Structural analysis of presynaptic architecture by cryoelectron tomography [Elektronische Ressource] / Rubén Fernández-Busnadiego

Ruben

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 30
• Langue: English
• Poids de l'ouvrage: 75 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Max-Planck-Institut für Biochemie
Abteilung für Molekulare Strukturbiologie





Structural Analysis of Presynaptic
Architecture by Cryoelectron Tomography



Rubén Fernández-Busnadiego









Vollständiger Abdruck der von der Fakultät für Chemie
der Technischen Universität München
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.








Vorsitzender: Univ.-Prof. Dr. Dr. h. c. H. Kessler

Prüfer der Dissertation: 1. Hon.-Prof. Dr. W. Baumeister
2. Univ.-Prof. Dr. S. Weinkauf



Die Dissertation wurde am 05.01.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 31.03.2010 angenommen.
Contents


Abstract..........................................................................................................................5
1. Introduction............................................................................................................9
1.1 Synaptic Transmission...................................................................................9
1.2 The Presynaptic Terminal............................................................................10
1.2.1 The synaptic vesicle cycle ...................................................................10
1.2.2 Synaptic vesicle exocytosis..................................................................12
1.2.3 The presynaptic cytomatrix..................................................................15
1.2.4 Electron microscopy analysis of presynaptic architecture...................16
2. Electron Microscopy and Tomography ...............................................................21
2.1 Transmission Electron Microscopy.............................................................21
2.1.1 Components of transmission electron microscopes.............................
a) Electron gun.............................................................................................21
b) Illumination system..................................................................................21
c) Imaging system........................................................................................23
d) Image detection system............................................................................23
e) Other TEM systems .................................................................................23
2.1.2 Image formation: Phase contrast..........................................................24
2.2 Sample preparation......................................................................................28
2.2.1 Radiation damage and noise ................................................................29
2.3 Cryo-Electron Tomography.........................................................................30
2.3.1 Automated data acquisition..................................................................31
2.3.2 3D reconstruction32
2.3.3 Tomogram analysis..............................................................................34
3. Aims of This Study ..............................................................................................37
4. Materials and Methods.........................................................................................41
4.1 Sample preparation41
4.1.1 Synaptosomal extraction......................................................................
4.1.2 Glutamate release assay .......................................................................41
4.1.3 Pharmacological treatments and vitrification of synaptosomes...........43
4.1.4 Hippocampal slice cultures, high pressure freezing and cryosectioning .
..............................................................................................................43
4.1.5 Hippocampal neurons in culture ..........................................................44
4.2 Cryo-Electron Tomography.........................................................................44
4.3 Data analysis................................................................................................45
5. Results..................................................................................................................51
5.1 Establishment of the experimental system...................................................51
5.2 The presynaptic cytomatrix in cortical synaptosomes and hippocampal
organotypic slices.....................................................................................................56
5.2.1 Morphology of frozen-hydrated presynaptic terminals .......................56
5.2.2 Inhomogeneity in the spatial distribution of synaptic vesicles ............59
5.2.3 Extensive interconnectivity of synaptic vesicles .................................61
5.2.4 Clusters of interconnected synaptic vesicles........................................64
5.2.5 Tethering of proximal synaptic vesicles to the active zone .................67
5.2.6 Synaptic vesicle size............................................................................69
5.2.7 Direct membrane contact between synaptic vesicles and the active
zone ..............................................................................................................72
5.3 Computational procedures for the analysis of presynaptic architecture ......74
5.3.1 Hierarchical classification of connectors and tethers...........................76
5.4 Observations on other neuronal structures...................................................80
5.4.1 The postsynaptic density and the synaptic cleft...................................80
5.4.2 Morphology of neurons in culture .......................................................82
6. Discussion............................................................................................................87
6.1 Morphology of frozen-hydrated neuronal preparations...............................87
6.2 Automated segmentation and analysis of presynaptic ultrastructure...........88
6.3 The role of synaptic vesicle connectors in vesicle mobilization..................89
6.4 Active zone organization, tethering and synaptic vesicle progression
towards fusion..........................................................................................................90
6.5 Synaptic vesicle distribution in resting synapses is not homogeneous........93
7. Outlook................................................................................................................97
Abbreviations...............................................................................................................99
Acknowledgments......................................................................................................101
References..................................................................................................................103


Abstract

Presynaptic terminals contain a large number of neurotransmitter-filled synaptic
vesicles embedded in a dense filamentous network. Although intensely studied over
the last decades, the organization and cellular role of this network remain unclear.
Combining cryo-electron tomography with pharmacological manipulations, this work
provided a quantitative assessment of the structural elements mediating synaptic
vesicle organization and release in mammalian central nervous system synapses. The
major conclusions of this study are the following:

1. Cortical synaptosomes and hippocampal organotypic slices are
complementary preparations that form an appropriate experimental system
for the study of the native presynaptic cytomatrix in vitreous frozen-hydrated
samples. Synaptosomes allow different pharmacological manipulations, whereas
slices provide a direct view into nervous tissue. In this work, presynaptic
morphology was comparable in both kinds of samples, pointing to a limited
influence of preparation artifacts.

2. An automated segmentation algorithm was developed to allow a quantitative
analysis of vesicle distribution and the presynaptic cytomatrix, introducing
quantitative tools for the analysis of tomographic data from pleomorphic
biological structures.

3. The short filaments that link synaptic vesicles to each other (connectors) and
to the active zone (tethers) are the main components of the presynaptic
cytomatrix. Longer filaments are much less abundant and therefore likely to play
secondary roles.

4. The rearrangement of connectors upon synaptic stimulation and inhibition of
phosphatases suggests that these connectors play an important role in vesicle
mobilization.

5. The comparison between synapses at rest and those stimulated by KCl or
hypertonic sucrose points to a link between the configuration of the tethering
assembly and vesicle availability for release. Short tethers were significantly
removed under treatments known to (a) release the readily releasable pool
(hypertonic sucrose) or (b) cleave synaptobrevin, one of the components of the
SNARE complex