Joanne Chia

Regulation of O-glycosylation through Golgi-to-ER relocation of initiation enzymes

Growth factor stimulation moves O-glycosylation initiation enzymes (GalNac-Ts) from the Golgi to the ER in a Src-dependent fashion, increasing protein O-glycosylation.

RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells

The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome‐ and phosphatome‐wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto‐myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness

Significance How cancer cells become invasive is key to understanding malignancy. Perturbations in O-glycosylation are strongly correlated with invasiveness. Here we report that tumor cells display relocation of O-glycosylation initiating glycosyltransferases from the Golgi apparatus to the endoplasmic reticulum (ER). ER-located O-glycosylation stimulates cell migration and invasiveness, which depend on cell surface O-glycoproteins. Inhibition of the glycosyltransferases in the ER reduces tissue invasion and metastasis formation in mice. Our study suggests that control of glycosylation via the subcellular localization of glycosyltransferases is a critical mechanism driving invasiveness in tumor cells. Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-density glycosylation, is critical for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.

WLS Retrograde Transport to the Endoplasmic Reticulum during Wnt Secretion

Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration

ER O-glycosylation can be induced through relocalisation GalNAc-Transferases from the Golgi. This process markedly stimulates cell migration and is constitutively activated in more than 60% of breast carcinomas. How this activation is achieved remains unclear. Here, we screened 948 signalling genes using RNAi and imaging. We identified 12 negative regulators of O-glycosylation that all control GalNAc-T sub-cellular localisation. ERK8, an atypical MAPK with high basal kinase activity, is a strong hit and is partially localised at the Golgi. Its inhibition induces the relocation of GalNAc-Ts, but not of KDEL receptors, revealing the existence of two separate COPI-dependent pathways. ERK8 down-regulation, in turn, activates cell motility. In human breast and lung carcinomas, ERK8 expression is reduced while ER O-glycosylation initiation is hyperactivated. In sum, ERK8 appears as a constitutive brake on GalNAc-T relocalisation, and the loss of its expression could drive cancer aggressivity through increased cell motility. DOI: http://dx.doi.org/10.7554/eLife.01828.001

VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed us to identify at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes.

Comment on “The GalNAc-T Activation Pathway (GALA) is not a general mechanism for regulating mucin-type O-glycosylation”

In the PLOS ONE article “The GalNAc-T Activation Pathway (GALA) is not a general mechanism for regulating mucin-type O-glycosylation”, Tabak and colleagues argue that they cannot reproduce part of the results we published in 2010 [1]. Specifically, the fact that EGF and PDGF growth factors stimulation of HeLa cells results in a relocation of the O-glycosylation initiation enzymes polypeptide N-acetylgalactosaminyltransferase (GALNTs) from the Golgi apparatus to the endoplasmic reticulum (ER). As O-GalNAc glycosylation concerns a large score of cell surface and secreted proteins and has been shown to affect biological functions in many cases, the question of the regulation of activity of GALNTs enzymes is of significant importance. One aspect of O-glycosylation regulation is the differential gene expression of GALNTs family members. Others have shown the differential glycosylation repertoire of GALNTs and the differential gene expression of some GALNTs family members depending on tissue or developmental stage [2,3]. We were the first to propose that signalling pathways and in particular, the tyrosine kinase Src regulate the GALNTs sub-cellular localisation and can induce a relocation from the Golgi apparatus to the ER. We found relocation to occur for the most abundantly expressed enzymes of the family (GALNT1, -T2, -T3) as well as for GALNT4 and -T6. We showed that this Golgi to ER relocation results in an upregulation of GALNTs activity, hence the name GALNTs Activation (GALA) pathway. GALNTs catalyse the formation of the Tn antigen, formed by a GalNAc residue alphalinked to a Serine or Threonine residue on a polypeptide. These GalNAc residues are usually biosynthetic intermediates for more complex glycans [4]. Tn can be recognised by lectins such as Vicia villosa lectin (VVL) and Helix pomatia lectin (HPL). GALA results in an increase in cellular Tn staining, because more Tn is formed and also because it is not capped in the ER as it is in the Golgi [5]. Over the last months, we have revisited the question of stimulation of GALNTs relocation by growth factors. We have been able to reproduce several of our 2010 results and have identified a couple of potential explanations for the discrepancy with Dr. Tabak’s group results.

Digging deep into Golgi phenotypic diversity with unsupervised machine learning

Structural alterations of the Golgi apparatus may lead to phenotypes that human vision cannot easily discriminate. In this work, we present a high-content analysis framework including an unsupervised clustering step to automatically uncover Golgi phenotypic diversity. We use this deep phenotyping to quantitatively compare the effects of gene depletion.

The GalNAc-T Activation (GALA) Pathway: Drivers and Markers

GALNTs are enzymes adding a GalNAc sugar to Ser and Thr residues in thousands of proteins in the secretory pathway, GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase. GALNTs relocation (aka GALA) drives high Tn levels, occurs frequently in liver tumors and is a key driver of tumor growth. Recently, Tabak and colleagues have contested that EGF stimulation can induce GALNTs relocation. Here, we show that, while moderate and sensitive to culture conditions, relocation induced by EGF is reproducible and detectable even in the images acquired by Tabak et al. EGF induced relocation is enhanced by expression of EGFR and strongly induced by depletion of ERK8. EGF also activates a novel, imaging-independent marker of GALA: the O-glycosylated form of ER resident protein PDIA4. In sum, we demonstrate that non-reproducibility was due to experimental errors and propose specific conditions to facilitate the study of the GALA pathway.