TAPT1-at the crossroads of extracellular matrix and signaling in Osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary skeletal disorder primarily affecting collagen type I structure and function, causing bone fragility and occasionally versatile extraskeletal symptoms. This study expands the spectrum of OI-causing TAPT1 mutations and links extracellular matrix changes to signaling regulation.

The factory, the antenna and the scaffold: the three-way interplay between the Golgi, cilium and extracellular matrix underlying tissue function.

The growth and development of healthy tissues is dependent on the construction of a highly specialised extracellular matrix (ECM) to provide support for cell growth and migration and to determine the biomechanical properties of the tissue. These scaffolds are composed of extensively glycosylated proteins which are secreted and assembled into well-ordered structures that can hydrate, mineralise, and store growth factors as required. The proteolytic processing and glycosylation of ECM components is vital to their function. These modifications are under the control of the Golgi apparatus, an intracellular factory hosting spatially organised, protein-modifying enzymes. Regulation also requires a cellular antenna, the cilium, which integrates extracellular growth signals and mechanical cues to inform ECM production. Consequently, mutations in either Golgi or ciliary genes frequently lead to connective tissue disorders. The individual importance of each of these organelles to ECM function is well-studied. However, emerging evidence points towards a more tightly linked system of interdependence between the Golgi, cilium and ECM. This review examines how the interplay between all three compartments underpins healthy tissue. As an example, it will look at several members of the golgin family of Golgi-resident proteins whose loss is detrimental to connective tissue function. This perspective will be important for many future studies looking to dissect the cause and effect of mutations impacting tissue integrity.

Multiple interactions of the dynein-2 complex with the IFT-B complex are required for effective intraflagellar transport.

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60 (also known as DYNC2I1) and the DYNC2H1-DYNC2LI1 dimer from dynein-2, and IFT54 (also known as TRAF3IP1) and IFT57 from IFT-B contribute to the dynein-2-IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection.

Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2.

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. The motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport, respectively, in the cilium. WDR34 (also known as DYNC2I2), a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome and asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases, making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34-knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity and Hedgehog signalling were also affected.

Supply chain logistics - the role of the Golgi complex in extracellular matrix production and maintenance.

The biomechanical and biochemical properties of connective tissues are determined by the composition and quality of their extracellular matrix. This, in turn, is highly dependent on the function and organisation of the secretory pathway. The Golgi complex plays a vital role in directing matrix output by co-ordinating the post-translational modification and proteolytic processing of matrix components prior to their secretion. These modifications have broad impacts on the secretion and subsequent assembly of matrix components, as well as their function in the extracellular environment. In this Review, we highlight the role of the Golgi in the formation of an adaptable, healthy matrix, with a focus on proteoglycan and procollagen secretion as example cargoes. We then discuss the impact of Golgi dysfunction on connective tissue in the context of human disease and ageing.

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

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.

Giantin is required for intracellular N-terminal processing of type I procollagen.

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

Cytoplasmic dynein-2 at a glance.

Cytoplasmic dynein-2 is a motor protein complex that drives the movement of cargoes along microtubules within cilia, facilitating the assembly of these organelles on the surface of nearly all mammalian cells. Dynein-2 is crucial for ciliary function, as evidenced by deleterious mutations in patients with skeletal abnormalities. Long-standing questions include how the dynein-2 complex is assembled, regulated, and switched between active and inactive states. A combination of model organisms, in vitro cell biology, live-cell imaging, structural biology and biochemistry has advanced our understanding of the dynein-2 motor. In this Cell Science at a Glance article and the accompanying poster, we discuss the current understanding of dynein-2 and its roles in ciliary assembly and function.

Planar Cell Polarity Effector Proteins Inturned and Fuzzy Form a Rab23 GEF Complex.

A subset of Rab GTPases have been implicated in cilium formation in cultured mammalian cells [1-6]. Rab11 and Rab8, together with their GDP-GTP exchange factors (GEFs), TRAPP-II and Rabin8, promote recruitment of the ciliary vesicle to the mother centriole and its subsequent maturation, docking, and fusion with the cell surface [2-5]. Rab23 has been linked to cilium formation and membrane trafficking at mature cilia [1, 7, 8]; however, the identity of the GEF pathway activating Rab23, a member of the Rab7 subfamily of Rabs, remains unclear. Longin-domain-containing complexes have been shown to act as GEFs for Rab7 subfamily GTPases [9-12]. Here, we show that Inturned and Fuzzy, proteins previously implicated as planar cell polarity (PCP) effectors and in developmentally regulated cilium formation [13, 14], contain multiple longin domains characteristic of the Mon1-Ccz1 family of Rab7 GEFs and form a specific Rab23 GEF complex. In flies, loss of Rab23 function gave rise to defects in planar-polarized trichome formation consistent with this biochemical relationship. In cultured human and mouse cells, Inturned and Fuzzy localized to the basal body and proximal region of cilia, and cilium formation was compromised by depletion of either Inturned or Fuzzy. Cilium formation arrested after docking of the ciliary vesicle to the mother centriole but prior to axoneme elongation and fusion of the ciliary vesicle and plasma membrane. These findings extend the family of longin domain GEFs and define a molecular activity linking Rab23-regulated membrane traffic to cilia and planar cell polarity.

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

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.

Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function.

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

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

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.

Giantin-knockout models reveal a feedback loop between Golgi function and glycosyltransferase expression.

The Golgi is the cellular hub for complex glycosylation, controlling accurate processing of complex proteoglycans, receptors, ligands and glycolipids. Its structure and organisation are dependent on golgins, which tether cisternal membranes and incoming transport vesicles. Here, we show that knockout of the largest golgin, giantin, leads to substantial changes in gene expression but only limited effects on Golgi structure. Notably, 22 Golgi-resident glycosyltransferases, but not glycan-processing enzymes or the ER glycosylation machinery, are differentially expressed following giantin ablation. This includes near-complete loss of function of GALNT3 in both mammalian cell and zebrafish models. Giantin-knockout zebrafish exhibit hyperostosis and ectopic calcium deposits, recapitulating phenotypes of hyperphosphatemic familial tumoral calcinosis, a disease caused by mutations in GALNT3. These data reveal a new feature of Golgi homeostasis: the ability to regulate glycosyltransferase expression to generate a functional proteoglycome.

Clathrin-mediated post-fusion membrane retrieval influences the exocytic mode of endothelial Weibel-Palade bodies.

Weibel-Palade bodies (WPBs), the storage organelles of endothelial cells, are essential to normal haemostatic and inflammatory responses. Their major constituent protein is von Willebrand factor (VWF) which, following stimulation with secretagogues, is released into the blood vessel lumen as large platelet-catching strings. This exocytosis changes the protein composition of the cell surface and also results in a net increase in the amount of plasma membrane. Compensatory endocytosis is thought to limit changes in cell size and retrieve fusion machinery and other misplaced integral membrane proteins following exocytosis; however, little is known about the extent, timing, mechanism and precise function of compensatory endocytosis in endothelial cells. Using biochemical assays, live-cell imaging and correlative spinning-disk microscopy and transmission electron microscopy assays we provide the first in-depth high-resolution characterisation of this process. We provide a model of compensatory endocytosis based on rapid clathrin- and dynamin-mediated retrieval. Inhibition of this process results in a change of exocytic mode: WPBs then fuse with previously fused WPBs rather than the plasma membrane, leading, in turn, to the formation of structurally impaired tangled VWF strings.This article has an associated First Person interview with the first authors of the paper.

Corrigendum: Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

This corrects the article DOI: 10.1038/sdata.2017.22.

The Golgi matrix protein giantin is required for normal cilia function in zebrafish.

The Golgi is essential for glycosylation of newly synthesised proteins including almost all cell-surface and extracellular matrix proteoglycans. Giantin, encoded by the golgb1 gene, is a member of the golgin family of proteins that reside within the Golgi stack, but its function remains elusive. Loss of function of giantin in rats causes osteochondrodysplasia; knockout mice show milder defects, notably a cleft palate. In vitro, giantin has been implicated in Golgi organisation, biosynthetic trafficking, and ciliogenesis. Here we show that loss of function of giantin in zebrafish, using either morpholino or knockout techniques, causes defects in cilia function. Giantin morphants have fewer cilia in the neural tube and those remaining are longer. Mutants have the same number of cilia in the neural tube but these cilia are also elongated. Scanning electron microscopy shows that loss of giantin results in an accumulation of material at the ciliary tip, consistent with a loss of function of retrograde intraflagellar transport. Mutants show milder defects than morphants consistent with adaptation to loss of giantin.

Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.

Feasibility of vocal fold abduction and adduction assessment using cine-MRI.

OBJECTIVE: Determine feasibility of vocal fold (VF) abduction and adduction assessment by cine magnetic resonance imaging (cine-MRI) METHODS: Cine-MRI of the VF was performed on five healthy and nine unilateral VF paralysis (UVFP) participants using an axial gradient echo acquisition with temporal resolution of 0.7 s. VFs were continuously imaged with cine-MRI during a 10-s period of quiet respiration and phonation. Scanning was repeated twice within an individual session and then once again at a 1-week interval. Asymmetry of VF position during phonation (VF phonation asymmetry, VFPa) and respiration (VF respiration asymmetry, VFRa) was determined. Percentage reduction in total glottal area between respiration and phonation (VF abduction potential, VFAP) was derived to measure overall mobility. An un-paired t-test was used to compare differences between groups. Intra-session, inter-session and inter-reader repeatability of the quantitative metrics was evaluated using intraclass correlation coefficient (ICC). RESULTS: VF position asymmetry (VFPa and VFRa) was greater (p=0.012; p=0.001) and overall mobility (VFAP) was lower (p=0.008) in UVFP patients compared with healthy participants. ICC of repeatability of all metrics was good, ranged from 0.82 to 0.95 except for the inter-session VFPa (0.44). CONCLUSION: Cine-MRI is feasible for assessing VF abduction and adduction. Derived quantitative metrics have good repeatability. KEY POINTS: • Cine-MRI is used to assess vocal folds (VFs) mobility: abduction and adduction. • New quantitative metrics are derived from VF position and abduction potential. • Cine-MRI able to depict the difference between normal and abnormal VF mobility. • Cine-MRI derived quantitative metrics have good repeatability.

TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion.

Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.