title: Electron microscopy and new technological approaches to investigate structural elements of the mitotic apparatus in Saccharomyces cerevisiae and Xenopus laevis
creator: Heiligenstein, Xavier
subject: ddc-570
subject: 570 Life sciences
description: The mitotic cell cycle is a complex process which leads to the chromosome segregation from the mother cell to the two daughter cells and transmit the full genetic background necessary to grow and adapt to the permanently evolving environment. The equal partitioning of the duplicated chromosomes is finely regulated by the cytoskeleton which on purpose organises into a mitotic spindle. In eukaryotes like Xenopus laevis and Saccharomyces cerevisiae the mitotic spindle is mainly composed of three components: first the microtubules (MT) are organised in a spindle emanating from the chromosomes and focusing at the poles. Second they connect to the chromosomes via the kinetochore complexes (KT) and third focus at the microtubule organising centres (MTOC), also called centrosomes. We have focused our interest on structural analysis of the S. cerevisiae centrosome, called the Spindle Pole Body (SPB), at the molecular level, and on the MTs organisation in the meiotic spindle midzone of X. laevis. With S. cerevisiae we have used cryo-electron tomography on vitreous sections (CETOVIS) to investigate the three dimensional (3D) molecular structure of the SPB. The samples were vitrified by high pressure freezing (HPF) and sectioned in vitreous state. Acquiring instant snapshots close to native state of the SPB in vivo, we confirmed previous observations done on plunge frozen or freeze substituted material. The Spc42 central crystal protein are organised with a defined spacing of 107Å, but the plaques composing the SPB could not all be identified. Mainly, the central and the outer plaque were visible, in contrast with the plastic tomography results where the inner plaque appeared dense and compact. Unfortunately, the very low signal to noise ratio prevented us from extracting details structural information about the SPB in its native state. The project concerning the X. laevis meiotic spindle focused on the 3D organisation of the MT within the spindle. Their interactions with the MTOC and the KT have been extensively studied over the past, but a high resolution structural map is still missing and has long been awaited by scientists to put all the knowledge into a 3D context. Furthermore, information about the individual MT is missing like their average length distribution, the existence of short MTs and the end morphologies distribution. To study this complex and large structure, we have developed a novel correlative light to electron microscopy (CLEM) approach combined with electron tomography. We cryo-fixed by HPF, for the first time to our knowledge, spindle assembled in X. laevis egg extracts and prepared them for a structural electron tomographic study. We reconstructed three quarter of a meiotic spindle midzone and identified three subcategories of MT bundle organisation. Finally, we have used our CLEM method to develop a new technology that should facilitate future CLEM work. Recent advances in plastic polymer transformation and micro-injection moulding (µIM) allowed us to create a cryocaspule designed for an easy correlative microscopy and transfer for HPF. 
date: 2011
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/12932/1/2011_Xavier_Heiligenstein_PhD_Thesis.pdf
identifier: DOI:10.11588/heidok.00012932
identifier: urn:nbn:de:bsz:16-opus-129329
identifier:   Heiligenstein, Xavier  (2011) Electron microscopy and new technological approaches to investigate structural elements of the mitotic apparatus in Saccharomyces cerevisiae and Xenopus laevis.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/12932/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng