collagens. A combination of imaging techniques, including super-resolution microscopy was used to show that on depleting cells of TFG, ER exit sites become more dispersed yet remain functional for the transport of small cargo such as Golgi membrane proteins. However, proteomics and cell imaging revealed a defect in the transport and secretion of extracellular matrix components including collagens. This work suggests a link between ER exit site organization and procollagen secretion that the lab are keen to follow up in the near future.

The work was conducted primarily by a MRC-funded postdoc, Vicky Miller, and a Masters student Janine McCaughey. Janine undertook this work as a part of her Master’s degree at the Leibniz University Hannover during a 6-month placement in the Stephens lab. Current postdoc Nikki Stevenson, as well as former lab members Anna Brown, and Annika Budnik contributed to the work. Key to the success of the project were the outstanding resources and support available within the Wolfson Bioimaging Facility (notably Dom Alibhai who wrote dedicated computer code for image analysis) and Proteomics Facility (where Facility Director Kate Heesom was key to the success of the project).

 

This work was funded by the Medical Research Council UK (grant numbers MR/J000604/1, MR/K018019/1, and G0801848). The Wolfson Bioimaging Facility has been funded generously by the Wolfson Foundation and University of Bristol; this work also benefited from additional equipment funded by the BBSRC (through BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre: grant number: L01386X) and ALERT 13 capital grant (BB/L014181/1).


Research Publications

Research papers and reviews (past 3 years):

McCaughey, J., Miller, V.J., Stevenson, N.L., Brown,  A.K., Budnik, A., Heesom,  K.J., Alibhai, D., and Stephens, D.J. (2016) TFG promotes organization of transitional ER and efficient collagen secretion. Cell Reports, Cell Reports, in press. doi: http://dx.doi.org/10.1016/j.celrep.2016.04.062

Boal, F. Hodgson, L.R., Reed, S.E., Yarwood, S.E., Just, V.J., Stephens, D.J., McCaffrey, M.W., and Tavaré, J.M. (2015) Insulin promotes Rip11 accumulation at the plasma membrane by inhibiting a dynamin- and PI3-kinase-dependent, but Akt-independent, internalisation event. Cellular Signalling, in press. doi:10.1016/j.cellsig.2015.10.014

Asante, D., Stevenson, N.L., and Stephens, D.J. (2014) Subunit composition of the human cytoplasmic dynein-2 motor. Journal of Cell Science, 127, 4774-4787. doi: 10.1242/jcs.159038

Miller, V.J. and Stephens, D.J. (2014) Artificially inducing close apposition of endoplasmic reticulum and mitochondria induces mitochondrial fragmentation. Manuscript submitted. Preprint available on BiorXix.

Bonavita, R., Walas, D., Townley, A.K., Luini, A., Stephens, D.J., and Colanzi, A. (2014) CEP126 is required for pericentriolar satellite localisation to the centrosome and for primary cilium formation. Biology of the Cell, 106(8):254-67. DOI: 10.1111/boc.201300087

Townley, A.K., Hunt, S.D., and Stephens, D.J. (2014) Opposing microtubule motors control motility, morphology, and cargo segregation during ER-to-Golgi transport. Biology Open, 3, 307-313  doi: 10.1242/bio.20147633

Fryer, L.G.D., Jones, B., Duncan, E.J., Hutchison, C.E., Ozkan, T., Williams, P.A., Alder, O., Nieuworp, M., Townley, A.K., Mensenkamp, A.R., Stephens, D.J., Dallinga-Thie G.M., and Shoulders, C.C (2013) The Endoplasmic Reticulum COPII-Vesicle Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis. Journal of  Biological. Chemistry, 289(7):4244-61.  doi: 10.1074/jbc.M113.479980

Miller V.J., McKinnon C.M., Mellor H., Stephens D.J. (2013) RNA interference approaches to examine Golgi function in animal cell culture. Methods in Cell Biology 118:15-34. doi: 10.1016/B978-0-12-417164-0.00002-1.

Asante, D., MacCarthy-Morrogh, L., Townley, A.K., Weiss, M.A., Katayama, K., Palmer, K.J., Suzuki, H., Westlake, C., and Stephens, D.J. (2013) A role for the Golgi matrix protein giantin in ciliogenesis through control of the localization of dynein-2. Journal of Cell Science. doi: 10.1242/jcs.131664.

Midgley R., Moffat K., Berryman S., Hawes P., Simpson J., Fullen D., Stephens D.J., Burman A., Jackson T. (2013) A role for endoplasmic reticulum exit sites (ERES) in foot-and-mouth disease virus infection.  Journal of General Virology doi: 10.1099/vir.0.055442-0.

Satchwell, T.J., Pellegrin, S., Bianchi, P., Hawley, B.R., Gampel, A., Mordue, K.E., Budnik, A., Fermo, E., Barcellini, W., Stephens DJ, van den Akker, E., Toye, A.M. (2013) Characteristic phenotypes associated with Congenital Dyserythropoietic Anemia (Type II) manifest at different stages of erythropoiesis. Haematologica In press. doi: 10.3324/haematol.2013.085522

Hunt, S.D., Townley, A.K., Danson, C.M., Cullen, P.J. and Stephens DJ (2013) Microtubule motors mediate endosomal sorting by maintaining functional domain organization.  Journal of Cell Science. 126, 2493-2501. doi: 10.1242/jcs.122317.

Miller, V.J. and Stephens DJ. (2013) Membrane contact sites: An interesting species, an interesting mix.
EMBO reports. 14, 396-7. doi:10.1038/embor.2013.43.

Schmidt, K., Cavodeassi, F., Feng, Y., and Stephens, D.J. (2013) Early stages of retinal development depend on Sec13 function.  Biology Open 2, 256–266. doi:10.1242/bio.20133251

Stephens, D.J. (2012) Functional coupling of microtubules to membranes: implications for membrane structure and dynamics. Journal of Cell Science. Invited review. doi: 10.1242/jcs.097675.

Stephens, D.J. (2012) News & Views “Cell Biology: Collagen secretion explained”. Nature 482, 474–475. doi:10.1038/482474a.

Townley, A.K., Schmidt, K, Hodgson, L., and Stephens, D.J. (2012) Cell autonomous COPII-dependent secretion is required for epithelial differentiation and cyst lumen expansion. Journal of Cell Science 125, 673-684. doi: 10.1242/jcs.091355

Hunt, S.D. and Stephens, D.J. (2011) The role of motor proteins in endosomal sorting. Invited Review. Biochemical Society Transactions 39, 1179-1184. http://www.biochemsoctrans.org/bst/039/bst0391179.htm

Budnik, A. and Stephens, D.J. (2011) Characterization of human Sec16B: indications of specialized, non-redundant functions. Scientific Reports 1, 77. http://www.nature.com/srep/2011/110830/srep00077/full/srep00077.html

Palmer, K.J., MacCarthy-Morrogh, L., Smyllie, N., and Stephens, D.J. (2011) A role for Tctex-1 (DYNLT1) in controlling primary cilium length. European Journal of Cell Biologydoi:10.1016/j.ejcb.2011.05.003
Final published version at EJCB/Science Direct:http://www.sciencedirect.com/science/article/pii/S0171933511001002

Schmidt, K.J. and Stephens, D.J. (2010) Cargo loading at the ER. Invited review. Molecular Membrane Biology 27, 398-411. http://informahealthcare.com/doi/abs/10.3109/09687688.2010.506203

Boal, F., Guetzoyan, L., Sessions, R.B., Spooner, R.A., Lord, J.M., Clarkson, G.J., Roberts, L.M., and Stephens, D.J. (2010) LG186: an inhibitor of GBF1 function that causes Golgi disassembly in human and canine cells. Traffic, 11, 1537-51. http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0854.2010.01122.x/abstract

Hughes, H. and Stephens, D.J. (2010) Sec16 defines the initiation site for vesicle budding from the endoplasmic reticulum on exit from mitosis. Journal of Cell Science, 123, 4032-8. http://jcs.biologists.org/cgi/content/abstract/123/23/4032.

Guetzoyan, L., Spooner, R., Boal, F., Stephens, D.J., Lord, J.M., Roberts, L.M., Clarkson, G.J. (2010) Fine tuning Exo2, a small molecule inhibitor of secretion and retrograde trafficking pathways in mammalian cells. Molecular BioSystems, DOI: 10.1039/c0mb00035c.
http://pubs.rsc.org/en/Content/ArticleLanding/2010/MB/C0MB00035C

Boal, F. and Stephens, D.J. (2010) Specific functions of BIG1, and BIG2 in endomembrane organization. PLoS One 5(3), e9898http://dx.plos.org/10.1371/journal.pone.0009898

Budnik, A., and Stephens, D.J. (2009) Export of secretory cargo from the ER. FEBS Letters. Invited review. Special Issue – “The Golgi”. FEBS Letters 583, 3796-3803 doi:10.1016/j.febslet.2009.10.038.

Stephens, D.J. (2009) Editorial:Imaging in cell and developmental biology. Seminars in Cell and Developmental Biology, Guest editor. doi:10.1016/j.semcdb.2009.09.003.

Townley, A.K., and Stephens, D.J. (2009) Vesicle coating and uncoating – controlling the formation of large COPII-coated carriers. Invited review. F1000 Biology Reports. 1, 65.

Hughes, H., Budnik, A., Schmidt, K.J., Johnson, A., Noakes, C., Carter, D.A., Verkade, P., Watson, P., and Stephens, D.J. (2009) Organization of human endoplasmic reticulum exit sites: requirements for the localization of Sec16 to transitional ER. Journal of Cell Science, 122, 2924-2934.

Wassmer, T., Attar, N., Harterink, M., van Weering, J.R.T., Traer, C.J., Oakley, J., Goud, B., Stephens, D.J., Verkade, P., Korswagen, H.C., and Cullen, P.J. (2009) Endosomal sorting by retromer is coupled to the molecular motor subunit dynactin 1 and requires the putative Golgi tether Rab6IP1. Developmental Cell, 17, 110-122.

Palmer, K.J., Hughes, H., and Stephens, D.J. (2009) Specificity of cytoplasmic dynein subunits in discrete membrane trafficking events. Molecular Biology of the Cell 20, 2885-2899.

Townley, A.K., Feng, Y., Schmidt, K., Carter, D.A., R. Porter, Verkade, P., and Stephens, D.J. (2008) Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. Journal of Cell Science 121, 3025-34. 

Gupta, V., Palmer, K.J., Spence, P., Hudson, A., and Stephens, D.J. (2008) Kinesin-1 (uKHC/KIF5B) is required for bidirectional motility of ER exit sites and efficient ER-to-Golgi transport. Traffic, 9, 1850-1866.

Spence, P., Gupta, V., Stephens, D.J. and Hudson, A. (2008) Optimizing the precision for localizing fluorescent proteins in living cells by 2D Gaussian fitting of digital images: Application to COPII-coated endoplasmic reticulum exit sites. European Biophysics Journal, 37, 1335-49.

Spooner, R.A., Watson, P., Smith, D.C., Boal, F., Amessou, M., Johannes, L., Clarkson, G., Lord, J.M., Stephens, D.J., and Roberts, L.M. (2008) The brefeldin A-like inhibitor Exo2 perturbs trafficking between endosomes and the trans-Golgi network. Biochemical Journal, 41, 471-84.

Wakana, Y., Takai, S., Nakajima, K., Tani, K., Yamamoto, A., Watson, P., Stephens, D.J., Hauri, H.-P. and Tagaya, M. (2008) Bap31 is an itinerant protein that moves between the peripheral ER and a juxtanuclear compartment related to ER-associated degradation. Molecular Biology of the Cell, 19, 1825-1836.

Hughes, H. and Stephens, D.J. (2008) Assembly, organization and function of the COPII coat. Histochemistry and Cell Biology, 129, 129-151.

Traer, C.J., Rutherford, A.C., Palmer, K.J., Wassmer, T., Oakley, J., Attar, N., Carlton, J.G., Kremerskothen, J., Stephens, D.J. and Cullen, P.J. (2007). Sorting nexin-4 co-ordinates endosomal sorting of transferrin receptor with dynein-mediated transport into the endocytic recycling compartment. Nature Cell Biology, 9, 1370-1380.

Haas, A.K., Yoshimura, S., Stephens, D.J., Preisinger, C., Fuchs, E. and Barr, F.A. (2007) Analysis of GTPase-activating proteins reveals that Rab1 and Rab43 are key Rabs required for the maintenance of a functional Golgi in human cells. Journal of Cell Science, 120, 2997-3010.

Arasaki, K., Tani, K., Yoshimori, T., Stephens, D.J. and Tagaya, M. (2007) Nordihydroguaiaretic Acid Affects Multiple Dynein-dynactin Functions in Interphase and Mitotic Cells.Molecular Pharmacology, 71 454-460.

Watson, P., Townley, A.K., Koka, P., Palmer, K.J., and Stephens, D.J. (2006) Sec16 defines ER exit sites and is required for secretory cargo export in mammalian cells. Traffic, 7 (12) 1678-1687.

Watson, P. and Stephens, D.J. (2006) Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells. Journal of Cell Science, 119 2758-2767.

Forster, R., Weiss, M., Zimmermann, T., Reynaud, E.G., Stephens , D.J. and Pepperkok, R. (2006) Secretory cargo regulates the turnover of COPII subunits at single ER-exit sites.Current Biology16 (2) 173-179.

Minogue, S., Waugh, M.G., De Matteis, M.A., Stephens, D.J. , Berditchevski, F., and Hsuan, J.J. (2006) Phosphatidylinositol-4-kinase is required for the endosomal trafficking and degradation of the EGF receptor. Journal of Cell Science 119 (3) 571-581.

Palmer, K.J., Konkel, J.E. and Stephens , D.J. (2005) PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. Journal of Cell Science, 118 (17) 3839-3847.

Watson, P., Forster, R., Palmer, K.J., Pepperkok, R, and Stephens , D.J. (2005) Coupling of ER exit to microtubules through direct interaction of COPII with dynactin. Nature Cell Biology , 7 (1) 48-55.
Highlighted by the Faculty of 1000 as a “Must read” paper.

Watson, P., Jones, A.T. and Stephens , D.J. (2005) Intracellular trafficking pathways and drug delivery: Fluorescence imaging of living and fixed cells. Advanced Drug Delivery Reviews57, 43-61. (Invited review).

Gumbleton, M. and Stephens , D.J. (2005) Coming out of the dark: The evolving role of fluorescence imaging in drug delivery research. Advanced Drug Delivery Reviews 57, 5-15 (Invited review).

Stephens , D.J. and Pepperkok, R. (2004) Differential requirements for efficient GTP hydrolysis by Sar1p direct selective export of cargo from the endoplasmic reticulum. Journal of Cell Science, 117, 3635-3644.

Shoulders, C.C., Stephens, D.J. and Jones, B. (2004) The Intracellular Transport of Chylomicrons requires the Small GTPase Sar1b. Current Opinion in Lipidology 15, 191-197 (Invited review).

Palmer, K.J. and Stephens, D.J. (2004) Biogenesis of ER-to-Golgi transport carriers: complex roles of COPII in ER export . Trends in Cell Biology 14, 57-61. (Invited review).

Stephens, D.J. and Allan, V.J. (2003) Light microscopy techniques for live cell imaging. Science 300, 82-86. (Invited review).

Stephens . D.J. (2003) De novo formation, fusion and fission of COPII coated ER exit sites in mammalian cells. EMBO reports 4, 210-217Highlighted by the Faculty of 1000 as a “Must read” paper.

Wakefield , J.G., Stephens , D.J. and Tavaré, J.M. (2003) A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment. J. Cell Science 116, 637-646.
Highlighted by the Faculty of 1000 as a “Must read” paper.

Rous, B.A., Reaves, B.J., Ihrke, G., Briggs, J.A.G., Gray, S.R., StephensD.J., Banting, G. and Luzio, J.P. (2002) The role of the adaptor complex AP-3 in targeting wild type and mutated CD63/LIMP-1 to lysosomes. Mol. Biol. Cell 13, 1071-1082Highlighted by the Faculty of 1000 as a “Must read” paper.

Stephens, D.J. and Pepperkok, R. (2002) Imaging of procollagen transport reveals COPI- dependent cargo sorting during ER-to-Golgi transport in mammalian cells. J. Cell Science 115, 1149-1160.

Stephens, D.J. and Pepperkok, R.(2001) Perspectives: The many ways to cross the plasma membrane. 
Proc. Natl. Acad. Sci. U.S.A. 98, 4295-4298. (Invited review).

Stephens, D.J. and Pepperkok, R.(2001) Illuminating The Secretory Pathway: When Do We Need Vesicles? 
J. Cell Science 114, 1053-1059. (Invited review).

Blum, R., Stephens, D.J. and Schultz, I. (2000) Lumenal targeted GFP used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network. J. Cell Science 113, 3151-3159.

Stephens, D.J., Lin-Marq, N., Pagano, A., Pepperkok, R. and Paccaud, J.-P. (2000) COPI coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites. J. Cell Science 113, 2177-2185.

Stephens D.J. and Banting, G. ( 2000 ) Application of the two-hybrid system to the study of membrane traffic. Traffic 1, 763-768 (Invited review).

Stephens, D.J. and Banting, G. (2000 ) In vivo dynamics of the F-actin binding protein, neurabin-II.
Biochem. J. 345, 185-194.

Stephens, D.J. and Banting, G. (1999) Direct interaction of the type-I transmembrane glycoprotein, TGN38, with the F-actin binding protein, neurabin. J. Biol. Chem. 274, (42) 30080-30087.

Frears, E.R., Stephens, D.J., Walters, C.E., Davies, H., and Austen, B.M. (1999). The role of cholesterol in the biosynthesis of b-amyloid. Neuroreport 10, (8) 1699-1705.

Stephens, D.J. and Banting, G. (1998) Specificity of interaction between adaptor complex medium chains and the tyrosine-based sorting motifs of TGN38 and lgp120. Biochem. J. 335, 567-572.

Crump, C.M., Williams, J.L., Stephens, D.J. and Banting G. (1998). Inhibition of the interaction between tyrosine-based sorting motifs and the medium chain subunit of the AP2 adaptor complex by specific tyrphostins. J. Biol. Chem. 273, 28073-28077.

Stephens D.J. and Austen B.M. (1998) Review: Characterization of b-secretase. Biochem. Soc. Trans. 26, 500-4.

Stephens, D.J. and Banting, G. (1997). Insulin-dependent tyrosine phosphorylation of TGN38 creates a specific SH2 domain binding site. FEBS Letts.416, 27-29.

Stephens, D.J., Crump, C.M., Clarke, A.R. and Banting, G. (1997). Serine-331 and Tyrosine-333 are both involved in the interaction between the cytosolic domain of TGN38 and the m2 subunit of the AP2 clathrin adaptor complex. J. Biol. Chem. 272, 14104-14109.

Stephens, D.J. and Austen, B.M. (1996). b-amyloidogenic fragments of the Alzheimer’s b -amyloid precursor protein accumulate in the trans-Golgi network and/or late endosome in 293 cells. J.Neurosci. Res.46, 211-225.

Stephens, D.J. (1995) Review; Apolipoprotein E and Alzheimer’s Disease. J. Royal Soc. Health 116, 41-43. (Invited review).

Books, book chapters and other publications

Stephens, D.J. and Perez, F. (2013) Analysis of Golgi Complex Functions: Methods in Cell Biology. Elsevier, London.

Miller, V.J., MacKinnon, C., Mellor, H., and Stephens, D.J. (2013) RNAi for the analysis of Golgi function. In, Analysis of Golgi Complex Functions: Methods in Cell Biology. Elsevier, London. Eds. Stephens, D.J. and Perez, F.

Stephens, D.J. (2006) An introduction to Cell Imaging. InCell Imaging: Method Express. (Ed. Stephens, D.J.) Scion Publishing, Bloxham, UK.

Palmer, K.J., Watson, P. and Stephens , D.J. (2005) The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus. Biochemical Society Symposia 72, 1-13. (Invited review).

Curnow, P., Mellor, H, Stephens , D.J. and Booth, P.J. (2005) Translocation of the Tat(48-60) peptide across artificial bilayers. Biochemical Society Symposia 72, in press. (Invited review).

Watson, P. Palmer, K.J. and Stephens , D.J. (2005) Immunofluorescence labelling and fluorescent dyes. In, Encyclopaedia of Genetics, Genomics, Proteomics and Bioinformatics. (Eds. Tavaré, J. and Dunn, M.), John Wiley and Sons, London .

Kowalczyk, A.M., Roeder, G.E., Green, K., Stephens, D.J. and Gaston, K. (2005) Measuring the induction or inhibition of apoptosis by HPV proteins. In,Methods in Molecular Biology; Humana Press, Totowa , USA . (Ed. Brady, H.J.M.), Humana Press, Totowa , USA.

Zimmerman, T., Pepperkok, R., Stephens, D., Girod, A. and Rietdorf, J. (2003) Basic Light Microscopy. In, Essential Cell Biology: A Practical Approach. (Ed. J. Davey and J.M. Lord), OUP, London. Available on Amazon.co.uk

Austen, B. and Stephens, D. (1995) Protein Targeting. In, Molecular Biology and Biotechnology, a Comprehensive Desk Reference. (Ed. RA Meyers), VCH Publishers, Cambridge ; pp 774-8.

Stephens, D ., Kulkarni, S. and Austen B. (1995) The Biosynthesis of Membrane Proteins.
In, Biomembranes Vol. 1 (Ed. Lee, A.G.), JAI Press, Connecticut ; pp107-135.

Stephens, D., Kulkarni, S. and Austen, B. (1994) Assembly of Membrane Proteins and their Movement to the Cell Surface. In, Animal Cell Biotechnology, Vol. 6 (Eds. Spier, R.E. and Griffiths , J.B.), Academic Press, London ; pp 24-47.

Stephens, D.J. (2003) Bristol and the organisation of the secretory pathway. Olympus Objectives Autumn 2003.

Stephens, D.J. (2003) Live cell imaging. The Biochemist vol. 25 no.3, pp15-17.

Stephens, D.J. and Pepperkok, R. (2001) Time lapse movies included in GFP In Motion II, a CD-ROM published with Trends in Cell Biology vol. 11(5).

Stephens, D. (2000-2002) “In Brief” articles for Trends in Cell Biology.