A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the “neck filaments” that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

Deciphering enzymes. Genetic selection as a probe of structure and mechanism.

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases. The insights gained from these investigations will aid future enzyme design efforts.     

Deletion of the Candida albicans PIR32 Results in Increased Virulence, Stress Response, and Upregulation of Cell Wall Chitin Deposition

Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.     

Deciphering tumor-suppressor signaling in flies: Genetic link between Scribble/Dlg/Lgl and the Hippo pathways

Loss of apico-basal polarity is one of the crucial factors that drives epithelial tumor progression.scribble/discs large/lethal giant larvae (scrib/dlg/lgl),a group of apico-basal polarity genes,were initially identified as members of “neoplastic” tumor-suppressors in flies.The components of the Hippo signaling pathway,which is crucial for organ size control and cancer development,were also identified through Drosophila genetic screens as members of “hyperplastic” tumor-suppressors.Accumulating evidence in recent studies implies that these two tumor-suppressor signaling pathways are not mutually exclusive but rather cooperatively act to give rise to highly malignant tumors.The interaction of these tumor-suppressor pathways could include deregulations of actin cytoskeleton,cell-cell contact,and apical-domain size of the epithelial cell.     

Deciphering the Mammary Epithelial Cell Hierarchy

Clonal assays offer a powerful approach to dissecting the many events involved in the generation and maintenance of complex tissues from an undifferentiated stem cell pool. The application of such quantitative functional methodologies to studies of the hematopoietic system have been key to defining the hierarchy of progenitor subsets that reflect an irreversible stepwise process of lineage restriction. Recent studies now suggest that a similar paradigm applies to the normal mammary gland. The adult mouse mammary gland maintains a population of stem cells that generate biologically distinct and physically separable subpopulations of mammary epithelial progenitors which, in turn, generate terminally differentiated cells. Suggestive parallels between mouse and human mammary cells point to the likelihood that a similarly structured multi-step differentiation program characterizes the mammary gland from both species.     

Vitamin D and cancer: Deciphering the truth

Vitamin D is a hormone-like micronutrient involved not only in calcium metabolism but also in a variety of biological activities (e.g., cell proliferation, apoptosis, angiogenesis, inflammation) that makes it a candidate anticancer agent. Preclinical studies support the therapeutic potential of vitamin D both alone and in combination with other therapeutics. Overall, epidemiological data suggest the existence of a link between vitamin D and cancer risk, whereas the results of clinical trials are quite conflicting.     

Deciphering the distribution of the savanna biome

? We aimed to identify the limits of savanna across Africa, Australia and South America. We based our investigation on the rich history of hypotheses previously examined: that the limits of savanna are variously determined by rainfall, rainfall seasonality, soil fertility and disturbance. ? We categorized vegetation on all continents as 'savanna' (open habitats with a C(4) grass layer) or 'not-savanna' (closed habitats with no C(4) grass layer) and used a combination of statistical approaches to examine how the presence of savanna varied as a function of five environmental correlates. ? The presence of savanna is constrained by effective rainfall and rainfall seasonality. Soil fertility is regionally important, although the direction of its effect changes relative to rainfall. We identified three continental divergences in the limits of savanna that could not be explained by environment. ? Climate and soils do not have a deterministic effect on the distribution of savanna. Over the range of savanna, some proportion of the land is always 'not-savanna'. We reconciled previous contradictory views of savanna limits by developing a new conceptual framework for understanding these limits by categorizing environmental factors into whether they had a positive or negative effect on woody growth and the frequency of disturbance.     

Deciphering the Probiotic Potential of Bacillus amyloliquefaciens COFCAU_P1 Isolated from the Intestine of Labeo rohita Through In Vitro and Genetic Assessment

Probiotics and Antimicrobial Proteins - In this study, a bacterial strain COFCAU_P1, isolated from the digestive tract of a freshwater teleost rohu (Labeo rohita), was identified as Bacillus...     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.