Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

Scaffold-mediated symmetry breaking by Cdc42p

Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking. In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally. The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization. We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments. Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour. Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch.     

Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage

The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.     

An image analysis toolbox for high-throughput C. elegans assays

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.     

EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways

Understanding host defense against microbes is key to developing new and more effective therapies for infection and inflammatory disease. However, how animals integrate multiple environmental signals and discriminate between different pathogens to mount specific and tailored responses remains poorly understood. Using the genetically tractable model host Caenorhabditis elegans and pathogenic bacterium Staphylococcus aureus, we describe an important role for hypoxia-inducible factor (HIF) in defining the specificity of the host response in the intestine. We demonstrate that loss of egl-9, a negative regulator of HIF, confers HIF-dependent enhanced susceptibility to S. aureus while increasing resistance to Pseudomonas aeruginosa. In our attempt to understand how HIF could have these apparently dichotomous roles in host defense, we find that distinct pathways separately regulate two opposing functions of HIF: the canonical pathway is important for blocking expression of a set of HIF-induced defense genes, whereas a less well understood noncanonical pathway appears to be important for allowing the expression of another distinct set of HIF-repressed defense genes. Thus, HIF can function either as a gene-specific inducer or repressor of host defense, providing a molecular mechanism by which HIF can have apparently opposing roles in defense and inflammation. Together, our observations show that HIF can set the balance between alternative pathogen-specific host responses, potentially acting as an evolutionarily conserved specificity switch in the host innate immune response.     

The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.     

Fine carbohydrate recognition of Euphorbia milii lectin

Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates.     

Correlative fine specificity of several Thomsen-Friedenreich disaccharide-binding proteins with an effect on tumor cell proliferation.

Epithelial cancer cells show increased cell surface expression of mucin antigens with aberrant O-glycosylation, notably type I core (Galbeta1-3GalNAcalpha), termed Thomsen-Friedenreich disaccharide (TFD), a chemically well-defined carbohydrate antigen with a proven link to malignancy. Several TFD-binding proteins influence the proliferation of cells to which they bind. We studied the fine specificity of TFD-binding proteins and its relationship with epithelial tumor cell proliferation. Competitive binding assays against asialoglycophorin showed that Agaricus bisporus lectin (ABL) and human anti-TFD monoclonal antibody (mAb) TF1 were inhibited only by TFD and its alpha-derivatives. Peanut agglutinin (PNA), mAb TF2, and mAb TF5 were also inhibited by other carbohydrates such as lacto-N-biose (Galbeta1-3GlcNAc), lactose, and (Mealpha or beta) Gal, indicating lower recognition of the axial C-4 hydroxyl group position of GalNAc from TFD, and the major relevance of the terminal Gal on interaction of these three TFD-binding proteins. In the direct glycolipid-binding assay, ABL bound mostly to alpha-anomeric TFD-bearing glycolipids, whereas PNA interacted mainly with beta-linked TFD. Of the three anti-TFD mAbs analyzed, all bound N5b (terminal beta-TFD), but only TF2 interacted with N6 (terminal alpha-TFD). These findings indicate that TFD-binding proteins that stimulate the proliferation of epithelial tumor cell lines recognize mainly a terminal beta-Gal region of beta-linked TFD, whereas ABL, which inhibits the proliferation of these tumor cells, binds mainly to subterminal GalNAc of alpha-anomeric TFD.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.