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Methods in Cell Biology

Helder Maiato, Melina Schuh
No abstract text is available yet for this article.
2018: Methods in Cell Biology
Ana C F Bolhaqueiro, Richard H van Jaarsveld, Bas Ponsioen, René M Overmeer, Hugo J Snippert, Geert J P L Kops
Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids...
2018: Methods in Cell Biology
Nike Walther, Jan Ellenberg
No abstract text is available yet for this article.
2018: Methods in Cell Biology
Norbert Lindow, Stefanie Redemann, Florian Brünig, Gunar Fabig, Thomas Müller-Reichert, Steffen Prohaska
Mitotic and meiotic spindles are microtubule-based structures to faithfully segregate chromosomes. Electron tomography is currently the method of choice to analyze the three-dimensional (3D) architecture of both types of spindles. Over the years, we have developed methods and software for automatic segmentation and stitching of microtubules in serial sections for large-scale reconstructions. 3D reconstruction of microtubules, however, is only the first step toward biological insight. The second step is the analysis of the structural data to derive measurable spindle properties...
2018: Methods in Cell Biology
Daniel Bollschweiler, Laura Radu, Luca Pellegrini
The synaptonemal complex (SC) forms during the early stages of meiotic prophase I, when it mediates the pairing of homologous chromosomes. Despite the crucial role of the SC in chromosome synapsis and genetic recombination, the molecular details of its function are still unclear. High-resolution information on the structure of SC proteins would be very valuable to elucidate the molecular basis of their function in meiosis. Here we show how cryo-electron tomography and subtomographic averaging can be usefully applied to provide insights into the structure of the helical SYCP3 protein in its filamentous state...
2018: Methods in Cell Biology
Katharina Schücker, Markus Sauer, Ricardo Benavente
This chapter describes how two different superresolution microscopy techniques, namely, structured illumination microscopy and direct stochastic optical reconstruction microscopy, can be used to analyze the molecular architecture of the synaptonemal complex. The experimental protocols provided allow the construction of precise localization maps for different synaptonemal complex proteins.
2018: Methods in Cell Biology
Agata P Zielinska, Melina Schuh
Human eggs frequently carry an incorrect number of chromosomes, which is a leading cause of pregnancy loss and congenital disorders. The origins of high aneuploidy rates in human eggs have remained largely unclear. This is due to two main reasons: first, the availability of human eggs is limited so that studies of fixed human eggs typically involve very small numbers and limited quantifications. Second, methods for studying meiosis in live human eggs have been missing. The ever rising prevalence of Assisted Reproductive Technologies has facilitated a recent breakthrough in the field...
2018: Methods in Cell Biology
Mariia Burdyniuk, Natalia Wesolowska, Michal Fleszar, Matthia A Karreman, Pedro Machado, Joana Borrego-Pinto, Bernhard Ruthensteiner, Yannick Schwab, Péter Lénárt
The rapid and synchronous divisions of large and transparent oocytes, eggs, and embryos of marine species are exceptionally well suited for microscopic observation. Consequently, these cells have been models for cell division research since its beginnings and contributed some of its first and most fundamental discoveries. While large size and rapid transitions render these cells ideal specimens for light microscopy, the same features constitute a challenge for electron microscopy. Here, we describe example protocols from our work on starfish oocyte meiosis, where we overcome these challenges by using live imaging of fluorescently labeled structures in combination with correlated electron microscopy...
2018: Methods in Cell Biology
Nicholas I Clarke, Stephen J Royle
The mitotic spindle is a complex structure that coordinates the accurate segregation of chromosomes during cell division. To understand how the mitotic spindle operates at the molecular level, high resolution imaging is needed. Serial block face-scanning electron microscopy (SBF-SEM) is a technique that can be used to visualize the ultrastructure of entire cells, including components of the mitotic spindle such as microtubules, kinetochores, centrosomes, and chromosomes. Although transmission electron microscopy (TEM) has higher resolution, the reconstruction of large volumes using TEM and tomography is labor intensive, whereas SBF-SEM takes only days to process, image, and segment samples...
2018: Methods in Cell Biology
Judith Reichmann, Manuel Eguren, Yu Lin, Isabell Schneider, Jan Ellenberg
Systematic studies of cell divisions at the beginning of mammalian life are of fundamental importance for our understanding of embryonic development and fertility. However, in the past the challenges of in vitro embryo culture and the embryo's pronounced light sensitivity have precluded a detailed investigation of preimplantation cell divisions. This protocol is based on recent technological breakthroughs in inverted light microscopy tailored for mouse embryology. Due to its reduced light dose, and therefore low phototoxicity, as well as higher acquisition speed, light-sheet microscopy allows extended 3D time-lapse imaging of early embryonic development with very high spatial and temporal resolution...
2018: Methods in Cell Biology
Hiroki Shibuya, Yoshinori Watanabe
For the analysis of the molecular mechanisms underlying mammalian meiosis, the establishment of a transient gene expression system for meiocytes has been long awaited. We have established an efficient in vivo electroporation method for live mouse testis and demonstrate short-term transgene expression in spermatocytes. By expressing specific marker proteins fused with GFP, this technique is applicable not only to fixed cell observations after transgene expression but also to live imaging to dissect dynamic cellular events in live spermatocytes...
2018: Methods in Cell Biology
James R LaFountain, Christopher S Cohan
This chapter covers methods that are useful for the in vitro culture and live-cell study of insect spermatocytes in general and of crane-fly spermatocytes in particular. The merits of crane-fly spermatocytes are detailed in the Introduction section. In the following sections, step-by-step instructions are given for optimizing visualization of meiotic events taking place within living spermatocytes by employing microaspiration to flatten cells and then in subsequent operations to manipulate them via microinjection...
2018: Methods in Cell Biology
Pierre Romé, Hiroyuki Ohkura
Studies using Drosophila have played pivotal roles in advancing our understanding of molecular mechanisms of mitosis throughout the past decades, due to the short generation time and advanced genetic research of this organism. Drosophila is also an excellent model to study female meiosis in oocytes. Pathways such as the acentrosomal assembly of the meiotic spindle in oocytes are conserved from fly to humans. Collecting and manipulating large Drosophila oocytes for microscopy and biochemistry are both time and cost efficient, offering advantages over mouse or human oocytes...
2018: Methods in Cell Biology
Kimberley Laband, Benjamin Lacroix, Frances Edwards, Julie C Canman, Julien Dumont
Caenorhabditis elegans is a self-fertilizing hermaphroditic worm. A single C. elegans worm therefore produces both male and female gametes that fuse to generate embryos. While sperm production stops at the end of the C. elegans larval development, oocytes are continuously generated and fertilized during the entire reproductive life of the adult worm. The molecular and cellular mechanisms involved in gametogenesis and the early embryonic divisions are highly conserved between worms and humans; thus C. elegans is a powerful model to study meiotic and mitotic cell divisions in a metazoan system...
2018: Methods in Cell Biology
Ana Milas, Mihaela Jagrić, Jelena Martinčić, Iva M Tolić
At the onset of mitosis, cells assemble the mitotic spindle, a dynamic micromachine made of microtubules and associated proteins. Although most of these proteins have been identified, it is still unknown how their collective behavior drives spindle formation and function. Over the last decade, RNA interference has been the main tool for revealing the role of spindle proteins. However, the effects of this method are evident only after a longer time period, leading to difficulties in the interpretation of phenotypes...
2018: Methods in Cell Biology
Yuta Shimamoto, Tarun M Kapoor
Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules...
2018: Methods in Cell Biology
Charles A Day, Jessica Hornick, Alyssa Langfald, Christopher Mader, Edward H Hinchcliffe
The use of microtechnique for studying cell division is well established (Begg & Ellis, 1979; Wadsworth, 1999; Zhang & Nicklas, 1999). The advantage of microinjection in cell division research is the timed delivery of a macromolecules at a particular stage of mitosis (for example, pre- vs postanaphase), which can circumvent the spindle assembly checkpoint (Hinchcliffe et al., 2016). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al...
2018: Methods in Cell Biology
Petra Vyplelová, Miroslav Ovečka, George Komis, Jozef Šamaj
Mitotic cell division in plants is a dynamic process playing a key role in plant morphogenesis, growth, and development. Since progress of mitosis is highly sensitive to external stresses, documentation of mitotic cell division in living plants requires fast and gentle live-cell imaging microscopy methods and suitable sample preparation procedures. This chapter describes, both theoretically and practically, currently used advanced microscopy methods for the live-cell visualization of the entire process of plant mitosis...
2018: Methods in Cell Biology
Iskra Yanakieva, Marija Matejčić, Caren Norden
Tissue growth and organismal development require orchestrated cell proliferation. To understand how cell division guides development, it is important to explore mitosis at the tissue-wide, cellular, and subcellular scale. At the tissue level this includes determining a tissue's mitotic index, at the cellular level the tracing of cell lineages, and at the subcellular level the characterization of intracellular components. These different tasks can be addressed by different imaging approaches (e.g., laser-scanning confocal, spinning disk confocal, and light-sheet fluorescence microscopy)...
2018: Methods in Cell Biology
Anna-Maria Olziersky, Chris A Smith, Nigel Burroughs, Andrew D McAinsh, Patrick Meraldi
Mitosis is a highly dynamic and choreographed process in which chromosomes are captured by the mitotic spindle and physically segregated into the two daughter cells to ensure faithful transmission of the genetic material. Live-cell fluorescence microscopy enables these dynamics to be analyzed over diverse temporal scales. Here we present the methodologies to study chromosome segregation at three timescales: we first show how automated tracking of kinetochores enables investigation of mitotic spindle and chromosome dynamics in the seconds-to-minutes timescale; next we highlight how new DNA live dyes allow the study of chromosome segregation over a period of several hours in any cell line; finally, we demonstrate how image sequences acquired over several days can reveal the fate of whole cell populations over several consecutive cell divisions...
2018: Methods in Cell Biology
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