1 |
Postmeiotische Expression und funktionelle Charakterisierung von Lamin B3 in der Spermatogenese der MausSchütz, Wolfgang. Unknown Date (has links) (PDF)
Universiẗat, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.
|
2 |
Postmeiotische Expression und funktionelle Charakterisierung von Lamin B3 in der Spermatogenese der Maus / Postmeiotic expression and functional characterisation of lamin B3 in mouse spermatogenesisSchütz, Wolfgang January 2006 (has links) (PDF)
Die Lamine gehören zu einer Familie von Proteinen, die als strukturelle Hauptelemente die Kernlamina ausbilden, einen wesentlichen Bestandteil der Kernhülle eukaryontischer Zellen. In Säugern exprimieren differenzierte somatische Zellen die Lamine A, C, B1 und B2. Die Kernhülle in Keimzellen unterscheidet sich in Bezug auf Struktur und Proteinzusammensetzung deutlich von der einer somatischen Zelle. So exprimieren Keimzellen Lamin B1 als einziges der somatischen Lamine und zwei kurze keimbahnspezifische Spleißvarianten, die Lamine C2 und B3. Die vorliegende Arbeit enthält eine detaillierte Analyse des Expressionsmusters und der zellulären Verteilung von Lamin B3 im Verlauf der Spermatogenese der Maus. Die Daten aus RT-PCR, Western Blot und Immunfluoreszenz belegen eindeutig, dass Lamin B3 ausschließlich in postmeiotischen Stadien während der Spermiogenese exprimiert wird. In runden Spermatiden konnte das Protein an der Kernhülle und überraschenderweise auch im Nukleoplasma nachgewiesen werden. Im weiteren Verlauf der Spermiogenese kommt es zu einer Umverteilung des Proteins, es konzentriert sich zunehmend am posterioren Pol des Spermatidenkerns. Damit ist die Lamina während der Säuger-Spermiogenese nur aus B-Typ-Laminen aufgebaut und Lamin B3 ist in Säugern das erste Beispiel für ein Lamin, das selektiv nur in postmeiotischen Stadien der Spermatogenese exprimiert wird. Die ektopische Expression von Lamin B3 in Kulturzellen führt zu einer Deformation der Zellkerne, die eine hakenförmige Gestalt annehmen. Mit Hilfe von Transfektionsexperimenten in COS-7-Zellen konnte eindeutig gezeigt werden, dass die auftretenden morphologischen Veränderungen der Kerne transfizierter Zellen auf die trunkierte zentrale Stäbchendomäne in Lamin B3 zurückzuführen ist. Darüber hinaus zeigte das Protein eine stark erhöhte Löslichkeit im Vergleich zu Lamin B2 und die Analyse transfizierter Kulturzellen mit „fluorescence recovery after photobleaching“ (FRAP) und „fluorescence loss in photobleaching“ (FLIP) ergab, dass ein erheblicher Anteil der Lamin-B3-Moleküle eine hohe Mobilität aufweist, die ebenfalls ausschließlich durch die kurze Stäbchendomäne begründet ist. Die Ergebnisse führen zu dem Schluss, dass Lamin B3 die Kernhülle in Keimzellen flexibler macht, was eine Voraussetzung für einige Vorgänge in der Spermiogenese sein könnte. Mit einem Fusionsprotein aus GST und dem 84 Aminosäuren umfassenden N-Terminus von Lamin B3 wurde über einen „Pull-Down-Assay“ nach möglichen Interaktionspartnern in Keimzellen gesucht. Mit MSY2, MSY2a und MSY4 wurden drei hoch interessante Kandidaten identifiziert. Sie gehören zu den Y-Box-Proteinen, DNA- und RNA-bindende Proteine, die bei der Speicherung und späteren Translation von mRNAs beteiligt sind, u.a. die mRNA von Protamin 1 (diese Form der Regulation von Genexpression hat in der Spermatogenese große Bedeutung). Die Interaktion von Lamin B3 mit diesen Proteinen muss noch überprüft werden, würde aber einen weiteren Bezug zwischen Kernhülle und Chromatinreorganisation in der Spermiogenese herstellen, wie es für die Kernhüllenproteine GCL und LBR bereits gezeigt werden konnte. Außerdem wäre es ein erster Hinweis auf eine funktionelle Bedeutung der N-terminalen Domäne von Lamin B3. / Lamins are members of a protein family that are the main structural elements of the nuclear envelope in eukaryotic cells. Differentiated mammalian somatic cells express lamins A, C, B1 and B2. The composition and structural organisation of the nuclear lamina in spermatogenic cells differ significantly from that of somatic cells: among the somatic lamins they only express lamin B1 but, additionally, two germ line-specific isoforms could be found, namely lamins C2 and B3. This study contains a detailed investigation of the expression pattern and localisation of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, it turned out, that lamin B3 is selectively expressed in postmeiotic stages during spermiogenesis. In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. The results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely lamin B1 and the germ line-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis. When ectopically expressed in culture cells, lamin B3 causes severe deformation of nuclei which adopt a hook-like configuration. Transfection experiments in COS-7 cells could prove that the observed nuclear deformations are due to the shortened rod domain of lamin B3. Cell fractionation experiments revealed that lamin B3 can be solubilised more easily than lamin B2. In addition, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses of transfected COS-7 cells showed that considerable amounts of lamin B3 molecules exhibit a significantly increased mobility compared to lamin B2. The increased solubility of lamin B3 compared to lamin B2 as well as the mobility of that protein is only determined by its shortened rod domain. Taken together, these data lead to the conclusion that lamin B3 reduces the stability of the nuclear periphery, what might be an important prerequisite for some reorganisation processes during spermiogenesis to occur. Via a pull-down assay using a fusion protein containing GST and the 84 amino acid long N-terminal domain of lamin B3 a screen for interaction partners of lamin B3 was performed. With MSY2, MSY2a and MSY4 three interesting candidates were found. These proteins belong to the large family of Y-box containing proteins, which are DNA and RNA binding proteins. They are involved in storage and subsequent translation of various mRNAs, e.g. the mRNA of protamine 1 (this mechanism for regulation of gene expression is a major principle in spermatogenesis). The interaction between lamin B3 and the Y-box proteins has to be verified but it would provide an additional link between reorganisation of chromatin and the nuclear envelope as it has already been reported for other proteins of the nuclear envelope like GCL or LBR. Besides that it could be a first evidence for a specific function of the lamin B3 N-terminal domain.
|
3 |
Discovery and restoration of aberrant nuclear structure and genome behaviour in breast cancer cellsHassan Ahmed, Mai January 2013 (has links)
The eukaryotic interphase nucleus is well organised and the genome positioned non-randomly. Nuclear structure is an important regulator of genome behaviour and function. Genome organisation and nuclear structure are compromised in diseases such as cancer and laminopathies. This study was to find out and to determine if there is any functional relationship between nuclear structure and genome mis-organisation in cancer cells. I have assessed the presence and distribution of specific nuclear structural proteins (A-type, B-type lamins and its receptor LBR, many of their binding proteins such as MAN1, LAP2α, LAP2, and Emerin and other nuclear proteins (PML, Nucleolin, and Ki67) using indirect immunofluorescence. From this study, it is found that the nuclear structure of breast cancer cells is often altered. The most severely affected proteins are the nuclear lamins B1 and B2 and they found as large foci within the nucleoplasm with little LBR expression to localise the lamin B. I also assessed the chromosome positioning (HSA 7, 10, 11, 14 and 17) and gene positioning (AKT1, CCND1, HSP90AA1, EGFR, ERRBB2/HER2 and PTEN) in breast cancer cell lines (T-47D, GI-101, Sk-Br-3 and BT-474) and in normal breast cell lines (MCF-10A) using 2D-FISH technique. I also assessed the position of the genes in nuclei and correlated with gene expression using qRT-PCR. Breast cell lines have treated with a drug named lovastatin and it was found that the cells have restored LBR expression and localisation of lamin B, leading to altered gene positioning and changed expression of breast cancer genes. Since the drug (lovastatin, 12 μM/48 hours) affects the prenylation as a post-translation modification process and lamins B biosythensis, it is found that B-type lamins and its receptor expression and distribution were improved and increased in expression by 2-fold in expression levels in the most affected cells (T-47D, and BT-474) compared to the normal cells (MCF-10A) and these cells also showed abnormal nuclei and dead cells. When analysing the nuclear positioning of the genes (AKT1, HSP90AA1 and ERRBB2/HER2), it is found that AKT1 was positioned periphery in BT-474 and T-47D cells and interiorly in the normal cells (MCF-10A) before treatment whereas the same gene was positioned periphery in T-47D and MCF-10A cells and interiorly in BT-474 after treatment with lovastatin. It is also found that HSP90AA1 was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 cells before and after treatment (no change). Moreover, ERRBB2/HER2 gene was positioned periphery in T-47D and BT-474 cells and interiorly in MCF-10A cells before treatment whereas the same gene was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 after treatment with the same drug. Regarding LMNB1, LMNB2, and LBR genes, the study focussed only on their expression levels and no work has done on their chromosome positioning as well as gene position before and after treatment. These three genes were over expressed when assessed by measuring the relative and fold changes in expression. Therefore, it is suggestive that 2D-FISH experiment to assess their localisation and their specific chromosome territories is required. The results shown in this thesis demonstrate the importance and roles of nuclear architecture specifically nuclear lamins and the integral nuclear membrane proteins (B-type lamins and LBR) in mediating correct genome organisation and function. The breast normal (immortalised cells) and cancerous cell lines showed different nuclear structures as lamin B affect the position of specific target chromosomes and genes. These results will strength the finding that the nuclear lamina is a significant nuclear structure which associates, organises, and regulates numerous vital nuclear processes and the stability of the genome.
|
4 |
Interakce hlavního kapsidového proteinu polyomavirů s jadernými laminy / Major capsid protein of polyomaviruses and its interactions with nuclear laminsŽáčková, Sandra January 2021 (has links)
In this study, we focused on interactions of structural proteins of mouse polyomavirus (MPyV) and BK virus (BKV) with the nuclear lamina. Our goal was to examine whether and how can the virus, hence viral structural proteins, interact with the nuclear lamina and how would these interactions affect its properties. We supposed, that the expression of viral proteins would induce disintegration of the structure of nuclear lamina, thus enabling nuclear egress of virions in the late phase of infection. Viral structural proteins were expressed transiently in cells transfected with an expression vector pMPyV LATE. In these cells, VP1 was localized in a likewise manner as it shows in infected cells - mostly in a perinuclear area. Concurrently, defects in staining of nuclear lamina were observed in these cells, similarly to infected cells. Also, another expression vector was used in our experiments, the pMPyV mut3 VP1 encoding for a mutated protein VP1. When transiently expressed in cells, the mutated VP1 protein showed mostly diffuse nuclear localization. However, we observed significant morphological deformations and defective staining of the nuclear lamina. These observations imply an important role of VP1 in mechanical and biochemical properties alterations of the nuclear lamina in transfected and...
|
5 |
DNA Methylation, Cellular Stress Response and Expression of Inner Nuclear Membrane ProteinsLevesque, Steve 04 May 2011 (has links)
Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.
|
6 |
DNA Methylation, Cellular Stress Response and Expression of Inner Nuclear Membrane ProteinsLevesque, Steve 04 May 2011 (has links)
Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.
|
7 |
DNA Methylation, Cellular Stress Response and Expression of Inner Nuclear Membrane ProteinsLevesque, Steve 04 May 2011 (has links)
Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.
|
8 |
DNA Methylation, Cellular Stress Response and Expression of Inner Nuclear Membrane ProteinsLevesque, Steve January 2011 (has links)
Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.
|
Page generated in 0.0531 seconds