V Malhotra is an Institucio Catalana de Recerca i Estudis Avancats professor at the Centre for Genomic Regulation, the work in his laboratory is funded by grants from the Ministerio de Economia, Industria y Competitividad Plan Nacional (ref. BFU2013-44188-P) and Consolider (CSD2009-00016). We acknowledge support of the Spanish Ministry of Economy and Competitiveness, through the Programmes ‘Centro de Excelencia Severo Ochoa 2013–2017’ (SEV-2012–0208) and Maria de Maeztu Units of Excellence in R and D (MDM-2015–0502). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya. F Campelo and M Garcia-Parajo acknowledge support by the Spanish Ministry of Economy and Competitiveness (‘Severo Ochoa’ Programme for Centres of Excellence in R and D (SEV-2015–240522) and FIS2014-56107-R), BFU2015-73288-JIN, AEI/FEDER; UE, Fundacion Privada Cellex, HFSP (GA RGP0027/2012), EC FP7-NANO-VISTA (GA 288263) and LaserLab 4 Europe (GA 654148). I. Raote and F. Campelo acknowledge support by the BIST Ignite Grant (eTANGO).

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TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes

ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Osteoporosis is metabolic bone disease caused by an altered balance between bone anabolism and catabolism. This dysregulated balance is responsible for fragile bones that fracture easily after minor falls. With an ageing population, the incidence is rising and as yet pharmaceutical options to restore this imbalance is limited, especially stimulating osteoblast bone-building activity. Excitingly, output from large genetic studies on people with high bone mass (HBM) cases and genome wide association studies (GWAS) on the population, yielded new insights into pathways containing osteo-anabolic players that have potential for drug target development. However, a bottleneck in development of new treatments targeting these putative osteo-anabolic genes is the lack of animal models for rapid and affordable testing to generate functional data and that simultaneously can be used as a compound testing platform. Zebrafish, a small teleost fish, are increasingly used in functional genomics and drug screening assays which resulted in new treatments in the clinic for other diseases. In this review we outline the zebrafish as a powerful model for osteoporosis research to validate potential therapeutic candidates, describe the tools and assays that can be used to study bone homeostasis, and affordable (semi-)high-throughput compound testing.

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Zebrafish as an emerging model for osteoporosis: a primary testing platform for screening new osteo-active compounds

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.

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COPII-coated membranes function as transport carriers of intracellular procollagen I.

Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the biology of collagen prolyl 4-hydroxylases and collagen prolyl 3-hydroxylases, the enzymes responsible for proline hydroxylation. Furthermore, we describe the potential roles of hydroxyproline residues in the complex interplay between collagens and other proteins, especially integrin and discoidin domain receptor type cell adhesion receptors. Qualitative and quantitative regulation of collagen hydroxylation may have remarkable effects on the properties of the extracellular matrix and consequently on the cell behaviour.

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Role of prolyl hydroxylation in the molecular interactions of collagens

Secretion and assembly of collagen are fundamental to the function of the extracellular matrix. Defects in the assembly of a collagen matrix lead to pathologies including fibrosis and osteogenesis imperfecta. Owing to the size of fibril-forming procollagen molecules it is assumed that they are transported from the endoplasmic reticulum to the Golgi in specialized large COPII-dependent carriers. Here, analyzing endogenous procollagen and a new engineered GFP-tagged form, we show that transport to the Golgi occurs in the absence of large (>350 nm) carriers. Large GFP-positive structures were observed occasionally, but these were nondynamic, are not COPII positive, and are labeled with markers of the ER. We propose a short-loop model of COPII-dependent ER-to-Golgi traffic that, while consistent with models of ERGIC-dependent expansion of COPII carriers, does not invoke long-range trafficking of large vesicular structures. Our findings provide an important insight into the process of procollagen trafficking and reveal a short-loop pathway from the ER to the Golgi, without the use of large carriers.

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

https://research-information.bris.ac.uk/files/72475353/PIIS221112471630506X.pdf

TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

The export of newly synthesized proteins from the endoplasmic reticulum is fundamental to the ongoing maintenance of cell and tissue structure and function. After co-translational translocation into the ER, proteins destined for downstream intracellular compartments or secretion from the cell are sorted and packaged into transport vesicles by the COPII coat protein complex. The fundamental discovery and characterization of the pathway has now been augmented by a greater understanding of the role of COPII in diverse aspects of cell function. We now have a deep understanding of how COPII contributes to the trafficking of diverse cargoes including extracellular matrix molecules, developmental signalling proteins, and key metabolic factors such as lipoproteins. Structural and functional studies have shown that the COPII coat is both highly flexible and subject to multiple modes of regulation. This has led to new discoveries defining roles of COPII in development, autophagy, and tissue organization. Many of these newly emerging features of the canonical COPII pathway are placed in a context of procollagen secretion because of the fundamental interest in how a coat complex that typically generates 80-nm transport vesicles can package a cargo reported to be over 300 nm. Here we review the current understanding of COPII and assess the current consensus on its role in packaging diverse cargo proteins.

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COPII-dependent ER export in animal cells: adaptation and control for diverse cargo

https://research-information.bris.ac.uk/ws/files/213783931/TCB_final_author_version_2_.pdf

ER-to-Golgi Transport: A Sizeable Problem

Background Cole-Carpenter syndrome (CCS) is commonly classified as a rare Osteogenesis Imperfecta (OI) disorder. This was following the description of two unrelated patients with very similar phenotypes who were subsequently shown to have a heterozygous missense mutation in P4HB . Objectives Here, we report a 3-year old female patient with severe OI who on exome sequencing was found to carry the same missense mutation in P4HB as reported in the original cohort. We discuss the genetic heterogeneity of CCS and underlying mechanism of P4HB in collagen production. Methods We undertook detailed clinical, radiological and molecular phenotyping in addition, to analysis of collagen in cultured fibroblasts and electron microscopic examination in the patient reported here. Results The clinical phenotype appears consistent in patients reported so far but interestingly, there also appears to be a definitive phenotypic clue (crumpling metadiaphyseal fractures of the long tubular bones with metaphyseal sclerosis which are findings that are uncommon in OI) to the underlying genotype ( P4HB variant). Discussion P4HB (Prolyl 4-hydroxylase, betasubunit) encodes for PDI (Protein Disulfide isomerase) and in cells, in its tetrameric form, catalyses formation of 4-hydroxyproline in collagen. The recurrent variant in P4HB , c.1178A>G, p.Tyr393Cys, sits in the C-terminal reactive centre and is said to interfere with disulphide isomerase function of the C-terminal reactive centre. P4HB catalyses the hydroxylation of proline residues within the X-Pro-Gly repeats in the procollagen helical domain. Given the inter-dependence of extracellular matrix (ECM) components in assembly of a functional matrix, our data suggest that it is the organisation and assembly of the functional ECM that is perturbed rather than the secretion of collagen type I per se. Conclusions We provide additional evidence of P4HB as a cause of a specific form of OI-CCS and expand on response to treatment with bisphosphonates in this rare disorder.

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Janine McCaughey

Papers

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

TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

COPII-dependent ER export in animal cells: adaptation and control for diverse cargo

ER-to-Golgi Transport: A Sizeable Problem

P4HB recurrent missense mutation causing Cole-Carpenter syndrome.