Duy T. Tran

Mucin-Type O-Glycosylation During Development

Mucin-type O-glycosylation is an evolutionarily conserved protein modification present on membrane-bound and secreted proteins. Aberrations in O-glycosylation are responsible for certain human diseases and are associated with disease risk factors. Recent studies have demonstrated essential roles for mucin-type O-glycosylation in protein secretion, stability, processing, and function. Here, we summarize our current understanding of the diverse roles of mucin-type O-glycosylation during eukaryotic development. Appreciating how this conserved modification operates in developmental processes will provide insight into its roles in human disease and disease susceptibilities.

An O-Glycosyltransferase Promotes Cell Adhesion during Development by Influencing Secretion of an Extracellular Matrix Integrin Ligand

Protein secretion and localization are crucial during eukaryotic development, establishing local cell environments as well as mediating cell interactions, signaling, and adhesion. In this study, we demonstrate that the glycosyltransferase, pgant3, specifically modulates integrin-mediated cell adhesion by influencing the secretion and localization of the integrin ligand, Tiggrin. We demonstrate that Tiggrin is normally O-glycosylated and localized to the basal matrix where the dorsal and ventral cell layers adhere in wild type Drosophila wings. In pgant3 mutants, Tiggrin is no longer O-glycosylated and fails to be properly secreted to this basal cell layer interface, resulting in disruption of integrin-mediated cell adhesion in the wing. pgant3-mediated effects are dependent on enzymatic activity, as mutations that form a stable protein yet abrogate O-glycosyltransferase activity result in Tiggrin accumulation within the dorsal and ventral cells comprising the wing. Our results provide the first in vivo evidence for the role of O-glycosylation in the secretion of specific extracellular matrix proteins, thus altering the composition of the cellular “microenvironment” and thereby modulating developmentally regulated cell adhesion events. As alterations in cell adhesion are a hallmark of cancer progression, this work provides insight into the long-standing association between aberrant O-glycosylation and tumorigenesis.

Multiple Members of the UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase Family Are Essential for Viability in Drosophila

Background: Protein O-glycosylation is an evolutionarily conserved modification that is initiated by a family of enzymes. Results: RNA interference to the genes encoding each enzyme of the family identified 4 genes that are essential for viability. Conclusion: Protein O-glycosylation is required for eukaryotic development and viability. Significance: Certain members of this enzyme family serve unique and essential functions in specific tissues during eukaryotic development. Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.

Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo.

The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo.

Glycosylation of α-Dystroglycan O-MANNOSYLATION INFLUENCES THE SUBSEQUENT ADDITION OF GalNAc BY UDP-GalNAc POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASES

Background: Proper glycosylation of α-dystroglycan is a complex process critical for function. Results: Pre-existing O-mannose sites can regulate O-GalNAc addition by the ppGalNAc-Ts. Conclusion: O-Mannosylation has a significant impact on the pattern of another form of glycosylation (O-GalNAc) in α-dystroglycan. Significance: Contributions to disease phenotypes associated with α-dystroglycan O-mannosylation defects may arise from its impact on other types of glycosylation. O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.

Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules.

Real-time insights into regulated exocytosis

ABSTRACT Real-time imaging of regulated exocytosis in secreting organs can provide unprecedented temporal and spatial detail. Here, we highlight recent advances in 3D time-lapse imaging in Drosophila salivary glands at single-granule resolution. Using fluorescently labeled proteins expressed in the fly, it is now possible to image the dynamics of vesicle biogenesis and the cytoskeletal factors involved in secretion. 3D imaging over time allows one to visualize and define the temporal sequence of events, including clearance of cortical actin, fusion pore formation, mixing of the vesicular and plasma membranes and recruitment of components of the cytoskeleton. We will also discuss the genetic tools available in the fly that allow one to interrogate the essential factors involved in secretory vesicle formation, cargo secretion and the ultimate integration of the vesicular and plasma membranes. We argue that the combination of high-resolution real-time imaging and powerful genetics provides a platform to investigate the role of any factor in regulated secretion. Summary: This Commentary focuses on recent developments in three-dimensional time-lapse imaging of regulated secretion occurring in living secreting organs.

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

Mucin-type O-glycosylation is initiated by the UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes. Their activity results in the GalNAc α1-O-Thr/Ser structure, termed the Tn antigen, which is further decorated with additional sugars. In neoplastic cells, the Tn antigen is often overexpressed. Because O-glycosylation is controlled by the activity of GalNAc-Ts, their regulation is of great interest. Previous reports suggest that growth factors, EGF or PDGF, induce Golgi complex-to-endoplasmic reticulum (ER) relocation of both GalNAc-Ts and Tn antigen in HeLa cells, offering a mechanism for Tn antigen overexpression termed “GALA”. However, we were unable to reproduce these findings. Upon treatment of HeLa cells with either EGF or PDGF we observed no change in the co-localization of endogenous GalNAc-T1, GalNAc-T2 or Tn antigen with the Golgi complex marker TGN46. There was also no enhancement of localization with the ER marker calnexin. We conclude that growth factors do not cause redistribution of GalNAc-Ts from the Golgi complex to the ER in HeLa cells.

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3− and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing–based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl− for oxalate and HCO3−, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl−/oxalate exchange. Of note, both Cl−/oxalate exchange and Cl−/HCO3− exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6−/− mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6−/− mice, but HCO3− secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl−/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

Members of the GalNAc-T family of enzymes utilize distinct Golgi localization mechanisms

Mucin-type O-glycosylation is an evolutionarily conserved and essential post-translational protein modification that is initiated in the Golgi apparatus by a family of enzymes known as the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). GalNAc-Ts are type II membrane proteins which contain short N-terminal tails located in the cytoplasm, a transmembrane domain that crosses the Golgi membrane, to which is connected a stem region that tethers the C-terminal catalytic and lectin domains that reside in the Golgi lumen. Although mucin-type O-glycans have been shown to play critical roles in numerous biological processes, little is known about how the GalNAc-Ts are targeted to their site of action within the Golgi complex. Here, we investigate the essential protein domains required for Golgi localization of four representative members of the GalNAc-T family of enzymes. We find that GalNAc-T1 and -T2 require their cytoplasmic tail and transmembrane domains for proper Golgi localization, while GalNAc-T10 requires its transmembrane and luminal stem domains. GalNAc-T7 can use either its cytoplasmic tail or its luminal stem, in combination with its transmembrane domain, to localize to the Golgi. We determined that a single glutamic acid in the GalNAc-T10 cytoplasmic tail inhibits its ability to localize to the Golgi via a cytoplasmic tail-dependent mechanism. We therefore demonstrate that despite their similarity, different members of this enzyme family are directed to the Golgi by more than one set of targeting signals.