Axonal swellings (AS) are one of the neuropathological hallmark of axonal injury in several disorders from trauma to neurodegeneration. Current evidence proposes a role of perturbed Ca2+ homeostasis in AS formation, involving impaired axonal transport and focal distension of the axons. Mechanisms of AS formation, in particular moments following injury, however, remain unknown. Here we show that AS form independently from intra-axonal Ca2+ changes, which are required primarily for the persistence of AS in time. We further show that the majority of axonal proteins undergoing de/phosphorylation immediately following injury belong to the cytoskeleton. This correlates with an increase in the distance of the actin/spectrin periodic rings and with microtubule tracks remodeling within AS. Observed cytoskeletal rearrangements support axonal transport without major interruptions. Our results demonstrate that the earliest axonal response to injury consists in physiological adaptations of axonal structure to preserve function rather than in immediate pathological events signaling axonal destruction.
- MeSH
- Actins metabolism MeSH
- Axonal Transport physiology MeSH
- Axons pathology MeSH
- Humans MeSH
- Spectrin * metabolism MeSH
- Brain Injuries, Traumatic * pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Proton detection in solid state NMR is continuously developing and allows one to gain new insights in structural biology. Overall, this progress is a result of the synergy between hardware development, new NMR methodology and new isotope labeling strategies, to name a few factors. Even though current developments are rapid, it is worthwhile to summarize what can currently be achieved employing proton detection in biological solids. We illustrate this by analysing the signal-to-noise ratio (SNR) for spectra obtained for a microcrystalline α-spectrin SH3 domain protein sample by (i) employing different degrees of chemical dilution to replace protons by incorporating deuterons in different sites, by (ii) variation of the magic angle spinning (MAS) frequencies between 20 and 110 kHz, and by (iii) variation of the static magnetic field B0. The experimental SNR values are validated with numerical simulations employing up to 9 proton spins. Although in reality a protein would contain far more than 9 protons, in a deuterated environment this is a sufficient number to achieve satisfactory simulations consistent with the experimental data. The key results of this analysis are (i) with current hardware, deuteration is still necessary to record spectra of optimum quality; (ii) 13CH3 isotopomers for methyl groups yield the best SNR when MAS frequencies above 100 kHz are available; and (iii) sensitivity increases with a factor beyond B0 3/2 with the static magnetic field due to a transition of proton-proton dipolar interactions from a strong to a weak coupling limit.
SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system.
- MeSH
- Genes, Dominant * MeSH
- Phenotype MeSH
- Genetic Predisposition to Disease * MeSH
- Genetic Variation * MeSH
- Genetic Association Studies methods MeSH
- Heterozygote MeSH
- Humans MeSH
- Mice MeSH
- Neurodevelopmental Disorders diagnosis genetics MeSH
- Spectrin genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.
Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.
- MeSH
- Actinin MeSH
- Actins analysis MeSH
- Cell Nucleus chemistry ultrastructure MeSH
- Financing, Organized MeSH
- HeLa Cells MeSH
- Nuclear Proteins analysis MeSH
- Humans MeSH
- Lymphocytes chemistry ultrastructure MeSH
- Microfilament Proteins analysis MeSH
- Paxillin MeSH
- GTPase-Activating Proteins MeSH
- Spectrin MeSH
- Tropomyosin MeSH
- Check Tag
- Humans MeSH
Phenylhydrazine and its derivatives were first given a medical application at the end of the 19th century but with with very little benefit. However, this compound seems to be very useful in models studying mechanisms of haemolytic anaemia. Phenylhydrazine induces a reactive oxygen species formation peroxidation of lipids and oxidative degradation of spectrin in the membrane skeleton. PHZ-induced haemolytic injury seems to be derived from oxidative alternations to red blood cell proteins. This compound can modulate immune reactions.
- MeSH
- Gene Expression immunology radiation effects MeSH
- Phenylhydrazines pharmacokinetics adverse effects therapeutic use MeSH
- Anemia, Hemolytic etiology blood MeSH
- Membrane Proteins physiology MeSH
- Lipid Peroxidation drug effects radiation effects MeSH
- Reactive Oxygen Species adverse effects MeSH
- Spectrin chemistry MeSH
- Mutagenicity Tests utilization MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Three major cytoskeletal proteins, actin, tubulin and spectrin, are present in the head of mammalian spermatozoa. Although cytoskeletal proteins are implicated in the regulation of capacitation and the acrosome reaction (AR), their exact role remains poorly understood. The aim of this study was to compare the distribution of the sperm head cytoskeleton before and after the AR in spermatozoa representing a range of acrosome size and shape. Spermatozoa from the human and three rodents (rat, hamster and grey squirrel) were fixed before and after the AR in appropriate medium in vitro. Indirect immunofluorescent localization of cytoskeletal proteins was undertaken with antibodies recognizing actin, spectrin and alpha-tubulin. Preparations were counterstained with propidium iodide and examined by epifluorescent and confocal microscopy. Our results clearly demonstrated changes in localization of cytoskeleton during the AR, mainly in the apical acrosome with further changes to the equatorial segment and post-acrosomal regions. The pattern of cytoskeletal proteins in the sperm head of all the species was similar in respect to various sub-compartments. These observations indicated that the sperm head cortical cytoskeleton exhibits significant changes during the AR and, therefore, support the image of cytoskeletal proteins as highly dynamic structures participating actively in processes prior to fertilization.
- MeSH
- Acrosome Reaction physiology MeSH
- Actins analysis MeSH
- Biomarkers analysis MeSH
- Cytoskeletal Proteins analysis ultrastructure MeSH
- Financing, Organized MeSH
- Microscopy, Fluorescence MeSH
- Fluorescent Antibody Technique, Indirect MeSH
- Sperm Head ultrastructure MeSH
- Microscopy, Confocal MeSH
- Cricetinae MeSH
- Rats MeSH
- Sciuridae MeSH
- Spectrin analysis MeSH
- Tubulin analysis MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Rats MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
143, [4] s. : il., tab. ; 32 cm + 1 volná příloha
Charakterizace mutací vedoucích k hereditární sferocytose za účelem pochopení patogenese hereditární sferocytosy a úlohy jednotlivých proteinů v erytrocytami menbráně.Korelace mezi typem a polohou mutaci a klinickým průběhem hereditarní sferocytosy.; Charcterization of mutation underlying hereditary spherocytosis in order to elucidate its pathogenesis and the role of the mutant proteins in red cell membrane.Correlation between the type of the mutations and the clinical course of spherocytosis.
- MeSH
- Ankyrins genetics MeSH
- Spherocytosis, Hereditary physiopathology MeSH
- Electrophoresis utilization methods MeSH
- Fluorescent Antibody Technique utilization MeSH
- Membrane Proteins MeSH
- Flow Cytometry utilization methods MeSH
- Spectrin genetics MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- hematologie a transfuzní lékařství
- chemie, klinická chemie
- NML Publication type
- závěrečné zprávy o řešení grantu IGA MZ ČR
- MeSH
- Actins MeSH
- Child MeSH
- Erythrocytes pathology MeSH
- Hyperbilirubinemia MeSH
- Humans MeSH
- Lipid Bilayers MeSH
- Infant, Low Birth Weight MeSH
- Infant, Newborn MeSH
- Jaundice, Neonatal MeSH
- Spectrin MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Infant, Newborn MeSH
- Publication type
- Comparative Study MeSH
