Nanoscale organization of multiple GPI-anchored proteins in living cell membranes

Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.     

Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.     

Rafts: scale-dependent, active lipid organization at the cell surface

Rafts have been conceptualized as lateral heterogeneities in the organization of cholesterol and sphingolipids, endowed with sorting and signaling functions. In this review we critically examine evidence for the main tenet of the 'raft hypothesis', namely lipid-dependent segregation of specific membrane components in the plasma membrane. We suggest that conventional approaches to studying raft organization wherein membranes are treated as passive, thermally equilibrated systems are unlikely to provide an adequate framework to understand the mechanisms of raft-organization in vivo. An emerging view of raft organization is that it is spatio-temporally regulated at different scales by the cell. This argues that rafts must be defined by simultaneous observation of components involved in particular functions. Recent evidence from the study of glycosylphosphatidyl inositol-anchored proteins, a common raft-marker, supports this picture in which larger scale, more stable rafts are induced from preexisting small-scale lipid-dependent structures actively maintained by cellular processes.     

Predicting anti-HIV activity of 2,3-diaryl-1,3-thiazolidin-4-ones: computational approach using reformed eccentric connectivity index

The eccentric connectivity index, which has recently been employed successfully for the development of numerous mathematical models for the prediction of biological activities of diverse nature, has been reformed to overcome its limitations caused by degeneracy and insensitivity towards heteroatoms. The reformed eccentric connectivity index, termed the eccentric connectivity topochemical index, overcomes the limitations of the eccentric connectivity index by exhibiting very low degeneracy and displaying sensitivity to both the presence and relative position of heteroatoms without compromizing the discriminating power of the eccentric connectivity index. The relationship of the eccentric connectivity topochemical index, eccentric connectivity index and Wieners index with regard to the anti-HIV activity of 2, 3-diaryl-1, 3-thiazolidin-4-one derivatives was subsequently investigated. The values of the eccentric connectivity topochemical index, the eccentric connectivity index and Wieners index of each of 31 analogues comprizing the data set were computed using in-house computer program. Resultant data was analyzed and suitable models developed after identification of active ranges. Subsequently, each derivative was assigned a biological activity using these models, which was then compared with the reported anti-HIV activity. The accuracy of prediction using these models was found to vary from 81 to 90%. The proposed index offers a vast potential for virtual screening of combinatorial libraries, structure property/activity studies and drug design.Figure Basic structure of 2,3-diaryl-1, 3-thiazoidin-4-ones.     

Hamilton goes empirical: estimation of inclusive fitness from life-history data

Hamilton's theory of kin selection is one of the most important advances in evolutionary biology since Darwin. Central to the kin-selection theory is the concept of inclusive fitness. However, despite the importance of inclusive fitness in evolutionary theory, empirical estimation of inclusive fitness has remained an elusive task. Using the concept of individual fitness, I present a method for estimating inclusive fitness and its components for diploid organisms with age-structured life histories. The method presented here: (i) allows empirical estimation of inclusive fitness from life-history data; (ii) simultaneously considers all components of fitness, including timing and magnitude of reproduction; (iii) is consistent with Hamilton's definition of inclusive fitness; and (iv) adequately addresses shortcomings of existing methods of estimating inclusive fitness. I also demonstrate the application of this new method for testing Hamilton's rule.     

Sociality and individual fitness in yellow-bellied marmots: insights from a long-term study (1962–2001)

Theoretical and empirical studies suggest that the age of first reproduction (the age at which reproduction begins) can have a substantial influence on population dynamics and individual fitness. Using complete survival and reproductive histories of 428 female yellow-bellied marmots (Marmota flaviventris) from a 40-year study (1962-2001), we investigated causes and fitness consequences of delayed maturity. Most females (86%) died without reproducing. The age of first reproduction of females that survived to reproduce at least once (n=60) ranged from 2 to 6 years. Females maturing later did not have a larger lifetime number of successful reproductive events or offspring production, nor did they experience improved survival. Females reproducing earlier had a higher fitness than those that delayed maturity. These results suggest that the net cost of early maturity was less than fitness benefits associated with early onset of reproduction, and that age of first reproduction in our study population is under substantial directional selection favoring early maturity. We conclude that female yellow-bellied marmots delay onset of reproduction not because of fitness benefits of foregoing reproduction at an earlier age, but due to the social suppression of reproduction by older, reproductive females, which enhances their own fitness to the detriment of the fitness of young females. Our results indicate that female yellow-bellied marmots that survive to reproduce may act to increase their own direct fitness, and that social suppression of reproduction of young females is a part of that strategy.     

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Differential action of iodine on mitochondria from human tumoral- and extra-tumoral tissue in inducing the release of apoptogenic proteins

Iodide is actively concentrated in the thyroid gland for thyroid hormone biosynthesis. Excess iodine has been observed to induce apoptosis in thyrocytes and mammary cells. The mechanism of iodine induced apoptosis is poorly understood. Among various cell organelles, mitochondria is known to provide conducive environment for the organification of iodine, i.e. iodination of different proteins. Mitochondria also play a central role in execution of apoptosis. To study the role of mitochondria in iodine induced apoptosis, we investigated the direct interaction of iodine and human breast mitochondria vis-a-vis its role in the initiation of apoptosis in vitro. We observed that mitochondria isolated from the tumor (TT) and extra-tumoral tissue (ET) of human breast display significant uptake of iodine. Mitochondrial proteins were observed to be predominantly iodinated in ET but not in TT mitochondria. Treatment with iodine showed an increase in mitochondrial permeability transition of TT and decrease in ET. Iodine induced released factor(s) other than cytochrome c from tumor mitochondria initiate(s) apoptosis in vitro, while those from ET mitochondria were non-apoptogenic in nature. To our knowledge, this is first report demonstrating that iodine acts differentially on mitochondria of tumor and extratumoral origin to release apoptogenic proteins from TT and has a protective effect on ET.     

The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.     

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.