Electron microscopy and new technological approaches to investigate structural elements of the mitotic apparatus in Saccharomyces cerevisiae and Xenopus laevis

The mitotic cell cycle is a complex process which leads to the chromosome segregation from the mother cell to the two daughter cells and transmit the full genetic background necessary to grow and adapt to the permanently evolving environment. The equal partitioning of the duplicated chromosomes is finely regulated by the cytoskeleton which on purpose organises into a mitotic spindle. In eukaryotes like Xenopus laevis and Saccharomyces cerevisiae the mitotic spindle is mainly composed of three components: first the microtubules (MT) are organised in a spindle emanating from the chromosomes and focusing at the poles. Second they connect to the chromosomes via the kinetochore complexes (KT) and third focus at the microtubule organising centres (MTOC), also called centrosomes. We have focused our interest on structural analysis of the S. cerevisiae centrosome, called the Spindle Pole Body (SPB), at the molecular level, and on the MTs organisation in the meiotic spindle midzone of X. laevis. With S. cerevisiae we have used cryo-electron tomography on vitreous sections (CETOVIS) to investigate the three dimensional (3D) molecular structure of the SPB. The samples were vitrified by high pressure freezing (HPF) and sectioned in vitreous state. Acquiring instant snapshots close to native state of the SPB in vivo, we confirmed previous observations done on plunge frozen or freeze substituted material. The Spc42 central crystal protein are organised with a defined spacing of 107Å, but the plaques composing the SPB could not all be identified. Mainly, the central and the outer plaque were visible, in contrast with the plastic tomography results where the inner plaque appeared dense and compact. Unfortunately, the very low signal to noise ratio prevented us from extracting details structural information about the SPB in its native state. The project concerning the X. laevis meiotic spindle focused on the 3D organisation of the MT within the spindle. Their interactions with the MTOC and the KT have been extensively studied over the past, but a high resolution structural map is still missing and has long been awaited by scientists to put all the knowledge into a 3D context. Furthermore, information about the individual MT is missing like their average length distribution, the existence of short MTs and the end morphologies distribution. To study this complex and large structure, we have developed a novel correlative light to electron microscopy (CLEM) approach combined with electron tomography. We cryo-fixed by HPF, for the first time to our knowledge, spindle assembled in X. laevis egg extracts and prepared them for a structural electron tomographic study. We reconstructed three quarter of a meiotic spindle midzone and identified three subcategories of MT bundle organisation. Finally, we have used our CLEM method to develop a new technology that should facilitate future CLEM work. Recent advances in plastic polymer transformation and micro-injection moulding (µIM) allowed us to create a cryocaspule designed for an easy correlative microscopy and transfer for HPF

Modélisation physique de l'interaction entre obstacles et avalanches de neige poudreuse

International audienceIn order to better understand the interaction between powder snow avalanches and defence structures, we carried out physical experiments on small-scale models. The powder snow avalanche was simulated by a heavy salt solution in a water tank. Quasi two-dimensional and three-dimensional experiments were carried out with different catching dam heights. For the reference avalanche, the velocity just behind the nose in the head was greater than the front velocity. For the 2-D configuration, the ratio Umax/Ufront was as high as 1.6, but it depends on the height. For the 3-D configuration, this ratio differed slightly and was even greater (up to 1.8). The vertical velocity rose to 106% of the front velocity for the 3-D simulation and 74% for the 2-D simulation. The reduction in front velocity due to the presence of dams was an increasing function of the dam height. But this reduction depended on topography: dams were more effective on an open slope avalanche (3-D configuration). The ratio Umax/Ufront was an increasing function of the dam's height and reached a value of 1.9. The obstacle led to a reduction in vertical velocity downstream of the vortex zone

Characterization of early ultrastructural changes in the cerebral white matter of CADASIL small vessel disease using high pressure freezing/freeze-substitution

AIMS: The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease. METHODS: We used high pressure freezing and freeze substitution (HPF/FS) in combination with high resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3R169C ) mice aged 4 to 15 months to study white matter lesions in the corpus callosum. RESULTS: We first optimized the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months, then vesiculation of the inner tongue and myelin sheath thinning at 15 months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age. CONCLUSION: We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism

Intensity-based matching and registration for 3D correlative microscopy with large discrepancies

International audienceCorrelative microscopy, especially light and electron microscopy (CLEM), enables the study of cells and subcellular elements in complementary ways, provided a reliable registration between images is efficiently achievable. We propose a general automatic registration method. Due to large discrepancies in appearance, field-of-view, resolution and position, a pre-alignment stage is required before any 3D fine registration stage. We define an intensity-based method for both stages, which leverages a common representation of the two involved image modalities. We report experimental results on different real datasets of 3D correlative microscopy, demonstrating time efficiency and overlay accuracy

The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated

Archeological Investigations at the Santa Maria Creek Site (41CW104) Caldwell County, Texas

The excavations by Atkins at the Santa Maria Creek site (41CW104) described in the following report have succeeded in bringing together a myriad of information regarding aboriginal occupations in eastern Central Texas at the dawn of the Historic period. The analysis of the materials recovered from National Register of Historic Places testing and data recovery has demonstrated that even a site buried in sandy, bioturbated sediments can still significantly add to the archeological record. This becomes even more important for areas such as Caldwell County, Texas, which have witnessed few such investigations. The report utilized a wide array of analytical techniques to unravel the site, including extensive ethnohistorical research, artifact analysis, special studies, and experimental archeology

Are we drawing the right conclusions from randomised placebo-controlled trials? A post-hoc analysis of data from a randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>Assumptions underlying placebo controlled trials include that the placebo effect impacts on all study arms equally, and that treatment effects are additional to the placebo effect. However, these assumptions have recently been challenged, and different mechanisms may potentially be operating in the placebo and treatment arms. The objective of the current study was to explore the nature of placebo versus pharmacological effects by comparing predictors of the placebo response with predictors of the treatment response in a randomised, placebo-controlled trial of a phytotherapeutic combination for the treatment of menopausal symptoms. A substantial placebo response was observed but no significant difference in efficacy between the two arms.</p> <p>Methods</p> <p>A <it>post hoc </it>analysis was conducted on data from 93 participants who completed this previously published study. Variables at baseline were investigated as potential predictors of the response on any of the endpoints of flushing, overall menopausal symptoms and depression. Focused tests were conducted using hierarchical linear regression analyses. Based on these findings, analyses were conducted for both groups separately. These findings are discussed in relation to existing literature on placebo effects.</p> <p>Results</p> <p>Distinct differences in predictors were observed between the placebo and active groups. A significant difference was found for study entry anxiety, and Greene Climacteric Scale (GCS) scores, on all three endpoints. Attitude to menopause was found to differ significantly between the two groups for GCS scores. Examination of the individual arms found anxiety at study entry to predict placebo response on all three outcome measures individually. In contrast, <it>low </it>anxiety was significantly associated with improvement in the active treatment group. None of the variables found to predict the placebo response was relevant to the treatment arm.</p> <p>Conclusion</p> <p>This study was a <it>post hoc </it>analysis of predictors of the placebo versus treatment response. Whilst this study does not explore neurobiological mechanisms, these observations are consistent with the hypotheses that 'drug' effects and placebo effects are not necessarily additive, and that mutually exclusive mechanisms may be operating in the two arms. The need for more research in the area of mechanisms and mediators of placebo versus active responses is supported.</p> <p>Trial Registration</p> <p>International Clinical Trials Registry ISRCTN98972974.</p

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures

Archeological Investigations at the Santa Maria Creek Site (41CW104) Caldwell County, Texas

Report on the excavations at the Santa Maria creek site in Caldwell County, Texas during 2006 and 2007. The report includes a discussion of research methods, analysis of the findings, and history of the area