A Common Image Representation And A Patch-Based Search For Correlative Light-Electron-Microscopy (Clem) Registration

International audienceCorrelative light-electron microscopy (CLEM) enables to relate dynamics (or functions) with structure for a better understanding of cell mechanisms. However, the LM and EM images are of very different size, spatial resolution, field of view, and appearance. Registration of LM and EM modalities is then a timely, important but difficult open problem, which still requires some manual assistance. We have designed an original automated CLEM retracing-and-registration method involving a common representation with an adaptive associated scale (or blurring), the determination of the EM patch geometry, and the specification of appropriate descriptors and similarity criterion for the EM patch search. Its efficiency is demonstrated on real CLEM images

Intensity-based matching and registration for 3D correlative microscopy with large discrepancies

International audienceCorrelative microscopy, especially light and electron microscopy (CLEM), enables the study of cells and subcellular elements in complementary ways, provided a reliable registration between images is efficiently achievable. We propose a general automatic registration method. Due to large discrepancies in appearance, field-of-view, resolution and position, a pre-alignment stage is required before any 3D fine registration stage. We define an intensity-based method for both stages, which leverages a common representation of the two involved image modalities. We report experimental results on different real datasets of 3D correlative microscopy, demonstrating time efficiency and overlay accuracy

Correlated Multimodal Imaging in Life Sciences:Expanding the Biomedical Horizon

International audienceThe frontiers of bioimaging are currently being pushed toward the integration and correlation of several modalities to tackle biomedical research questions holistically and across multiple scales. Correlated Multimodal Imaging (CMI) gathers information about exactly the same specimen with two or more complementary modalities that-in combination-create a composite and complementary view of the sample (including insights into structure, function, dynamics and molecular composition). CMI allows to describe biomedical processes within their overall spatio-temporal context and gain a mechanistic understanding of cells, tissues, diseases or organisms by untangling their molecular mechanisms within their native environment. The two best-established CMI implementations for small animals and model organisms are hardware-fused platforms in preclinical imaging (Hybrid Imaging) and Correlated Light and Electron Microscopy (CLEM) in biological imaging. Although the merits of Preclinical Hybrid Imaging (PHI) and CLEM are well-established, both approaches would benefit from standardization of protocols, ontologies and data handling, and the development of optimized and advanced implementations. Specifically, CMI pipelines that aim at bridging preclinical and biological imaging beyond CLEM and PHI are rare but bear great potential to substantially advance both bioimaging and biomedical research. CMI faces three mai

Automated Correlative Light and Electron Microscopy using FIB-SEM as a tool to screen for ultrastructural phenotypes

In Correlative Light and Electron Microscopy (CLEM), two imaging modalities are combined to take advantage of the localization capabilities of light microscopy (LM) to guide the capture of high-resolution details in the electron microscope (EM). However, traditional approaches have proven to be very laborious, thus yielding a too low throughput for quantitative or exploratory studies of populations. Recently, in the electron microscopy field, FIB-SEM (Focused Ion Beam -Scanning Electron Microscope) tomography has emerged as a flexible method that enables semi-automated 3D volume acquisitions. During my thesis, I developed CLEMSite, a tool that takes advantage of the semi-automation and scanning capabilities of the FIB-SEM to automatically acquire volumes of adherent cultured cells. CLEMSite is a combination of computer vision and machine learning applications with a library for controlling the microscope ( product from a collaboration with Carl Zeiss GmbH and Fibics Inc.). Thanks to this, the microscope was able to automatically track, find and acquire cell regions previously identified in the light microscope. More specifically, two main modules were implemented. First, a correlation module was designed to detect and record reference points from a grid pattern present on the culture substrate in both modalities (LM and EM). Second, I designed a module that retrieves the regions of interest in the FIB-SEM and that drives the acquisition of image stacks between different targets in an unattended fashion. The automated CLEM approach is demonstrated on a project where 3D EM volumes are examined upon multiple siRNA treatments for knocking down genes involved in the morphogenesis of the Golgi apparatus. Additionally, the power of CLEM approaches using FIB-SEM is demonstrated with the detailed structural analysis of two events: the breakage of the nuclear envelope within constricted cells and an intriguing catastrophic DNA Damage Response in binucleated cells. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible and that EM can provide incredible insights to guide new biological discoveries

In situ architecture of the ER–mitochondria encounter structure

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1,2,3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum–mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4,5,6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.<br/

Development and application of electron microscopy methods for endocytic-secretory pathway studies in cells

Electron microscopy (EM) and tomography provides the most detailed view of the cellular ultrastructure and can be further extended to the structural level using cryogenic electron tomography. Fluorescent microscopy (FM) is used to complement this data with molecular identity information in a wide range of correlative microscopy techniques. In this thesis I present my work on the development of the correlative microscopy methods as well as the application of new and established electron microscopy methods to the study of the endocytic and secretory pathways. I developed a sample parallelization approach based on the fluorescent barcoding of budding yeast cells that allows high-throughput screening of yeast mutants using EM. Cells from different strains or under different conditions are grown in parallel and then subjected to combinatorial labeling with fluorescent dyes. Labeled cells are mixed together to generate a single sample which is subjected to high-pressure freezing, freeze-substitution and sectioning. The sections are imaged with FM and EM. FM data is used to determine the fluorescent barcode of each cell and thus its strain identity or experimental conditions, and high-resolution EM data can be collected in parallel for each of the strains or conditions. The total time spent on embedding and sectioning can be reduced up to 30 times using the developed protocol. I demonstrate the utility of the method by analyzing the variation of total multivesicular body volume (MVB) in different yeast strains. As a part of the collaborative project investigating the role of the ATPase Vps4 in the formation of MVBs, I performed correlative FM and electron tomography of MVBs containing Vps4. It showed that MVBs correlating with the Vps4 signal usually form clusters of more than one organelle and that the Vps4 signal correlates with MVBs actively forming intraluminal vesicles. Finally, I used subtomogram averaging to determine the COPI coat structure in situ, within its native cellular environment. I analyzed a tomographic dataset of cryo-lamella prepared by collaborators using focused ion beam milling of vitrified Chlamydomonas reinhardtii cells. I determined the COPI coat structure de novo and analyzed its variability during uncoating and within the Golgi stack. The COPI coat preserved its structure and stoichiometry during uncoating and in different Golgi regions. However the density of bound dilysine cargo and membrane thickness varied along the stack. In this thesis I have applied different EM methods to investigate morphological and structural aspects of the endocytic-secretory pathway. In the future such an integrative EM approach, ranging from functional genetic screens to structure determination, can be used to address multiple questions in cell biology

Actin synchronizes chromosome capture by microtubules in starfish oocyte meiosis

Chromosome capture by microtubules is an early step of cell division essential for alignment and subsequent separation of sister chromatids. Failure to transmit even one chromosome results in aneuploidy, a common cause of infertility, genetic disorders or cancer. The canonical mechanism of ‘search and capture’ by dynamic astral microtubules has been validated by recent studies, and additionally revealed mechanisms that facilitate microtubule search, ensuring rapid and efficient capture of chromosomes in somatic cells. However, in specialized cell types such as oocytes with large nucleus chromosomes are located much further from microtubule asters. In these cells, the models that work in small somatic cells are insufficient to explain chromosome capture. Recently, the Lénárt group has shown that in starfish oocytes an actin-driven mechanism facilitates chromosome congression and is required to prevent chromosome loss: a contractile actin meshwork transports chromosomes to within the capture range of microtubule asters of approximately 30 µm. How these actin- and microtubule-driven mechanisms of chromosome capture are coordinated remained an open question. Here, I investigated the cooperation between the actin meshwork transporting chromosomes and capture by microtubules in meiosis of starfish oocytes using high spatio-temporal resolution tracking of chromosome motion in 3D combined with drug-perturbation experiments. This assay allowed me to characterize chromosome capture kinetics during the two-staged chromosome congression under different conditions. I find that the actin meshwork, while transporting the distal chromosomes to the vicinity of microtubule asters, also synchronizes their capture. I show that this synchronizing effect is due to an actin-dependent block of chromosome capture active for approx. 5 minutes after NEBD. As a result, chromosomes close to microtubule asters – that in principle could be captured immediately after NEBD – are captured simultaneously with chromosomes transported from distal nuclear locations by the actin meshwork at approx. 5-15 minutes after NEBD and independent of their distance from the asters. I show that this delay in the capture of the proximally located chromosomes cannot be explained by altered microtubule dynamics when growing through the actin meshwork. The delay is also not the consequence of physical entrapment in the actin network ‘holding back’ chromosomes, because capture is not delayed in slowed or even fully stabilized actin networks. Together, my results point to an actin-dependent mechanism, which prevents the formation of lateral kinetochore-microtubule attachments. Synchronous disassembly of these F-actin structures exposes kinetochores and thereby synchronizes chromosome capture. This is a first description of a mechanism by which the actin cytoskeleton directly affects spindle assembly, and which actively controls and coordinates chromosome search and capture. I show how this mechanism coordinates chromosome congression in the specialized oocyte nucleus, but it is interesting to speculate whether such mechanisms may have a broader relevance for example to synchronize mitotic events such as cell rounding mediated by the actin cytoskeleton with spindle assembly. The detailed molecular mechanism of how F-actin prevents chromosome-microtubule attachment remains an exciting open question for the future studies

Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

© 2020. Published by The Company of Biologists Ltd.The yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome–ER contacts. Upon deletion of PEX24 or PEX32 – and to a much lesser extent, of PEX23 or PEX29 – peroxisome–ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome–ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome–ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.This work was supported by a grant from the FP7 People: Marie-Curie Actions Initial Training Networks (ITN) program PerFuMe (Grant Agreement Number 316723) to N.B., D.P.D. and I.J.v.d.K., from the China Scholarship Council (CSC) to F.W., and from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek/Chemical Sciences (NWO/CW) to A.A. (711.012.002)