Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells.

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.

Cryptococcal Meningitis in an HIV-Negative Puerperal Woman.

Cryptococcal meningitis is a common opportunistic infection in HIV-infected patients and other immunocompromised people. Pregnancy, which is a state of relative immunosuppression, can also be a risk factor for the development of cryptococcal meningitis. We report a clinical case of a 41-year-old woman who developed a severe meningeal syndrome after an otherwise normal pregnancy. Cerebrospinal fluid (CSF) cytochemical analysis presented hypoglycorrhachia, high protein levels, and pleocytosis. Cryptococcal antigen tested positive in serum and CSF, and Cryptococcus neoformans was identified in the CSF culture. The diagnosis of cryptococcal meningitis was confirmed, and antifungal induction therapy was started with liposomal amphotericin B and flucytosine. After clinical improvement, induction therapy was discontinued, and the patient was discharged under maintenance therapy with fluconazole. While under antifungal maintenance therapy, the patient presented worsening of symptoms and a new brain magnetic resonance showed the development of multiple cryptococcoma. Despite sterile CSF cultures, there was a deterioration of the cytochemical parameters. The diagnosis of immune reconstitution inflammatory syndrome was assumed, and after initiation of corticotherapy, the patient improved considerably. This is a rare case of cryptococcal meningitis in a puerperal woman with a challenging management.

Epidermal PAR-6 and PKC-3 are essential for larval development of C. elegans and organize non-centrosomal microtubules.

The cortical polarity regulators PAR-6, PKC-3, and PAR-3 are essential for the polarization of a broad variety of cell types in multicellular animals. In C. elegans, the roles of the PAR proteins in embryonic development have been extensively studied, yet little is known about their functions during larval development. Using inducible protein degradation, we show that PAR-6 and PKC-3, but not PAR-3, are essential for postembryonic development. PAR-6 and PKC-3 are required in the epidermal epithelium for animal growth, molting, and the proper pattern of seam-cell divisions. Finally, we uncovered a novel role for PAR-6 in organizing non-centrosomal microtubule arrays in the epidermis. PAR-6 was required for the localization of the microtubule organizer NOCA-1/Ninein, and defects in a noca-1 mutant are highly similar to those caused by epidermal PAR-6 depletion. As NOCA-1 physically interacts with PAR-6, we propose that PAR-6 promotes non-centrosomal microtubule organization through localization of NOCA-1/Ninein.

Mapping the Polarity Interactome.

Interactions between proteins are an essential part of biology, and the desire to identify these interactions has led to the development of numerous technologies to systematically map protein-protein interactions at a large scale. As in most cellular processes, protein interactions are central to the control of cell polarity, and a full understanding of polarity will require comprehensive knowledge of the protein interactions involved. At its core, cell polarity is established through carefully regulated mutually inhibitory interactions between several groups of cortical proteins. While several interactions have been identified, the dynamics and molecular mechanisms that control these interactions are not well understood. Cell polarity also needs to be integrated with cellular processes including junction formation, cytoskeletal organization, organelle positioning, protein trafficking, and functional specialization of membrane domains. Moreover, polarized cells need to respond to external cues that coordinate polarity at the tissue level. Identifying the protein-protein interactions responsible for integrating polarity with all of these processes remains a major challenge, in part because the mechanisms of polarity control vary in different contexts and with developmental times. Because of their unbiased nature, systematic large-scale protein-protein interaction mapping approaches can be particularly helpful to identify such mechanisms. Here, we discuss methods commonly used to generate proteome-wide interactome maps, with an emphasis on advances in our understanding of cell polarity that have been achieved through application of such methods.

Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly.

The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod-Zw10-Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod's N-terminal ß-propeller and the associated Zwilch subunit bind Spindly's C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod ß-propeller-Zwilch complex in vitro. Spindly's N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein-dynactin-Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein-dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.

The ULTRAPETALA1 trxG factor contributes to patterning the Arabidopsis adaxial-abaxial leaf polarity axis.

The SAND domain protein ULTRAPETALA1 (ULT1) functions as a trithorax group factor that regulates a variety of developmental processes in Arabidopsis. We have recently shown that ULT1 regulates developmental patterning in the gynoecia and leaves. ULT1 acts together with the KANADI1 (KAN1) transcription factor to pattern the apical-basal axis during gynoecium formation, whereas the 2 genes act antagonistically to pattern the adaxial-abaxial axis during both gynoecium and leaf formation. In particular, our data showed that ULT1 is necessary for the kan1 adaxialized organ phenotype. Here, we observe the internal structure of ult1, kan1 and ult1 kan1 rosette leaves to better understand the suppression of the kan1 adaxialized leaf polarity defect by ult1 mutations. Our results indicate that ULT1 and KAN1 act antagonistically to pattern the adaxial-abaxial axis in leaves by establishing the asymmetry of the internal cell layers.

ULTRAPETALA trxG genes interact with KANADI transcription factor genes to regulate Arabidopsis gynoecium patterning.

Organ formation relies upon precise patterns of gene expression that are under tight spatial and temporal regulation. Transcription patterns are specified by several cellular processes during development, including chromatin remodeling, but little is known about how chromatin-remodeling factors contribute to plant organogenesis. We demonstrate that the trithorax group (trxG) gene ULTRAPETALA1 (ULT1) and the GARP transcription factor gene KANADI1 (KAN1) organize the Arabidopsis thaliana gynoecium along two distinct polarity axes. We show that ULT1 activity is required for the kan1 adaxialized polarity defect, indicating that ULT1 and KAN1 act oppositely to regulate the adaxial-abaxial axis. Conversely, ULT1 and KAN1 together establish apical-basal polarity by promoting basal cell fate in the gynoecium, restricting the expression domain of the basic helix-loop-helix transcription factor gene SPATULA. Finally, we show that ult alleles display dose-dependent genetic interactions with kan alleles and that ULT and KAN proteins can associate physically. Our findings identify a dual role for plant trxG factors in organ patterning, with ULT1 and KAN1 acting antagonistically to pattern the adaxial-abaxial polarity axis but jointly to pattern the apical-basal axis. Our data indicate that the ULT proteins function to link chromatin-remodeling factors with DNA binding transcription factors to regulate target gene expression.

The ULT1 and ULT2 trxG genes play overlapping roles in Arabidopsis development and gene regulation.

The epigenetic regulation of gene expression is critical for ensuring the proper deployment and stability of defined genome transcription programs at specific developmental stages. The cellular memory of stable gene expression states during animal and plant development is mediated by the opposing activities of Polycomb group (PcG) factors and trithorax group (trxG) factors. Yet, despite their importance, only a few trxG factors have been characterized in plants and their roles in regulating plant development are poorly defined. In this work, we report that the closely related Arabidopsis trxG genes ULTRAPETALA1 (ULT1) and ULT2 have overlapping functions in regulating shoot and floral stem cell accumulation, with ULT1 playing a major role but ULT2 also making a minor contribution. The two genes also have a novel, redundant activity in establishing the apical­basal polarity axis of the gynoecium, indicating that they function in differentiating tissues. Like ULT1 proteins, ULT2 proteins have a dual nuclear and cytoplasmic localization, and the two proteins physically associate in planta. Finally, we demonstrate that ULT1 and ULT2 have very similar overexpression phenotypes and regulate a common set of key development target genes, including floral MADS-box genes and class I KNOX genes. Our results reveal that chromatin remodeling mediated by the ULT1 and ULT2 proteins is necessary to control the development of meristems and reproductive organs. They also suggest that, like their animal counterparts, plant trxG proteins may function in multi-protein complexes to up-regulate the expression of key stage- and tissue-specific developmental regulatory genes.

Analyzing floral meristem development.

Flowers contain the male and female sexual organs that are critical for plant reproduction and survival. Each individual flower is produced from a floral meristem that arises on the flank of the shoot apical meristem and consists of four organ types: sepals, petals, stamens, and carpels. Because floral meristems contain a transient stem-cell pool that generates a small number of organs composed of a limited number of cell types, they are excellent model systems for studying stem-cell maintenance and termination, cell fate specification, organ morphogenesis, and pattern formation.

Phylogenetic relationship of potato CAT1 and CAT2 genes, their differential expression in non-photosynthetic organs and during leaf development, and their association with different cellular processes.

Plants contain multiple forms of catalase (CAT) and their specific functions remain uncertain. We cloned two potato cDNAs corresponding to CAT1 and CAT2 genes, analysed their phylogenetic relationship, and studied their expression and activity in different organs to gain clues to their functions. Phylogenetic trees and the alignment of CAT cDNA sequences provided evidence that CAT1 and CAT2 genes have high identity to catalases of other solanaceous species, but are not phylogenetically closely related to one another, which contradicts the phylogenetic closeness ascribed to these genes. Northern blot analyses revealed that expression of CAT genes is controlled by leaf developmental phase. CAT2 expression was higher in both very young and senescent leaves, whereas CAT1 mRNA accumulated mainly in mature leaf, where the lowest CAT2 expression occurred. CAT1 and CAT2 are also differentially expressed in root, sprout and petal. Expression and activity patterns are consistent with different physiological roles for CAT1 and CAT2 isoforms. CAT1 is considered to be associated with photorespiration whereas CAT2 would fulfill physiological roles unrelated to this process. CAT2 appears to be a multifunctional isoform, associated with glyoxysomal activity in leaf senescence, other processes in non-photosynthetic organs and defence, functions that in other solanaceous species are fulfilled by two different isoforms.

Effect of hydrogen peroxide on catalase gene expression, isoform activities and levels in leaves of potato sprayed with homobrassinolide and ultrastructural changes in mesophyll cells.

The effect of hydrogen peroxide (H2O2) on catalase (CAT) isoform activities and amounts and on mRNA levels was studied in leaves from potato plants untreated and treated with homobrassinolide (HBR). Northern blot analysis revealed that 100 mm H2O2 supplied through the leaf petiole for 4 h did not induce CAT expression. In contrast, CAT1 and CAT2 responded differently to longer treatment, as CAT2 transcript levels increased markedly whereas CAT1 transcript levels remained unchanged. Western blot analysis showed disparity between the level of CAT1 transcript and CAT1 amount, which actually decreased after 28 h. CAT2 amount correlated well with transcript accumulation and CAT2 activity as visualised by zymogram analysis. H2O2 modified the relative importance of CAT isoforms. After 4 h, CAT1 was prevalent in untreated and H2O2-treated leaves. After 28 h, CAT2 was prevalent in H2O2-treated leaves; therefore, the quantified increase in total CAT activity in these leaves was due to the rise in CAT2. HBR pre-treatment increased CAT2 basal level not changing the pattern of CAT responses to H2O2, only lowering its amplitude. Even so, ultrastructural studies showed that HBR significantly reduced H2O2 negative effects on cellular sub-structures, allowing better recovery of affected structures and reducing the macroscopic injury symptoms on leaves, thus data point to a HBR protective role.