Coupling actin dynamics to the endocytic process in Saccharomyces cerevisiae

Summary. Endocytosis is an essential eukaryotic process that, in many systems, has been reported to require a functional actin cytoskeleton. The process of endocytosis is critical for controlling the protein–lipid composition of the plasma membrane and uptake of nutrients as well as pathogens and also plays an important role in regulation of cell signalling. While several distinct pathways for endocytosis have been characterised, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly supported by studies in mammalian cells. Current evidence suggests that cortical patches are sites of endocytosis in Saccharomyces cerevisiae and that these sites are composed of sequentially forming protein complexes. Distinct stages in complex formation are characterised by the presence of different activators of F-actin polymerisation. Disassembly of the complexes is also essential for the endocytosis to proceed. Mutants lacking the kinases Ark1 and Prk1 accumulate actin and endocytic machinery in a single large clump in cells. Phosphorylation of endocytic proteins including Sla1p is proposed to cause their removal from the complex and allow later stages of the invagination process to occur. Dephosphorylation of endocytic components may then allow subsequent reincorporation into new sites of endocytic complex assembly. Correspondence and reprints: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.     

Change in spatial distribution patterns of a biennial plant between growth stages and generations in a patchy habitat

BACKGROUND AND AIMS: The aim of the study was to evaluate factors causing change in spatial distribution patterns of plants between growth stages and generations for a monocarpic biennial plant, Lysimachia rubida. It was assumed that habitat heterogeneity was a primary factor determining spatial patterns of plants, and a randomization procedure was developed for testing the null hypothesis that only spatial association with ground surface conditions determined spatial patterns of plants. METHODS: A 5-year demographic census was conducted on an open dry habitat that was heterogeneous with regard to the ground surface conditions. KEY RESULTS: There was significant habitat association in that plants at vegetative and reproductive stages were denser in areas with smaller gravel than with larger gravel. Point process analyses rejected the null hypothesis of the spatial association with ground surface conditions. CONCLUSIONS: The results suggest that other factors, such as patchy seed dispersal, secondary dispersal of the seeds and life-history variation at various spatial scales, also affected spatial patterns of individuals in a population of L. rubida. Spatial structures and dynamics of a local population in a patchy habitat represent various performances of plants within patches and seed dispersal within a patch and beyond the patch.     

Fine-scale clonal structure and diversity within patches of a clone-forming dioecious shrub, Ilex leucoclada (Aquifoliaceae)

BACKGROUND AND AIMS: The mode of reproduction (sexual vs. asexual) is likely to have important effects on genetic variation and its spatial distribution within plant populations. An investigation was undertaken of fine-scale clonal structure and diversity within patches of Ilex leucoclada (a clone-forming dioecious shrub). METHODS: Six patches were selected in a 1-ha plot previously established in an old-growth beech forest. Two of the selected patches were composed predominantly of stems with male flowers (male patch), and two contained stems with predominantly female flowers (female patch). The remaining two patches contained stems with male flowers and stems with female flowers in more or less equal proportions (mixed patch). Different genets were distinguished using random amplified polymorphic DNA (RAPD) markers. KEY RESULTS: One hundred and fifty-six genets with different RAPD phenotypes were identified among 1928 stems from the six patches. Among the six patches, the male patches had the lowest clonal diversity, and the mixed patches had the highest. Distribution maps of the genets showed that they extended downhill, reflecting natural layering that occurred when stems were pressed to the ground by heavy snow. In every patch, there were a few large genets with many stems and many small genets with a few stems. CONCLUSION: The differences in clonal diversity among patches may be due to differences in seedling recruitment frequencies. The skewed distribution of genet size (defined as the number of stems per genet) within patches may be due to differences in the timing of germination, or age (with early-establishing genets having clear advantages for acquiring resources) and/or intraspecific competition.     

In silico discovery of enzyme-substrate specificity-determining residue clusters

The binding between an enzyme and its substrate is highly specific, despite the fact that many different enzymes show significant sequence and structure similarity. There must be, then, substrate specificity-determining residues that enable different enzymes to recognize their unique substrates. We reason that a coordinated, not independent, action of both conserved and non-conserved residues determine enzymatic activity and specificity. Here, we present a surface patch ranking (SPR) method for in silico discovery of substrate specificity-determining residue clusters by exploring both sequence conservation and correlated mutations. As case studies we apply SPR to several highly homologous enzymatic protein pairs, such as guanylyl versus adenylyl cyclases, lactate versus malate dehydrogenases, and trypsin versus chymotrypsin. Without using experimental data, we predict several single and multi-residue clusters that are consistent with previous mutagenesis experimental results. Most single-residue clusters are directly involved in enzyme-substrate interactions, whereas multi-residue clusters are vital for domain-domain and regulator-enzyme interactions, indicating their complementary role in specificity determination. These results demonstrate that SPR may help the selection of target residues for mutagenesis experiments and, thus, focus rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.     

Patch exploitation strategies of parasitic wasps under intraspecific competition

The optimal residence time of a forager exploiting a resourcepatch in the absence of competitors has been much studied sincethe development of the marginal value theorem. However, severalforagers are frequently observed exploiting the same patch simultaneously,and patch residence time has been surprisingly little studiedin such competitive situations. The few theoretical models developedon this topic predict that foragers should engage in a war ofattrition and stay in the patch longer than when foraging alone.We tested this prediction in Pachycrepoideus vindemmiae (Hymenoptera:Pteromalidae), a solitary parasitoid species in which femalesare known to defend the hosts they are exploiting via intraspecificfighting. By measuring the effect of direct (i.e., presenceof conspecifics) and indirect (i.e., presence of already-parasitizedhosts) competition on patch exploitation strategies, we revealedan apparent polymorphism of strategies. Indeed, in competitivesituations, some members of the population tended to retreatalmost immediately from the patch whereas other members tendedto remain, further exploit the patch, and be more involved ininteractions with conspecifics. The proportion of "retreaters"strongly depended on competition intensity and prior experienceof competition. The forager's physiological state (age) alsoaffects patch exploitation strategy and the intensity of interactionsbetween competing females. Our results highlight the necessityfor further theoretical studies that consider cases where contestsbetween foragers are costly and take into account prior experienceof competition and the forager's physiological state.     

A Mathematical Model for Invasion Range of Population Dispersion Through a Patchy Environment

We focus on the question of how the dispersion of an invading population is affected by the spatial distribution of patches that have resource available for the population’s settlement and reproduction. We have developed and analyzed a mathematical model with a simple stochastic process. The patches are grouped into three classes – free, occupied and abandoned – depending on the state of the patch used by the population. We especially consider the range expanded by invaded patches, the invaded range R, assuming a certain generalized relation between R and the total number of invaded patches k, making use of an index, a sort of fractal dimension, to characterize the spatial distribution of invaded patches. We show that the expected velocity is significantly affected by the nature of spatial distribution of resource patches, and is temporally variable. When the invading population finally becomes extinct at a certain moment, the terminal size of the invaded range at that the moment is closely related to the nature of the spatial distribution of resource patches, which is explicitly demonstrated by our analysis.     

吗啡对新生鼠尾核神经元钾离子通道电流的作用

目的 :研究吗啡对新生鼠尾核神经元钾离子通道电流的作用。方法 :应用全细胞膜片钳技术在培养的尾核神经元上 ,观察吗啡急性与慢性处理对尾核神经元电压门控钾离子通道电流的影响。结果 :吗啡急性处理尾核神经元诱发钾离子通道电流增大 ,电流从加吗啡前的 (2 .6± 0 .4 )nA增高到 (3.3± 0 .5 )nA ,加纳洛酮后电流下降为 (2 .4± 0 .4 )nA ;吗啡慢性处理尾核神经元的钾离子通道电流从对照组的 (2 .6± 0 .4 )nA增高到 (3.1± 0 .5 )nA ,加纳洛酮后电流下降为 (2 .4± 0 .4 )nA。结论 :在吗啡急性或慢性处理尾核神经元后 ,吗啡经 μ受体介导 ,诱发尾核神经元钾离子通道电流增大 ,使神经元处于超极化状态 ,导致神经元活动的抑制     

Gating transitions in bacterial ion channels measured at 3 microns resolution

Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes. Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125-300 pA. To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 microns (10-20 times faster than usual). Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 micros. There is a clear trend toward longer transition times for the larger transitions. In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite. We detected a short-lived (approximately 20 microns) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution. Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents ("anticipations" or "regrets"). Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open. Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution. High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements.     

Presence of a conspecific increases superparasitism but not infanticide under self‐ and conspecific superparasitism in a semisolitary parasitoid,Echthrodelphax fairchildii (Hymenoptera: Dryinidae)

Female parasitoids are expected to avoid superparasitism (ovipositing in and/or on parasitized hosts) when unparasitized hosts are available. However, when the supply of unparasitized hosts is restricted, they are expected to self‐ as well as conspecifically superparasitize. One of the cues of a reduced availability of unparasitized hosts is the presence of a conspecific. Moreover, if the focal species can perform infanticide, after encountering a conspecific female, the females are expected to kill eggs existing in and/or on hosts when superparasitizing, because the eggs are more likely to be laid by others. In this study we investigated whether females of an infanticidal semisolitary parasitoid, Echthrodelphax fairchildii, increase their frequencies of superparasitism and infanticide after encountering a conspecific female. Echthrodelphax fairchildii females are capable of discriminating between self‐ and conspecific superparasitism until up to 0.75 h after the first egg was laid (self‐superparasitism frequency < conspecific superparasitism frequency). As expected, the female parasitoids were more likely to perform self‐ and conspecific superparasitism after they had encountered a conspecific. In particular, the self‐superparasitism frequency increased highly within a short period after the first oviposition, so that no difference between the self‐ and conspecific superparasitism frequencies was found. In contrast, the infanticidal‐probing frequency remained extremely low, irrespective of whether or not the female parasitoids had encountered a conspecific. Moreover, when superparasitizing, females usually laid female eggs. Possible causes for the low frequency of infanticidal probing and the female‐biased sex ratio are discussed.