Molecular analysis of the unconventional export machinery of HASPB, a component of the surface coat of Leishmania parasites [Elektronische Ressource] / presented by Carolin Stegmayer

Nicklab

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 21 Mo

Extrait

Molecular Analysis of the Unconventional
Export Machinery of HASPB,
a component of the surface coat of
Leishmania parasites



Dissertation submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto Carola University of Heidelberg, Germany










for the degree of
Doctor of Natural Sciences


presented by

Diplom-Biologin Carolin Stegmayer
born in Bad Kreuznach


DISSERTATION

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto Carola University of Heidelberg, Germany





for the degree of
Doctor of Natural Sciences




presented by

Diplom-Biologin Carolin Stegmayer
born in Bad Kreuznach


Oral examination:



Molecular Analysis of the Unconventional
Export Machinery of HASPB,
a component of the surface coat of
Leishmania parasites


















Referees:
Prof. Dr. rer. nat. Walter Nickel
Prof. Dr. rer. nat. Michael Brunner
List of Publications

Engling, A., Backhaus, R., Stegmayer, C., Zehe, C., Seelenmeyer, C.,
Kehlenbach, A., Schwappach, B., Wegehingel, S., and Nickel, W. (2002).
Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following
translocation to the extracellular surface of CHO cells. J Cell Sci 115, 3619-
3631.

Stegmayer, C., Kehlenbach, A., Tournaviti, S., Wegehingel, S., Zehe, C.,
Denny, P., Smith, D. F., Schwappach, B., and Nickel, W. (2005). Direct
transport across the plasma membrane of mammalian cells of Leishmania
HASPB as revealed by a CHO export mutant. J Cell Sci 118, 517-527.

Tournaviti, S., Hannemann, S., Terjung, S., Kitzing, T. M., Stegmayer, C.,
Ritzerfeld, J., Grosse, R., Nickel, W., and Fackler, O. T. (2006 submitted).
SH4 Domain-induced Plasma Membrane Blebbing Implicated in 3D Cell
Motility.Contents I

Contents
ABBREVIATION .............................................................................................................................1
ZUSAMMENFASSUNG..................................................................................................................5
SUMMARY ......................................................................................................................................7
1 INTRODUCTION ......................................................................................................................9
1.1 CLASSICAL OR ER-GOLGI-MEDIATED PROTEIN SECRETION................................................10
1.2 NON-CLASSICAL OR UNCONVENTIONAL PROTEIN SECRETION.............................................17
1.3 HYDROPHILIC ACYLATED SURFACE PROTEIN B (HASPB) ..................................................21
1.3.1 Dual acylation mediated by myristoylation and palmitoylation.............................22
1.3.2 Leishmania parasites ..............................................................................................26
1.3.3 Life Cycle of Leishmania parasites ........................................................................27
1.3.4 Composition and assembly of the Leishmania surface coat................................29
1.3.5 Non-classical export of HASPB..............................................................................31
1.3.6 HASPB is of exceptional biomedical relevance ....................................................36
1.4 FIBROBLAST GROWTH FACTOR-1 AND- 2 (FGF-1 AND -2)................................................37
1.5 GALECTIN-1.......................................................................................................................40
1.6 AIM OF THE CURRENT STUDY .............................................................................................43
2 MATERIAL AND METHODS.................................................................................................44
2.1 MATERIAL..........................................................................................................................44
2.1.1 Chemicals ................................................................................................................44
2.1.2 Plasmids...................................................................................................................48
2.1.3 DNA modifying enzymes ........................................................................................49
2.1.4 Primers and oligonucleotides .................................................................................49
2.1.5 Bacteria and bacterial media..................................................................................51
2.1.6 Eukaryotic cell lines.................................................................................................52
2.1.7 Eukaryotic cell culture media (Wilson et al., 1994) ...............................................52
2.1.8 Primary antibodies...................................................................................................53
2.1.9 Secondary antibodies .............................................................................................54
2.2 MOLECULAR BIOLOGICAL METHODS ...................................................................................54
2.2.1 Bacterial transformation..........................................................................................54
2.2.2 Selection and amplification of plasmids.................................................................55
2.2.3 Plasmid preparation ................................................................................................55
2.2.4 Isolation of genomic DNA from cultured cells .......................................................56
2.2.5 Isolation of RNA from cultured cells.......................................................................56
2.2.6 Determination of DNA/RNA concentrations ..........................................................57
2.2.7 Agarose gel electrophoresis...................................................................................58
2.2.8 DNA sample buffer/loading buffer..........................................................................59 Contents II

2.2.9 DNA marker .............................................................................................................59
2.2.10 Polymerase chain reaction ...................................................................................60
2.2.11 RT-PCR..................................................................................................................62
2.2.12 PCR purification ....................................................................................................64
2.2.13 Gel extraction of DNA fragments .........................................................................64
2.2.14 Restriction digests.................................................................................................65
2.2.15 Elongation of DNA using the Klenow fragment...................................................65
2.2.16 DNA dephosphorylation........................................................................................66
2.2.17 Ligation of DNA fragments ...................................................................................66
2.2.18 DNA sequencing ...................................................................................................67
2.2.19 Short interfering RNAs (siRNAs) in mammalian cells ........................................67
2.3 EUKARYOTIC CELL CULTURE TECHNIQUES .........................................................................68
2.3.1 Maintaining cell lines...............................................................................................68
2.3.2 Freezing of eukaryotic cells ....................................................................................69
2.3.3 Thawing of eukaryotic cells ....................................................................................70
2.3.4 Retroviral transduction ............................................................................................70
2.3.5 Doxicycline-dependent protein expression............................................................72
2.4 BIOCHEMICAL METHODS ....................................................................................................72
2.4.1 Generation of cell lines expressing various HASPB-GFP fusion proteins ..........72
2.4.2 Sample preparation for SDS polyacrylamide gel electrophoresis (SDS-PAGE) 73
2.4.3 SDS polyacrylamide gel electrophoresis...............................................................73
2.4.4 SDS-PAGE protein molecular weight standards...................................................75
2.4.5 Silver staining ..........................................................................................................76
2.4.6 Western blot analysis..............................................................................................77
2.4.7 Immunochemical protein detection using the ECL system ..................................78
2.4.8 Immunochemical protein