The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.

Glycans and glycan-binding proteins are central to a properly functioning immune system. Perhaps the best known example of this is the selectin family of surface proteins that are primarily found on leukocytes, and which bind to endothelial glycans near sites of infection or inflammation and enable extravasation into tissues. In the past decade, however, a number of other immune pathways that are dependent on or sensitive to changes in glycan-mediated mechanisms have been revealed. These include antibody function, apoptosis, Th1 versus Th2 skewing, T cell receptor signaling, and MHC class II antigen-presentation. Here, we highlight how regulated changes in protein glycosylation both at the cell surface and on secreted glycoproteins can positively and negatively modulate the immune response.

Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling.

N-linked glycans on class II MHC molecules are required for the presentation of glycoantigens, but not peptide antigens.

Epitope selection is an important consideration in the design of cancer vaccines, but factors affecting selection are not fully understood. We compared the immune responses to peptides and glycopeptides from the common human tumor antigen MUC1, a mucin that is coated with O-linked carbohydrates in its variable number of tandem repeats (VNTR) region. MUC1 expressed on tumor cells is characteristically underglycosylated, creating peptide and glycopeptide neoepitopes that are recognized by the immune system. The response to VNTR peptides is weaker in MUC1-transgenic mice (MUC1-Tg mice) than in wild-type (WT) mice, whereas the response to VNTR glycopeptides is equally strong in the two strains. Thus, glycopeptides seem to be recognized as foreign, whereas peptides, although immunogenic, are perceived as self. To explore this further, we generated MUC1 peptide-and glycopeptide-specific T-cell receptor transgenic mice and studied the function of their CD4 T cells when adoptively transferred into MUC1-Tg or WT mice. Peptide-specific T-cell precursors were not centrally deleted in MUC1-Tg mice and did not acquire a T regulatory phenotype. However, their response to the cognate peptide was reduced in MUC1-Tg mice compared with WT mice. In contrast, glycopeptide-specific CD4 T cells responded equally well in the two hosts and, when simultaneously activated, also enhanced the peptide-specific T-cell responses. Our data show that the immune system differentially recognizes various epitopes of tumor-associated antigens either as self or as foreign, and this controls the strength of antitumor immunity. This represents an important consideration for designing safe and effective cancer vaccines. Cancer Res; 70(14); 5788-96. ©2010 AACR.

The major histocompatibility complex (MHC) glycoprotein family, also referred to as human leukocyte antigens, present endogenous and exogenous antigens to T lymphocytes for recognition and response. These molecules play a central role in enabling the immune system to distinguish self from non-self, which is the basis for protective immunity against pathogenic infections and disease while at the same time representing a serious obstacle for tissue transplantation. All known MHC family members, like the majority of secreted, cell surface, and other immune-related molecules, carry asparagine (N)-linked glycans. The immune system has evolved increasing complexity in higher-order organisms along with a more complex pattern of protein glycosylation, a relationship that may contribute to immune function beyond the early protein quality control events in the endoplasmic reticulum that are commonly known. The broad MHC family maintains peptide sequence motifs for glycosylation at sites that are highly conserved across evolution, suggesting importance, yet functional roles for these glycans remain largely elusive. In this review, we will summarize what is known about MHC glycosylation and provide new insight for additional functional roles for this glycoprotein modification in mediating immune responses.

Human adenocarcinomas overexpress a hypoglycosylated, tumor-associated form of the mucin-like glycoprotein MUC1 containing abnormal mono- and disaccharide antigens, such as Tn, sialyl-Tn, and TF, as well as stretches of unglycosylated protein backbone in the variable number of tandem repeats (VNTR) region. Both peptide and glycopeptide epitopes generated from the VNTR are candidates for cancer vaccines and we performed experiments to evaluate their relative potential to elicit tumor-MUC1-specific immunity. We show here that immunization with the 100 amino acid-long VNTR peptide (MUC1p) elicits weaker responses in MUC1 transgenic mice compared to wild type mice suggesting self-tolerance. In contrast, when glycosylated with tumor-associated Tn antigen (GalNAc-O-S/T), TnMUC1 induces glycopeptide-specific T cell and antibody responses in both strains of mice and helps enhance responses to MUC1p in MUC1 transgenic mice. Using newly derived MUC1-specific mouse T cell hybridomas we show that the only antigen-presenting cells able to cross-present TnMUC1 glycopeptide are dendritic cells (DCs). This is likely due to their exclusive expression of receptors capable of binding TnMUC1. We conclude that MUC1 glycopeptides induce stronger immunity in MUC1-Tg mice because they are recognized as ‘foreign’ rather than ‘self’ and because they are cross-presented preferentially by DCs.

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pull down is lost by mutation of the activation domain.