Cav1.4 calcium channels control cytokine production by human peripheral TH17 cells and psoriatic skin-infiltrating T cells.

BACKGROUND: Type-17 inflammation characterizes psoriasis, a chronic skin disease. Because several inflammatory cytokines contribute to psoriasis pathogenesis, inhibiting the simultaneous production of these cytokines in TH17 cells may be beneficial in psoriasis. We found that Cav1.4, encoded by CACNA1F, was the only Cav1 calcium channel expressed in TH17 cells. OBJECTIVE: We sought to investigate the role of Cav1.4 expression in early TH17-activation events and effector functions, as well as its association with TH17 signature genes in lesional psoriatic (LP) skins. METHODS: Transcriptional gene signatures associated with CACNA1F expression were examined in LP skins by RT-PCR and in situ hybridization. Cav1 inhibitor and/or shRNA lentivectors were used to assess the contribution of Cav1.4 in TH17 activation and effector functions in a 3-dimensional skin reconstruction model. RESULTS: CACNA1F expression correlated with inflammatory cytokine expression that characterizes LP skins and was preferentially associated with RORC expression in CD4+ and CD4- cells from LP biopsies. Nicardipine, a Cav1 channel antagonist, markedly reduced inflammatory cytokine production by TH17 cells from blood or LP skin. This was associated with decreased TCR-induced early calcium events at cell membrane and proximal signaling events. The knockdown of Cav1.4 in TH17 cells impaired cytokine production. Finally, Cav1 inhibition reduced the expression of the keratinocyte genes characteristic of TH17-mediated psoriasis inflammation in human skin equivalents. CONCLUSIONS: Cav1.4 channels promote TH17-cell functions both at the periphery and in inflammatory psoriatic skin.

Arp2/3-dependent mechanical control of morphogenetic robustness in an inherently challenging environment.

Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, we show that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. We found that knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, our data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations.

Using Xenopus Neural Crest Explants to Study Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to posttranslational modifications, to protein turnover. It involves remodeling of polarity and adhesion and enhances migratory capabilities. During physiological events such as embryogenesis or wound healing EMT is used to initiate cell migration, but EMT can also occur in pathological settings. In particular, EMT has been linked to fibrosis and cancer. Neural crest (NC) cells, an embryonic stem cell population whose behavior recapitulates the main steps of carcinoma progression, are a great model to study EMT. In this chapter, we provide a fully detailed protocol to extract NC cells from Xenopus embryos and culture them to study the dynamics of cell-cell adhesion, cell motility, and dispersion.

Super-resolved live-cell imaging using random illumination microscopy.

Current super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.

Divergent mechanisms specify chordate motoneurons: evidence from ascidians.

Ascidians are members of the vertebrate sister group Urochordata. Their larvae exhibit a chordate body plan, which forms by a highly accelerated embryonic strategy involving a fixed cell lineage and small cell numbers. We report a detailed analysis of the specification of three of the five pairs of motoneurons in the ascidian Ciona intestinalis and show that despite well-conserved gene expression patterns and embryological outcomes compared with vertebrates, key signalling molecules have adopted different roles. We employed a combination of cell ablation and gene manipulation to analyse the function of two signalling molecules with key roles in vertebrate motoneuron specification that are known to be expressed equivalently in ascidians: the inducer Sonic hedgehog, produced ventrally by the notochord and floorplate; and the inhibitory BMP2/4, produced on the lateral/dorsal side of the neural plate. Our surprising conclusion is that neither BMP2/4 signalling nor the ventral cell lineages expressing hedgehog play crucial roles in motoneuron formation in Ciona. Furthermore, BMP2/4 overexpression induced ectopic motoneurons, the opposite of its vertebrate role. We suggest that the specification of motoneurons has been modified during ascidian evolution, such that BMP2/4 has adopted a redundant inductive role rather than a repressive role and Nodal, expressed upstream of BMP2/4 in the dorsal neural tube precursors, acts as a motoneuron inducer during normal development. Thus, our results uncover significant differences in the mechanisms used for motoneuron specification within chordates and also highlight the dangers of interpreting equivalent expression patterns as indicative of conserved function in evo-devo studies.

Association of marine viral and bacterial communities with reference black carbon particles under experimental conditions: an analysis with scanning electron, epifluorescence and confocal laser scanning microscopy.

Black carbon (BC), the product of incomplete combustion of fossil fuels and biomass, constitutes a significant fraction of the marine organic carbon pool. However, little is known about the possible interactions of BC and marine microorganisms. Here, we report the results of experiments using a standard reference BC material in high concentrations to investigate basic principles of the dynamics of natural bacterial and viral communities with BC particles. We assessed the attachment of viral and bacterial communities using scanning electron, epifluorescence and confocal laser scanning microscopy and shifts in bacterial community composition using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE). In 24-h time-course experiments, BC particles showed a strong potential for absorbing viruses and bacteria. Total viral abundance was reduced, whereas total bacterial abundance was stimulated in the BC treatments. Viral and bacterial abundance on BC particles increased with particle size, whereas the abundances of BC-associated viruses and bacteria per square micrometer surface area decreased significantly with BC particle size. DGGE results suggested that BC has the potential to change bacterial community structure and favour phylotypes related to Glaciecola sp. Our study indicates that BC could influence processes mediated by bacteria and viruses in marine ecosystems.

Complementary roles for dynein and kinesins in the Xenopus egg cortical rotation.

Aligned vegetal subcortical microtubules in fertilized Xenopus eggs mediate the "cortical rotation", a translocation of the vegetal cortex and of dorsalizing factors toward the egg equator. Kinesin-related protein (KRP) function is essential for the cortical rotation, and dynein has been implicated indirectly; however, the role of neither microtubule motor protein family is understood. We examined the consequence of inhibiting dynein--dynactin-based transport by microinjection of excess dynamitin beneath the vegetal egg surface. Dynamitin introduced before the cortical rotation prevented formation of the subcortical array, blocking microtubule incorporation from deeper regions. In contrast, dynamitin injected after the microtubule array was fully established did not block cortical translocation, unlike inhibitory-KRP antibodies. During an early phase of cortical rotation, when microtubules showed a distinctive wavy organization, dynamitin disrupted microtubule alignment and perturbed cortical movement. These findings indicate that dynein is required for formation and early maintenance of the vegetal microtubule array, while KRPs are largely responsible for displacing the cortex once the microtubule tracks are established. Consistent with this model for the cortical rotation, photobleach analysis revealed both microtubules that translocated with the vegetal cytoplasm relative to the cortex, and ones that moved with the cortex relative to the cytoplasm.

Fertilisation calcium signals in the ascidian egg.

The egg of ascidians (urochordate), as virtually all animal and plant species, displays Ca2+ signals upon fertilisation. These Ca2+ signals are repetitive Ca2+ waves that initiate in the cortex of the egg and spread through the whole egg interior. Two series of Ca2+ waves triggered from two distinct Ca2+ wave pacemakers entrain the two meiotic divisions preceding entry into the first interphase. The second messenger inositol (1,4,5) trisphosphate (IP3) is the main mediator of these global Ca2+ waves. Other Ca2+ signalling pathways (RyR and NAADPR) are functional in the egg but they mediate localised cortical Ca2+ signals whose physiological significance remains unclear. The meiosis I Ca2+ wave pacemaker is mobile and relies on intracellular Ca2+ release from the endoplasmic reticulum (ER) induced by a large production of IP3 at the sperm aster site. The meiosis II Ca2+ wave pacemaker is stably localised in a vegetal protrusion called the contraction pole. It is probable that a local production of IP3 in the contraction pole determines the site of this second pacemaker while functional interactions between ER and mitochondria regulate its activity. Finally, a third ectopic pacemaker can be induced by a global increase in IP3, making the ascidian egg a unique system where three different Ca2+ wave pacemakers coexist in the same cell.

Analysis of microtubule movement on isolated Xenopus egg cortices provides evidence that the cortical rotation involves dynein as well as Kinesin Related Proteins and is regulated by local microtubule polymerisation.

In amphibians, the cortical rotation, a translocation of the egg cortex relative to the cytoplasm, specifies the dorsoventral axis. The cortical rotation involves an array of subcortical microtubules whose alignment is mediated by Kinesin-related proteins (KRPs), and stops as M-phase promoting factor (MPF) activation propagates across the egg. To dissect the role of different motor proteins in the cortical rotation and to analyse their regulation, we have developed an open cell assay system involving reactivation of microtubule movement on isolated cortices. Microtubule movements were dependent on ATP and consisted mainly of wriggling and flailing without net displacement, consistent with a tethering of microtubules to the cortex. Reactivated movements were inhibited by anti-KRP and anti-dynein antibodies perfused together but not separately, the KRP antibody alone becoming fixed to the cortex. Neither antibody could inhibit movement in the presence of MPF, indicating that arrest of the cortical rotation is not due to MPF-dependent inhibition of motor molecules. In contrast, D(2)O treatment of live eggs to protect microtubules from progressive depolymerisation prolonged the cortical rotation. We conclude that the cortical rotation probably involves cytoplasmic dynein as well as cortical KRPs and terminates as a result of local MPF-dependent microtubule depolymerisation.

Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion.

Fertilization increases both cytosolic Ca(2+) concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca(2+) waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P(3)-induced Ca(2+) transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca(2+) during sperm-triggered Ca(2+) waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca(2+) wave pacemaker PM2. Strikingly, the activity of the Ca(2+) wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca(2+), the egg's mitochondria are stimulated by Ins(1,4,5)P(3)-mediated Ca(2+) signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca(2+) wave pacemaker PM2.