Mechanoreceptive organelles (MOs) are specialized subcellular entities in mechanoreceptors that transform extracellular mechanical stimuli into intracellular signals. Their ultrastructures are key to ...understanding the molecular nature and mechanics of mechanotransduction. Campaniform sensilla detect cuticular strain caused by muscular activities or external stimuli in Drosophila. Each campaniform sensillum has an MO located at the distal tip of its dendrite. Here we analyzed the molecular architecture of the MOs in fly campaniform mechanoreceptors using electron microscopic tomography. We focused on the ultrastructural organization of NompC (a force-sensitive channel) that is linked to the array of microtubules in these MOs via membrane-microtubule connectors (MMCs). We found that NompC channels are arranged in a regular pattern, with their number increasing from the distal to the proximal end of the MO. Double-length MMCs in nompC29+29ARs
confirm the ankyrin-repeat domain of NompC (NompC-AR) as a structural component of MMCs. The unexpected finding of regularly spaced NompC-independent linkers in nompC³ suggests that MMCs may contain non-NompC components. Localized laser ablation experiments on mechanoreceptor arrays in halteres suggest that MMCs bear tension, providing a possible mechanism for why the MMCs are longer when NompC-AR is duplicated or absent in mutants. Finally, mechanical modeling shows that upon cuticular deformation, sensillar architecture imposes a rotational activating force, with the proximal end of the MO, where more NOMPC channels are located, being subject to larger forces than the distal end. Our analysis reveals an ultrastructural pattern of NompC that is structurally and mechanically optimized for the sensory functions of campaniform mechanoreceptors.
Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis 1. It is assumed that ...sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions 2. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a “dry, yet alive” larva from a “dry and dead” one.
► Caenorhabditis elegans dauer is a true anhydrobiote; it can resist severe desiccation ► Trehalose is essential for desiccation tolerance in C. elegans ► Desiccation damages cellular membranes and membrane-bound organelles ► Trehalose protects membranes by preserving native lipid packing during desiccation
Lamp illuminants evidence the exploitation of natural resources, animal and plant domestication, commerce, religious practices and nutrition of ancient populations. However, the physicochemical ...analysis of their major constituent-burned, degraded and aged mixture of triacylglycerols is imprecise and may lead to ambiguous interpretations. We applied proteomics to analyze fuel deposits from eight lamps dated by 6th to 8th centuries AD that were excavated at the Astana necropolis (Xinjiang, China) and determined their origin by identifying organism-specific proteins. Proteomics evidence corroborated and detailed the assignments of source organism relying upon comparative profiling of intact triacylglycerols by shotgun lipidomics. We found that ruminant (mostly, sheep) fat, cattle ghee and sesame oil were common combustibles in Astana and concluded that sesame as an oilseed appeared in China under Tang Dynasty concomitantly with the expansion of Buddhism.
During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the ...amount of soluble and polymeric tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimeric and polymeric tubulin at mitotic centrosomes in early
embryos. We show that a
one-cell stage centrosome at metaphase contains >10,000 microtubules with a total polymer concentration of 230 µM. Centrosomes concentrate soluble α/β tubulin by about 10-fold over the cytoplasm, reaching peak values of 470 µM, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 µM. These findings support in vitro data suggesting that microtubule nucleation in
centrosomes is driven in part by concentrating soluble tubulin.
Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly ...due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.
Life of sexually reproducing organisms starts with the fusion of the haploid egg and sperm gametes to form the genome of a new diploid organism. Using the newly fertilized
zygote, we show that the ...mitotic Polo-like kinase PLK-1 phosphorylates the lamin LMN-1 to promote timely lamina disassembly and subsequent merging of the parental genomes into a single nucleus after mitosis. Expression of non-phosphorylatable versions of LMN-1, which affect lamina depolymerization during mitosis, is sufficient to prevent the mixing of the parental chromosomes into a single nucleus in daughter cells. Finally, we recapitulate lamina depolymerization by PLK-1 in vitro demonstrating that LMN-1 is a direct PLK-1 target. Our findings indicate that the timely removal of lamin is essential for the merging of parental chromosomes at the beginning of life in
and possibly also in humans, where a defect in this process might be fatal for embryo development.
The ability to rapidly assess microtubule number in 3D image stacks from electron tomograms is essential for collecting statistically meaningful data sets. Here we implement microtubule tracing using ...3D template matching. We evaluate our results by comparing the automatically traced centerlines to manual tracings in a large number of electron tomograms of the centrosome of the early Caenorhabditis elegans embryo. Furthermore, we give a qualitative description of the tracing results for three other types of samples. For dual-axis tomograms, the automatic tracing yields 4% false negatives and 8% false positives on average. For single-axis tomograms, the accuracy of tracing is lower (16% false negatives and 14% false positives) due to the missing wedge in electron tomography. We also implemented an editor specifically designed for correcting the automatic tracing. Besides, this editor can be used for annotating microtubules. The automatic tracing together with a manual correction significantly reduces the amount of manual labor for tracing microtubule centerlines so that large-scale analysis of microtubule network properties becomes feasible.
Throughout mammal erythroid differentiation, erythroblasts undergo enucleation and organelle clearance becoming mature red blood cell. Organelles are cleared by autophagic pathways non-specifically ...targeting organelles and cytosolic content or by specific mitophagy targeting mitochondria. Mitochondrial functions are essential to coordinate metabolism reprogramming, cell death, and differentiation balance, and also synthesis of heme, the prosthetic group needed in hemoglobin assembly. In mammals, mitochondria subcellular localization and mitochondria interaction with other structures as endoplasmic reticulum and nucleus might be of importance for the removal of the nucleus, that is, the enucleation. Here, we aim to characterize by electron microscopy the changes in ultrastructure of cells over successive stages of human erythroblast differentiation. We focus on mitochondria to gain insights into intracellular localization, ultrastructure, and contact with other organelles. We found that mitochondria are progressively cleared with a significant switch between PolyE and OrthoE stages, acquiring a rounded shape and losing contact sites with both ER (MAM) and nucleus (NAM). We studied intracellular vesicle trafficking and found that endosomes and MVBs, known to be involved in iron traffic and heme synthesis, are increased during BasoE to PolyE transition; autophagic structures such as autophagosomes increase from ProE to OrthoE stages. Finally, consistent with metabolic switch, glycogen accumulation was observed in OrthoE stage.
In this paper we address the difficult problem of parameter-finding in image segmentation. We replace a tedious manual process that is often based on guess-work and luck by a principled approach that ...systematically explores the parameter space. Our core idea is the following two-stage technique: We start with a sparse sampling of the parameter space and apply a statistical model to estimate the response of the segmentation algorithm. The statistical model incorporates a model of uncertainty of the estimation which we use in conjunction with the actual estimate in (visually) guiding the user towards areas that need refinement by placing additional sample points. In the second stage the user navigates through the parameter space in order to determine areas where the response value (goodness of segmentation) is high. In our exploration we rely on existing ground-truth images in order to evaluate the "goodness" of an image segmentation technique. We evaluate its usefulness by demonstrating this technique on two image segmentation algorithms: a three parameter model to detect microtubules in electron tomograms and an eight parameter model to identify functional regions in dynamic Positron Emission Tomography scans.
The water and solute transporting properties of the epidermis have been proposed to be important determinants of skin moisture content and barrier properties. The water/small solute-transporting ...protein aquaporin-3 (AQP3) was found by immunofluorescence and immunogold electron microscopy to be expressed at the plasma membrane of epidermal keratinocytes in mouse skin. We studied the role of AQP3 in stratum corneum (SC) hydration by comparative measurements in wild-type and AQP3 null mice generated in a hairless SKH1 genetic background. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries but were polyuric because of defective urinary concentrating ability. AQP3 deletion resulted in a >4-fold reduced osmotic water permeability and >2-fold reduced glycerol permeability in epidermis. Epidermal, dermal, and SC thickness and morphology were not grossly affected by AQP3 deletion. Surface conductance measurements showed remarkably reduced SC water content in AQP3 null mice in the hairless genetic background (165 ± 10 versus 269 ± 12 microsiemens (μS), p < 0.001), as well as in a CD1 genetic background (209 ± 21 versus 469 ± 11 μS). Reduced SC hydration was seen from 3 days after birth. SC hydration in hairless wild-type and AQP3 null mice was reduced to comparable levels (90–100 μS) after a 24-h exposure to a dry atmosphere, but the difference was increased when surface evaporation was prevented by occlusion or exposure to a humidified atmosphere (179 ± 13versus 441 ± 34 μS). Conductance measurements after serial tape stripping suggested reduced water content throughout the SC in AQP3 null mice. Water sorption-desorption experiments indicated reduced water holding capacity in the SC of AQP3 null mice. The impaired skin hydration in AQP3 null mice provides the first functional evidence for the involvement of AQP3 in skin physiology. Modulation of AQP3 expression or function may thus alter epidermal moisture content and water loss in skin diseases.
PDF
