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).

/pdf/tango1-builds-a-machine-for-collagen-export-by-recruiting-vugy9bu0se.pdf

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.

/pdf/zebrafish-as-an-emerging-model-for-osteoporosis-a-primary-2mx74i5wwp.pdf

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.

/pdf/copii-coated-membranes-function-as-transport-carriers-of-587l0vie0l.pdf

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.

/pdf/role-of-prolyl-hydroxylation-in-the-molecular-interactions-1e9gzrsacs.pdf

Role of prolyl hydroxylation in the molecular interactions of collagens

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function.

/pdf/regulator-of-calcineurin-2-is-a-centriolar-protein-with-a-4n2jx535vx.pdf

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER-Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.

/pdf/a-general-role-for-tango1-encoded-by-mia3-in-secretory-44sl4qwdsy.pdf

A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function.

Secretion and assembly of collagen is 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 specialised large COPII-dependent carriers. Here, analysing endogenous procollagen and a new engineered GFP-tagged form, we show that transport to the Golgi occurs in the absence of large carriers. Large GFP-positive structures are observed occasionally but these are non-dynamic, are not COPII-positive, and label with markers of the ER. We propose a short-loop model of 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.

https://www.biorxiv.org/content/early/2018/06/05/339804.full.pdf

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

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies characterised by debilitating developmental defects affecting many tissues. Here we report a new role for regulator of calcineurin 2, RCAN2 in primary cilia function. It localises to centrioles and cilia and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia on serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in cilia length control seen on RNAi of giantin itself. Together these data define RCAN2 as a regulator of cilia function that can compensate for loss of giantin function.

https://www.biorxiv.org/content/biorxiv/early/2018/04/04/188946.full.pdf

Background

With increased access to genetic testing, variants of uncertain significance (VUS) where pathogenicity is uncertain are being increasingly identified. More than 85% Osteogenesis Imperfecta (OI) patients have pathogenic variants in COL1A1/A2. However, when a VUS is identified, there are no pathways in place for determining significance.



Objective

Define a diagnostic pathway to confirm pathogenicity, providing patients with definitive genetic diagnosis, accurate recurrence risks, and prenatal testing options.



Methods

Functional studies on collagen secretion from cultured patient fibroblasts combined with detailed phenotyping and segregation family studies.



Results

We demonstrate data from a family with a VUS identified in type I collagen.



Family‐1

Six‐year‐old boy with failure‐to‐gain weight, talipes, fractures, on and off treatment with Pamidronate as diagnosis of OI uncertain. Transiliac bone biopsy at 2 years of age demonstrated active new bone formation within periosteum; bone cortices were normal thickness but increased porosity. Trabecular bone showed features of advanced osteoporosis. Genetic testing identified a de novo COL1A1 c.206_208delTGT, p.Leu69del variant. Sibling with similar phenotype but no fractures as yet, tested positive for variant raising concerns regarding her diagnosis, and management.



Results from three independent experiments (cell immunofluorescence, collagen secretion assay by Western Blot, and unbiased proteomics) from cultured patient fibroblasts demonstrate COL1A1 c.206_208delTGT, p.Leu69del variant causing a substantial defect to collagen extracellular matrix assembly confirming variant pathogenicity.



Conclusion

Access to genetic testing in OI is increasing as advances in genetic technologies decreases cost; a clinical diagnostic pathway needs to be implemented for managing variants identified by such testing.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/mgg3.912

Janine McCaughey

Papers

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control

A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function.

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

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control

Developing Pathways to Clarify Pathogenicity of Unclassified Variants in Osteogenesis Imperfecta Genetic Analysis