Linking acetylated α-Tubulin redistribution to α-Synuclein pathology in brain of Parkinson's disease patients.

Highly specialized microtubules in neurons are crucial to both health and disease of the nervous system, and their properties are strictly regulated by different post-translational modifications, including α-Tubulin acetylation. An imbalance in the levels of acetylated α-Tubulin has been reported in experimental models of Parkinson's disease (PD) whereas pharmacological or genetic modulation that leads to increased acetylated α-Tubulin successfully rescues axonal transport defects and inhibits α-Synuclein aggregation. However, the role of acetylation of α-Tubulin in the human nervous system is largely unknown as most studies are based on in vitro evidence. To capture the complexity of the pathological processes in vivo, we analysed post-mortem human brain of PD patients and control subjects. In the brain of PD patients at Braak stage 6, we found a redistribution of acetylated α-Tubulin, which accumulates in the neuronal cell bodies in subcortical structures but not in the cerebral cortex, and decreases in the axonal compartment, both in putamen bundles of fibres and in sudomotor fibres. High-resolution and 3D reconstruction analysis linked acetylated α-Tubulin redistribution to α-Synuclein oligomerization and to phosphorylated Ser 129 α-Synuclein, leading us to propose a model for Lewy body (LB) formation. Finally, in post-mortem human brain, we observed threadlike structures, resembling tunnelling nanotubes that contain α-Synuclein oligomers and are associated with acetylated α-Tubulin enriched neurons. In conclusion, we support the role of acetylated α-Tubulin in PD pathogenesis and LB formation.

Experimental protocol to investigate cortical, muscular and body representation alterations in adolescents with idiopathic scoliosis.

BACKGROUND: Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis. AIS is a three-dimensional morphological spinal deformity that affects approximately 1-3% of adolescents. Not all factors related to the etiology of AIS have yet been identified. OBJECTIVE: The primary aim of this experimental protocol is to quantitatively investigate alterations in body representation in AIS, and to quantitatively and objectively track the changes in body sensorimotor representation due to treatment. METHODS: Adolescent girls with a confirmed diagnosis of mild (Cobb angle: 10°-20°) or moderate (21°-35°) scoliosis as well as age and sex-matched controls will be recruited. Participants will be asked to perform a 6-min upright standing and two tasks-named target reaching and forearm bisection task. Eventually, subjects will fill in a self-report questionnaire and a computer-based test to assess body image. This evaluation will be repeated after 6 and 12 months of treatment (i.e., partial or full-time brace and physiotherapy corrective postural exercises). RESULTS: We expect that theta brain rhythm in the central brain areas, alpha brain rhythm lateralization and body representation will change over time depending on treatment and scoliosis progression as a compensatory strategy to overcome a sensorimotor dysfunction. We also expect asymmetric activation of the trunk muscle during reaching tasks and decreased postural stability in AIS. CONCLUSIONS: Quantitatively assess the body representation at different time points during AIS treatment may provide new insights on the pathophysiology and etiology of scoliosis.

Body-Related Attentional Bias in Adolescents Affected by Idiopathic Scoliosis.

Attentional biases toward body-related information increase body dissatisfaction. This can lead at-risk populations to develop psychopathologies. This phenomenon has not been extensively studied in girls affected by idiopathic scoliosis. This work aimed to study the cognitive processes that could contribute to the worsening and maintaining of body image disorders in adolescent idiopathic scoliosis. Twenty-eight girls were recruited and tested for body image dissatisfaction through the Scoliosis-Research-Society-22-revised (SRS-22r) questionnaire. Attentional biases towards disease-related body parts were assessed using a computerized visual match-to-sample task: girls were asked to answer as fast and accurately as possible to find the picture matching a target by pressing a button on a computer keyboard. Reaction times (RTs) and accuracy were collected as outcome variables and compared within and between groups and conditions. Lower scores in SRS-22r self-image, function, and total score were observed in scoliosis compared to the control group (p-value < 0.01). Faster response times (p-value = 0.02) and higher accuracy (p-value = 0.02) were detected in the scoliosis group when processing shoulders and backs (i.e., disease-relevant body parts). A self-body advantage effect emerged in the scoliosis group, showing higher accuracy when answering self-body stimuli compared to others' bodies stimuli (p-value = 0.04). These results provide evidence of body image dissatisfaction and attentional bias towards disease-relevant body parts in girls with scoliosis, requiring clinical attention as highly predisposing to psychopathologies.

Is higher compliance to brace therapy associated with poorer quality of life and self-image? A 36-months follow-up study.

BACKGROUND: Brace treatment is the most effective tool for avoiding curve progression in moderate adolescent idiopathic scoliosis and high adherence is required to achieve therapeutic success. Despite this, the compliance often is impaired by the concern about the psychological well-being of adolescents. OBJECTIVE: This 36-month follow-up study investigated if the patients most adherent to brace treatment could report a stronger impairment in the quality of life and body image. METHODS: 64 adolescents with idiopathic scoliosis responded to the Scoliosis Research Society-22 revised Patient Questionnaire at 12, 24, and 36 months after prescription of a TLSO rigid brace. Retrospectively, participants who wore a brace for more than 75% of the prescribed time were assigned to the good-compliance group (GC); the others formed the poor-compliance group (PC). RESULTS: At 12 months the GC group showed higher scores in treatment satisfaction and at 36 months they did not differ from the PC group in the overall SRS-22r score. Moreover, they achieved a statistically significant improvement in the scoliosis severity, although they showed lower scores in the self-image domain. CONCLUSION: In our patient's cohort, increased brace adherence does not compromise QoL and provides better treatment outcomes. However, more attention is needed to maintain good self-perception.

Brain oscillatory activity in adolescent idiopathic scoliosis.

Pathophysiology of Adolescent Idiopathic Scoliosis (AIS) is not yet completely understood. This exploratory study aims to investigate two aspects neglected in clinical practice: a defective postural central nervous system control in AIS, and alterations of body schema due to scoliosis spinal deformities. We recorded EEG data and balance data in four different standing positions in 14 adolescents with AIS and in 14 controls. A re-adaptation of the Image Marking Procedure (IMP) assessed body schema alterations on the horizontal (Body Perception Indices (BPIs)) and vertical direction (interacromial and bisiliac axes inclinations). Our results revealed no differences in balance control between groups; higher EEG alpha relative power over sensorimotor areas ipsilateral to the side of the curve and a significant increase of theta relative power localized over the central areas in adolescents with AIS. The difference in BPI shoulder and BPI waist significantly differed between the two groups. The inclinations of the perceived interacromial axes in adolescents with AIS was opposite to the real inclination. Increased theta activity and alpha lateralization observed may be a compensatory strategy to overcome sensorimotor dysfunction mirrored by altered body schema. Scoliosis onset might be preceded by sensorimotor control impairments that last during curve progression.

Influence of pain-related psychological factors on therapeutic outcomes in patients with chronic low back pain after oxygen-ozone treatment: a case-series.

The aim of the study was to identify the variables related to therapeutic success of intramuscular oxygen-ozone (O2O3) in patients with chronic low back pain (LBP). Twenty-one patients underwent an eight-session intramuscular-paravertebral O2O3 treatment with a weekly frequency. Numeric Rating Score for pain (NRSp), anxiety (NRSa), mood (NRSm), Oswestry Disability Index (ODI) and Physical and Mental Component Summary scores (PCS and MCS) of Short Form 12 (SF-12) were assessed baseline, after the treatment and at six-months follow-up. The ODI and NRSp scores showed significant improvement at the end of the treatment: the improvement in pain was maintained also at 6 months' follow-up. A significant correlation between baseline NRSa and the variation of the NRSp and ODI was observed. There was a correlation between NRSm at baseline and the variation of the ODI. A moderate correlation between MCS-12 at baseline and the variation of the NRSp score was found. Surprisingly, patients with a more compromised psychological well-being due to LBP had better results after oxygen-ozone therapy. Therefore, NRSa, NRSm and MCS-12 scores could be useful predictors of good therapeutic outcome.

A model to differentiate WAD patients and people with abnormal pain behaviour based on biomechanical and self-reported tests.

The prevalence of malingering among individuals presenting whiplash-related symptoms is significant and leads to a huge economic loss due to fraudulent injury claims. Various strategies have been proposed to detect malingering and symptoms exaggeration. However, most of them have been not consistently validated and tested to determine their accuracy in detecting feigned whiplash. This study merges two different approaches to detect whiplash malingering (the mechanical approach and the qualitative analysis of the symptomatology) to obtain a malingering detection model based on a wider range of indices, both biomechanical and self-reported. A sample of 46 malingerers and 59 genuine clinical patients was tested using a kinematic test and a self-report questionnaire asking about the presence of rare and impossible symptoms. The collected measures were used to train and validate a linear discriminant analysis (LDA) classification model. Results showed that malingerers were discriminated from genuine clinical patients based on a greater proportion of rare symptoms vs. possible self-reported symptoms and slower but more repeatable neck motions in the biomechanical test. The fivefold cross-validation of the LDA model yielded an area under the curve (AUC) of 0.84, with a sensitivity of 77.8% and a specificity of 84.7%.

An Analysis of Eating Disorders in Adolescent Idiopathic Scoliosis: A Prospective Cross-sectional Study in a Female Population.

STUDY DESIGN: Prospective cross-sectional study. OBJECTIVE: We designed this study to investigate whether adolescent girls with idiopathic scoliosis show a predisposition for eating disorders (EDs) and alterations of the quality of life and body image self-perception, compared to same-age healthy females. SUMMARY OF BACKGROUND DATA: Idiopathic scoliosis is the most common spinal deformity of adolescence. Recent findings about the impairment of the self-body image in adolescents with idiopathic scoliosis provide a common trait of scoliosis and EDs and could lead to the suspicion of an association between these two pathological conditions. Despite this, current literature shows the lack of evident results about the impact of adolescent idiopathic scoliosis (AIS) on the possibility to develop of EDs. METHODS: One hundred forty-four females with diagnosis of AIS (aged 10-18 years) formed the scoliosis group. One hundred forty-six same-age healthy girls were enrolled in the control group. For all subjects, we considered sport practice. Only for Scoliosis Group, we also considered the severity of scoliosis, the use of brace and the practice of physiotherapy scoliosis-specific exercises. The participants were asked to answer to the Scoliosis Research Society-22 revised Patient Questionnaire (SRS-22r) and the Eating Disorders Inventory (EDI). RESULTS: In the scoliosis group, significantly lower scores on the SRS-22r total and in the self-image domain were detected. The two groups showed differences in the total EDI score and in the body dissatisfaction EDI's domain. Severity of scoliosis was correlated with worse SRS-22r total score and SRS-22r self-image domain score. There were no differences in the scores of the SRS-22r and EDI between braced and nonbraced subjects. Lower scores in SRS-22r total and self-image domain were found in girls who practiced physiotherapy exercises. Subjects who practiced a sport showed higher SRS-22r total scores. CONCLUSION: The AIS cohort in our study demonstrated lower levels of eating psychopathology than healthy controls. Surprisingly, eating behavior does not seem to be affected by orthotic management. However, quality of life and self-body image could be impaired in scoliotic girls, especially when they practice physiotherapy exercises, whereas those who practice sport seem to be preserved in this regard.Level of Evidence: 4.

WHIRLY2 plays a key role in mitochondria morphology, dynamics, and functionality in Arabidopsis thaliana.

WHIRLY2 is a single-stranded DNA binding protein associated with mitochondrial nucleoids. In the why 2-1 mutant of Arabidopsis thaliana, a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild-type and why 2-1 mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the why 2-1 mutant. In line with that, the expression level of why 2 in seeds of wild-type plants is higher than that of why 3, whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the why 2-1 mutant, although not in seeds, the expression level of why 3 is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the why 2-1 mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, in organello protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of why 2 and why 3 predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant.

The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione.

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.

α-Synuclein is a Novel Microtubule Dynamase.

α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2ß2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

Two classes of short intraflagellar transport train with different 3D structures are present in Chlamydomonas flagella.

Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these 'narrow' and 'wide', and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16 nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.

Three-Dimensional Reconstruction, by TEM Tomography, of the Ultrastructural Modifications Occurring in Cucumis sativus L. Mitochondria under Fe Deficiency.

BACKGROUND: Mitochondria, as recently suggested, might be involved in iron sensing and signalling pathways in plant cells. For a better understanding of the role of these organelles in mediating the Fe deficiency responses in plant cells, it is crucial to provide a full overview of their modifications occurring under Fe-limited conditions. The aim of this work is to characterize the ultrastructural as well as the biochemical changes occurring in leaf mitochondria of cucumber (Cucumis sativus L.) plants grown under Fe deficiency. METHODOLOGY/RESULTS: Mitochondrial ultrastructure was investigated by transmission electron microscopy (TEM) and electron tomography techniques, which allowed a three-dimensional (3D) reconstruction of cellular structures. These analyses reveal that mitochondria isolated from cucumber leaves appear in the cristae junction model conformation and that Fe deficiency strongly alters both the number and the volume of cristae. The ultrastructural changes observed in mitochondria isolated from Fe-deficient leaves reflect a metabolic status characterized by a respiratory chain operating at a lower rate (orthodox-like conformation) with respect to mitochondria from control leaves. CONCLUSIONS: To our knowledge, this is the first report showing a 3D reconstruction of plant mitochondria. Furthermore, these results suggest that a detailed characterization of the link between changes in the ultrastructure and functionality of mitochondria during different nutritional conditions, can provide a successful approach to understand the role of these organelles in the plant response to Fe deficiency.

Electron tomography of IFT particles.

Cilia and flagella play very important roles in eukaryotic cells, ranging from cell motility to chemo- and mechanosensation with active involvement in embryonic development and control of cell division. Cilia and flagella are highly dynamic organelles undergoing constant turnover at their tip, where multiprotein precursors synthesized in the cell cytoplasm are assembled, turnover products are released and carried back for recycling. Such bidirectional trafficking is maintained by an ATP-dependent active transport that is carried out by intraflagellar transport (IFT) particles. Despite our knowledge of the cell biology, the genomic, and the biochemistry of IFT, high-resolution 3D models for IFT are still missing. To date, the only information on the 3D structure of IFT come from our analysis of full-length flagella from the biflagellate green alga Chlamydomonas reinhardtii: the model organism where IFT was discovered and first characterized. In this chapter, we describe and discuss the strategy we implemented to produce the first 3D models of in situ IFT trains in flat-embedded flagella. We provide detailed information about the acquisition of tomographic images, the simultaneous alignment of the double-tilt tomographic series, and the analysis of the tomograms by subtomogram averaging for the generation of detailed 3D models of IFT particles.

Simultaneous alignment of dual-axis tilt series.

We present a strategy for the alignment of dual-axis tomographic series, based on reference points and simultaneous alignment of both series. Each series is first aligned individually, an affine transformation is determined to bring the two series in a unique reference system, and all experimental coordinates are combined in a single system of equations. In case of severe shrinkage, a global and a local refinement of the orientation parameters are performed to correct all minors misalignments. The strategy is illustrated on tomographic experiments performed on sections from plastic-embedded biological samples. The efficiency in correcting the misalignment of gold particles and in improving the quality of the reconstruction is documented both visually and quantitatively. In our approach every region of the tomogram is associated with its own orientation parameters and can be eventually reconstructed with the preferred algorithm. This is convenient in the computation of 3D averages of equivalent structures. A simulation experiment is presented to show that the performances of this approach are superior to those of the method of rotation in direct space.

Electron-tomographic analysis of intraflagellar transport particle trains in situ.

Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

Conical electron tomography of a chemical synapse: polyhedral cages dock vesicles to the active zone.

In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical "average" synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of approximately 3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct "synaptic units" comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured approximately 60 nm in diameter, with a density of approximately 44/microm2 and were associated with vesicles at the active zone ("first tier"). Vesicles in this first-tier position represented approximately 7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (approximately 4% of total), or fully fused (approximately 0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release.

JUST (Java User Segmentation Tool) for semi-automatic segmentation of tomographic maps.

We are presenting a program for interactive segmentation of tomographic maps, based on objective criteria so as to yield reproducible results. The strategy starts with the automatic segmentation of the entire volume with the watershed algorithm in 3D. The watershed regions are clustered successively by supervised classification, allowing the segmentation of known organelles, such as membranes, vesicles and microtubules. These organelles are processed with topological models and input parameters manually derived from the tomograms. After known organelles are extracted from the volume, all other watershed regions can be organized into homogeneous assemblies on the basis of their densities. To complete the process, all voxels in the volume are assigned either to the background or individual structures, which can then be extracted for visualization with any rendering technique. The user interface of the program is written in Java, and computational routines are written in C. For some operations, involving the visualization of the tomogram, we refer to existing software, either open or commercial. While the program runs, a history file is created, that allows all parameters and other data to be saved for the purposes of comparison or exchange. Initially, the program was developed for the segmentation of synapses, and organelles belonging to these structures have thus far been the principal targets modeled with JUST. Since each organelle is clustered independently from the rest of the volume, however, the program can accommodate new models of different organelles as well as tomograms of other types of preparations of tissue, such as cytoskeletal components in vitreous ice.

Local refinement: an attempt to correct for shrinkage and distortion in electron tomography.

A critical problem in electron tomography is the deformation of the specimen due to radiation, or "shrinkage," which interferes with image alignment and thereby limits resolution. Here, we describe a general strategy for refining preliminary reconstructions which allows the damage due to the shrinkage of plastic-embedded thin sectioned specimens (50-80 nm) to be corrected. The basic steps of the strategy involve: (a) the partition of the preliminary reconstruction into sub-volumes; (b) the extraction of corresponding sub-areas for each sub-volume from the micrographs of the tilt series; (c) the re-projection of each sub-volume according to the orientation parameters; and (d) the refinement of these parameters by correlating each sub-area to the corresponding computed projection. We tested the strategy by refining chemical synapses reconstructed from series imaged with conical, double and single tilt geometries. The results gathered with local refinement were evaluated by visually inspecting the structure of biological membranes in the maps. In an effort to quantify these improvements, we studied the refined maps using correlation criteria and mapped the corrections applied to the orientation parameters in each sub-volume of the reconstruction. Simulation experiments complemented the data gathered by correlation analysis. Based on these criteria, we concluded that local refinement significantly improves the overall quality of the reconstructions of chemical synapses calculated from series imaged with conical and double tilt geometries.

Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography.

We present here for the first time a 3D reconstruction of in situ axonemal outer dynein arms. This reconstruction has been obtained by electron tomography applied to a series of tilted images collected from metal replicas of rapidly frozen, cryofractured, and metal-replicated sperm axonemes of the cecidomid dipteran Monarthropalpus flavus. This peculiar axonemal model consists of several microtubular laminae that proved to be particularly suitable for this type of analysis. These laminae are sufficiently planar to allow the visualization of many dynein molecules within the same fracture face, allowing us to recover a significant number of equivalent objects and to improve the signal-to-noise ratio of the reconstruction by applying advanced averaging protocols. The 3D model we obtained showed the following interesting structural features: First, each dynein arm has two head domains that are almost parallel and are obliquely oriented with respect to the longitudinal axis of microtubules. The two heads are therefore positioned at different distances from the surface of the A-tubule. Second, each head domain consists of a series of globular subdomains that are positioned on the same plane. Third, a stalk domain originates as a conical region from the proximal head and ends with a small globular domain that contacts the B-tubule. Fourth, the stem region comprises several globular subdomains and presents two distinct points of anchorage to the surface of the A-tubule. Finally, and most importantly, contrary to what has been observed in isolated dynein molecules adsorbed to flat surfaces, the stalk and the stem domains are not in the same plane as the head.