3-D Shape Estimation of DNA Molecules from Stereo Cryo-Electron Micro-Graphs Using a Projection-Steerable Snake

We introduce a three-dimensional (3-D) parametric active contour algorithm for the shape estimation of DNA molecules from stereo cryo-electron micrographs. We estimate the shape by matching the projections of a 3-D global shape model with the micrographs; we choose the global model as a 3-D filament with a B-spline skeleton and a specified radial profile. The active contour algorithm iteratively updates the B-spline coefficients, which requires us to evaluate the projections and match them with the micrographs at every iteration. Since the evaluation of the projections of the global model is computationally expensive, we propose a fast algorithm based on locally approximating it by elongated blob-like templates. We introduce the concept of projection-steerability and derive a projection-steerable elongated template. Since the two-dimensional projections of such a blob at any 3-D orientation can be expressed as a linear combination of a few basis functions, matching the projections of such a 3-D template involves evaluating a weighted sum of inner products between the basis functions and the micrographs. The weights are simple functions of the 3-D orientation and the inner-products are evaluated efficiently by separable filtering. We choose an internal energy term that penalizes the average curvature magnitude. Since the exact length of the DNA molecule is known a priori, we introduce a constraint energy term that forces the curve to have this specified length. The sum of these energies along with the image energy derived from the matching process is minimized using the conjugate gradients algorithm. We validate the algorithm using real, as well as simulated, data and show that it performs well

Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development

Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Parametric shape processing in biomedical imaging

In this thesis, we present a coherent and consistent approach for the estimation of shape and shape attributes from noisy images. As compared to the traditional sequential approach, our scheme is centered on a shape model which drives the feature extraction, shape optimization, and the attribute evaluation modules. In the first section, we deal with the detection of image features that guide the shape-extraction process. We propose a general approach for the design of 2-D feature detectors from a class of steerable functions, based on the optimization of a Canny-like criterion. As compared to previous computational designs, our approach is truly 2-D and yields more orientation selective detectors. We then address the estimation of the global shape from an image. Specifically, we propose to use cubic-spline-based parametric active contour models to solve two shape-extraction problems: (i) the segmentation of closed objects and (ii) the 3-D reconstruction of DNA filaments from their stereo cryo-electron micrographs. We present several enhancements of existing snake algorithms for segmentation. For the detection of 3-D DNA filaments from their orthogonal projections, we introduce the concept of projection-steerable matched filtering. We then use a 3-D snake algorithm to reconstruct the shape. Next, we analyze the efficiency of curve representations using refinable basis functions for the description of shape boundaries. We derive an exact expression for the error when we approximate a periodic signal in a scaling-function basis. Finally, we present a method for the exact computation of the area moments of such shapes

Effects of base-pair sequence, nicks and gaps on DNA minicircle shapes:analysis and experiment

DNA is a long polymer with the form of a double helix of about two nanometers diameter (2.10-9 meters). It is composed of nucleotides whose sequence carries the information of heredity. The four possible nucleotides have slightly different geometries, and their sequence along the DNA molecule are thought to influence the shape and the stiffness of the double helix on a scale of few tens to a few hundred base pairs, and thereby its biological activity. DNA minicircles (or miniplasmids) are closed loops with lengths of the order of a few tens or hundreds of base pairs in which the double helix bends around to close on its own tail with some number of twists. Its minimal energy shape and its energy of formation depend on the sequence of base pairs, and can be efficiently computed by polymer and rod models, if shape and stiffness parameters of DNA are provided as inputs. This structure is therefore an interesting experimental motif to test sequence-dependent mechanical properties of the DNA molecule. The purpose of this thesis is to explore the experimental methods to determine the shape and free energy of formation of DNA minicircles. The shapes have been be determined from cryo-electron micrographs. Efficiency of formation has been investigated by a novel method called annealing-cyclization. Cryo-electron microscopy allows observation of very small molecules (here 17 or 11 nm diameter) in vitrified water, in order to keep the 3D shape of the molecules as close as possible to the shape they had in solution. In Chapter 1, I used stereo cryo-electron micrographs to determine and compare the three-dimensional shape of 95 individual DNA minicircles of 158 base pairs, which were identical in sequence except within a 18 bp block which contained either a TATA box sequence or a CAP site. I defined the notion of shape-distance which I used to estimate the error of reconstruction, and I detected clusters of shapes using an appropriate sorting algorithm. However the cluster did not seem to be associated with the variable sequence (TATA or CAP). I then analyzed (in Chapter 2) two-dimensional shapes of shorter DNA minicircles (94 bp) determined by negative staining electron microscopy, designed with either two nicks (breaks in one of the strands) or two gaps (missing nucleotides) at diametrically opposite sites of the minicircles. I observed that the gapped minicircles have an elongated shape with respect to the nicked minicircles, and I used this result together with the results of atomic level molecular dynamics simulations to conclude that the gap flexibility, perhaps together with base unpairing at the gap site, is responsible for this elongated minicircle shape. Finally, I proposed a ligase-free assay to measure the minicircle formation efficiency, which can give a high yield of nicked minicircles. The method avoids the use of ligase and the associated concerns about the effect of ligase concentration on the measurements. I determined an equation from a chemical model of the reaction that fits the experimental data, and I defined the Ja factor which gives a measure of the cyclization yield independent of DNA initial concentration. This method seems to confirm that minicircles with two gaps cyclize more efficiently that minicircles with two nicks, probably because of the gap flexibility. As a perspective, the conclusions of this thesis could be used for the design of minicircle constructs whose shape would be sensitive enough to sequence mutation in order to be detected by electron microscopy. Such shapes could be then compared, thanks to the shape-distance tool defined herein, to shapes computed with known or putative DNA models