Francesca Cantele

Electron-tomographic analysis of intraflagellar transport particle trains in situ

Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains.

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 ∼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 ∼60 nm in diameter, with a density of ∼44/μm2 and were associated with vesicles at the active zone (“first tier”). Vesicles in this first-tier position represented ∼7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (∼4% of total), or fully fused (∼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.

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

ABSTRACT 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. Highlighted article: In-depth analyses of IFT train ultrastructure in regenerating Chlamydomonas flagella shows the occurrence of three train types with different 3D structure, implicating differences in function.

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.

The variance of icosahedral virus models is a key indicator in the structure determination: a model-free reconstruction of viruses, suitable for refractory particles

A model-free method to determine the three-dimensional structure of icosahedral viruses is described. The novel strategy is based upon the approximate principle that correct virus structures have high variance as do all other well-detailed structures, even wrong ones. The original projections of individual particles are reduced to a radius of 25 pixels and are used to compute single particle reconstruction models by assigning them 1800 different Euler triads. The variance of the models obtained from all projections is stored in maps and a decimation process is carried out. In a first stage, thresholds are adopted for the variance values, and in a second stage, carried out by correspondence analysis and classification, 30 clusters of models are sorted out. The clusters are refined to yield models contained in boxes of 64(3) voxels. The refined models with highest variance and closest similarity represent the correct solution. Once enlarged, these models can be used to align all available projections in their original scale in a customary projection-matching process. The method has proved successful in determining the structures of poliovirus, of the empty and filled capsids of L-A virus, and of a modified capsid of hepatitis B virus.

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 α2β2 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.

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.

Symmetry embedding in the reconstruction of macromolecular assemblies via the discrete Radon transform

In this paper we discuss the embedding of symmetry information in an algorithm for three-dimensional reconstruction, which is based on the discrete Radon transform. The original algorithm was designed for randomly oriented and in principal asymmetric particles. The expanded version presented here covers all symmetry point groups which can be exhibited by macromolecular protein assemblies. The orientations of all symmetry equivalent projections, based on the orientation of an experimental projection, are obtained using global group operators. Further, an improved interpolation scheme for the recovery of the three-dimensional discrete Radon transform has been designed for greater computational efficiency. The algorithm has been tested on phantom structures as well as on real data, a virus structure possessing icosahedral symmetry.

Correspondence analysis of sinogram lines. Sinogram trajectories in factor space replace raw images in the orientation of projections of macromolecular assemblies

The lines of a large group of sinograms of projections with random orientation can be submitted to correspondence analysis. Since the number of samples per line is small, a small matrix is accumulated which is quick to orthogonalise. In the eigenvector space, the lines of a sinogram are represented by points describing a closed trajectory. Two trajectories of different sinograms intersect in the position of their common line. Determining where two trajectories cross each other is a problem of minimum chi2 distance in the space of 5-7 eigenvectors. An algorithm to determine common lines has been implemented and tested with phantom projections oriented at random, and corrupted with noise. The images were simulating a set collected with the two exposures technique, already proposed by the authors for three dimensional reconstruction from random projections. The preliminary models obtained with the new algorithm have been refined by a projection matching based on trajectories. This step requires determining which trajectory, in a set representing computed projections, matches at best with that of an experimental projection. This is a problem of minimum distance in a space with low dimensionality. The present algorithms, based on chi2 distances, run much faster than those based on correlation analysis and the quality of the reconstructed phantoms looks satisfactory.

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.