Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells

BACKGROUND: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. RESULTS: We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. CONCLUSIONS: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.     

Ex vivo imaging of T cells in murine lymph node slices with widefield and confocal microscopes

Naïve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay ¹, originally set up by neurobiologists and transposed recently to murine thymus ². In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.     

Redox Interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in Mixed Culture under Enological Conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells

Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.     

Topical glucocorticoid therapy directly induces up-regulation of functional CXCR4 on primed T lymphocytes in the aqueous humor of patients with uveitis

Overexpression of the constitutive chemokine receptor CXCR4 has been shown to contribute to the accumulation of leukocytes at sites of chronic inflammation. Glucocorticoids are widely used to treat inflammatory disorders such as uveitis to considerable effect, yet paradoxically have been reported to increase CXCR4 expression in vitro. We show here that ocular lymphocytes isolated from patients with uveitis who had been treated with topical glucocorticoids expressed highly elevated levels of CXCR4. The up-regulation of CXCR4 could be reproduced in vitro by culture of CD4(+) T cells with aqueous humor (AqH), indicating a role for the ocular microenvironment rather than preferential recruitment of CXCR4(+) cells. Untreated uveitis and noninflammatory AqH up-regulated CXCR4 to a limited extent; this was dependent on TGF-beta2. However, the highest levels of CXCR4 both in vivo and in vitro were found in the glucocorticoid-treated patients. Glucocorticoids appeared to be directly responsible for the induction of CXCR4 in treated patients, as the glucocorticoid receptor antagonist RU38486 inhibited the in vitro up-regulation by AqH from these patients. Dexamethasone selectively up-regulated CXCR4 in vitro, but not any of a wide range of other chemokine receptors. CXCL12, the ligand for CXCR4, was present in AqH under noninflammatory conditions, but the levels were low in untreated uveitis and undetectable in treated uveitis AqH. The importance of these results for the treatment of HIV patients with glucocorticoids is discussed as well as a role for glucocorticoid-induced CXCR4 up-regulation and CXCL12 down-regulation in controlling the migration of lymphocyte populations, resulting in resolution of inflammation.     

Circadian preference modulates the neural substrate of conflict processing across the day

Human morning and evening chronotypes differ in their preferred timing for sleep and wakefulness, as well as in optimal daytime periods to cope with cognitive challenges. Recent evidence suggests that these preferences are not a simple by-product of socio-professional timing constraints, but can be driven by inter-individual differences in the expression of circadian and homeostatic sleep-wake promoting signals. Chronotypes thus constitute a unique tool to access the interplay between those processes under normally entrained day-night conditions, and to investigate how they impinge onto higher cognitive control processes. Using functional magnetic resonance imaging (fMRI), we assessed the influence of chronotype and time-of-day on conflict processing-related cerebral activity throughout a normal waking day. Sixteen morning and 15 evening types were recorded at two individually adapted time points (1.5 versus 10.5 hours spent awake) while performing the Stroop paradigm. Results show that interference-related hemodynamic responses are maintained or even increased in evening types from the subjective morning to the subjective evening in a set of brain areas playing a pivotal role in successful inhibitory functioning, whereas they decreased in morning types under the same conditions. Furthermore, during the evening hours, activity in a posterior hypothalamic region putatively involved in sleep-wake regulation correlated in a chronotype-specific manner with slow wave activity at the beginning of the night, an index of accumulated homeostatic sleep pressure. These results shed light into the cerebral mechanisms underlying inter-individual differences of higher-order cognitive state maintenance under normally entrained day-night conditions.