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ER-to-Golgi trafficking of procollagen in the absence of large carriers.

Janine McCaughey, Nicola Stevenson, Stephen Cross, David Stephens

Preprint posted on June 05, 2018 https://www.biorxiv.org/content/early/2018/06/05/339804

Light at the end of the tunnel? A new reporter for pulsed collagen secretion provides support for a direct route from ER to Golgi

Selected by Gautam Dey

Context

Multicellularity brings with it myriad advantages but also a problem of scales- millimetre-scale tissue architecture must be wrought by cells themselves often only tens of microns across. Nowhere is this more evident than at one of the early steps in the creation of the extra-cellular matrix (ECM). Collagen, typically the major ECM component (and the most abundant protein in the human body), is folded and processed in the ER and subsequently the Golgi before transport to the cell surface1.

The first steps in collagen secretion at ER exit sites are orchestrated by a protein called TANGO12 and its interactors, with the participation of the canonical ER-Golgi carrier coat protein complex COPII. One major wrinkle in the story, though: ER-localised procollagen takes the form of a gigantic, rigid rod 300 nm long. How on earth do procollagens fit into COPII-coated vesicles less than 100 nm across? Competing models for how this might reasonably occur- giant vesicular carriers3 or direct tunnels/channels1,4,5 between the ERGIC and Golgi- have both received some experimental support.

 

Key advance

The authors designed a novel live-cell reporter for human COL1A1, placing an N-terminal monomeric GFP tag upstream of the pro-peptide cleavage site (to eliminate signals from extracellular collagen) in combination with a Streptavidin Binding Peptide (SBP, to utilise in combination with an ER-resident streptavidin “hook”6) and selecting for stable, low expression. The critical assay involved monitoring synchronised pulses of collagen transport by confocal imaging from ER to Golgi by treating cells with ascorbate (to assist in collagen folding) and biotin (to release the COL1A1 from the hook).

The authors were able to track emptying of the ER and filling of the Golgi on a 20-30-minute timescale with high time and spatial resolution but were unable to detect any large vesicular carriers. Any large (or small) COL1A1 labelled structures not classified as bona-fide Golgi colocalised only with ER markers.

Figure 1: Panels reproduced from Figure 2D of McCaughey et al., 2018 under a CC-BY-NC-ND 4.0 international license, representing the collagen reporter (GFP-COL) and a trans-Golgi marker (ST-Cherry) during a pulse-chase assay.

 

 

Why I chose this preprint  

Recent advances in light microscopy and labelling techniques7 will have a broad impact on the way we study the cell biology of cargo flux between dynamic eukaryotic membrane compartments. Super-resolution imaging (albeit largely in fixed cells) has already made its mark on the collagen secretion field3,8. This preprint seems like another step in the right direction!

 

Questions for the future?

I was fascinated by the way the filling of the Golgi with collagen (as in Figure 2D excerpted above and in many other examples in the preprint) appears to actually physically reshape the Golgi stacks over time. Could the authors’ assay double up as a way to investigate the role of cargo in determining compartment structure, and could such experiments be aided by a more quantitative approach to tracking the dynamic evolution of compartments in space (e.g. machine learning)?

 

References:

  1. Malhotra, V. & Erlmann, P. The Pathway of Collagen Secretion. Annu. Rev. Cell Dev. Biol. 31, 109–124 (2015).
  2. Saito, K. et al. TANGO1 Facilitates Cargo Loading at Endoplasmic Reticulum Exit Sites. Cell 136, 891–902 (2009).
  3. Gorur, A. et al. COPII-coated membranes function as transport carriers of intracellular procollagen I. J. Cell Biol. 216, 1745–1759 (2017).
  4. Beznoussenko, G. V et al. Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae. Elife 3, (2014).
  5. Kurokawa, K., Okamoto, M. & Nakano, A. Contact of cis-Golgi with ER exit sites executes cargo capture and delivery from the ER. Nat. Commun. 5, 3653 (2014).
  6. Boncompain, G. et al. Synchronization of secretory protein traffic in populations of cells. Nat. Methods 9, 493–498 (2012).
  7. Pickard, A. et al. Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag. bioRxiv 331496 (2018). doi:10.1101/331496
  8. Raote, I. et al. TANGO1 assembles into rings around COPII coats at ER exit sites. J. Cell Biol. 216, 901–909 (2017).

Tags: er-golgi transport, membrane trafficking

Posted on: 26th July 2018 , updated on: 30th July 2018

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  • Author's response

    Janine McCaughey, Nicola Stevenson, and David Stephens shared

    We appreciate Gautam highlighting our work. We do think that the new biology that we define in this paper will be of broad interest. Our new controllable trafficking tool provides an opportunity to define many different aspects of procollagen trafficking. The observation that the trafficking of our mGFP-procollagen through the Golgi might “physically reshape the Golgi stacks over time” is very intriguing. The Golgi is highly dynamic and so we are not drawing that conclusion at present. This would of course require extensive analysis by both light and electron microscopy and we are hoping to work towards that in the near future. Certainly, the possibility of applying machine learning to such image analysis methods is an exciting opportunity and we would love to talk to anyone working in that area.

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