A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

The nuclear envelope is a barrier comprised of outer and inner membranes that separate the cytoplasm from the nucleoplasm. The outer (ONM) and inner (INM) membranes have different physical characteristics and protein compositions. In contrast to the extensive data available on the protein quality control processes operating in the cytoplasm, endoplasmic reticulum and the nucleoplasm, the mechanisms controlling protein turnover at the INM are poorly documented. The work presented in this thesis focuses on Asi1, Asi2 and Asi3, three bona-fide integral INM proteins of the yeast Saccharomyces cerevisiae. By contrast to mammalian cells, yeast progress through the cell cycle with a closed mitosis, that is cells divide in the absence of the cyclical fragmentation/reassembly of the nuclear membrane. Consequently, examining the processes affecting the stability of the Asi proteins in yeast may provide useful paradigms for understanding the turnover of INM components in non-dividing, terminally differentiated and post-mitotic cells of metazoan origin.The results have contributed to the elucidation of the biological function of Asi1 and Asi3, which are homologous proteins with C-terminal RING domains. Asi1 and Asi3 function together as a dimeric E3 ubiquitin ligase complex that operates with ubiquitin conjugating enzymes Ubc6 and Ubc7. The Asi1/3 complex ubiquitylates transcription factors Stp1 and Stp2 when they gain inappropriate access to the nucleus in the absence of SPS-sensor activation. Intriguingly, the Asi1/3 complex also mediates the turnover of multiple membrane proteins that primarily localize to other cell membranes. This latter finding indicates that the barrier function of nuclear pore complexes is not as tight as previously thought. Consistently, asi1 null mutations are synthetic lethal when introduced into hrd1Δ ire2Δ cells with compromised ER-associated degradation (ERAD) and unfolded protein response (UPR) pathways. Together the results define Asi1/3 as components of a novel quality control pathway operating in association with the INM that acts to safeguard the identity and maintain the function of the nuclear compartment. Asi1 and Asi2 exhibit rapid turnover and their turnover is ubiquitin-dependent, exhibiting a clear requirement for Ubc7. The ubiquitylated forms of Asi1 and Asi2 are degraded by nuclear-localized proteasomes; the ubiquitylated forms exhibit enhanced stability in sts1-2 mutants. Asi1 turnover requires Cue1, the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs unimpeded in cells lacking a functional Asi1/3 complex and in cells lacking Doa10, an E3 ligase complex also known to function at the INM. Consequently, Asi1 is subject to a quality control pathway associated with INM but that is distinct from the Asi1/3 and Doa10 INM- associated degradative (INMAD) pathways. This thesis documents work that clearly demonstrates that the INM is a highly dynamic structure that possesses multiple and active quality control pathways. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

No description available.

Gene positioning changes with respect to the nuclear periphery correlate with their activation in a number of tissues during development. However, the determination of the function this serves or the mechanism through which this was achieved has been remarkably difficult to resolve. It may now be possible to address these questions due to the recent identification of a number of tissue-specific nuclear envelope transmembrane proteins (NETs) which are capable of promoting the repositioning of specific subsets of chromosomes and concomitantly inducing changes to gene expression (Zuleger et al,. 2013). In this thesis I describe the role of NETs in the positioning of genes to the nuclear envelope (NE) during muscle differentiation and the role this activity plays in the optimisation of myogenic gene expression in as myoblasts (MTs) differentiate to myotubes (MTs). To do this I identified four NETs with the capacity to reposition a chromosome to the periphery that are present specifically in the NEs of skeletal muscle. Using a combination of genome-wide gene expression analysis and DamID I determined that depletion of these NETs disrupted myogenic gene expression and, more significantly, prevented the targeting to and silencing of normally repressed genes at the NE. I also investigated an analogous role for the blood-specific NET TAPBPL in the regulation of the critical T-cell regulator interleukin 2 (IL-2) at the NE in T-cells. Depletion of this NET caused release of the IL2 locus from the periphery and promoted its inappropriate and long-term activation. Interestingly, depletion of TAPBPL also prevented IL2 silencing following the end of T-cell activation, suggesting this genome organisation activity is critical for the maintenance of normal T-cell function. Collectively, the results discussed herein describe a new role for NETs in the regulation of gene expression through the manipulation of spatial genome organisation and may serve as an additional layer of higher order tissue-specific gene regulation in higher organisms.

Lamine bilden zusammen mit laminassoziierten Proteinen die nukleäre Lamina. Diese ist notwendig für die mechanische Stabilität von Zellen, die Organisation des Chromatins, der Genexpression, dem Fortgang des Zellzyklus und der Zellmigration. Die vielfältigen Funktionen der Lamine werden durch die Pathogenese von Laminopathien belegt. Zu diesen Erkrankungen, welche ihre Ursache in Mutationen innerhalb der laminkodierenden Gene, oder der Gene laminassoziierter bzw. laminprozessierender Proteine haben, zählen unter anderem das „Hutchinson-Gilford Progerie Syndrom“, die „Emery-Dreifuss“ Muskeldystrophie und die dilatierte Kardiomyopathie. Trotz der fundamentalen Bedeutung der Lamine, wurden diese bisher nur in Metazoen und nicht in einzelligen Organismen detektiert. Der amöbide Organismus Dictyostelium discoideum ist ein haploider Eukaryot, der häufig als Modellorganismus in den verschiedensten Bereichen der Zellbiologie eingesetzt wird. Mit der Entdeckung von NE81, einem Protein das mit der inneren Kernhülle von Dictyostelium discoideum assoziiert ist, wurde erstmals ein Protein identifiziert, dass man aufgrund seiner Eigenschaften als laminähnliches Protein in einem niederen Eukaryoten bezeichnen kann. Diese Merkmale umfassen die Existenz lamintypischer Sequenzen, wie die CDK1-Phosphorylierungsstelle, direkt gefolgt von einer zentralen „Rod“-Domäne, sowie eine typische NLS und die hoch konservierte CaaX-Box. Für die Etablierung des NE81 als „primitives“ Lamin, wurden im Rahmen dieser Arbeit verschiedene Experimente durchgeführt, die strukturelle und funktionelle Gemeinsamkeiten zu den Laminen in anderen Organismen aufzeigen konnten. Die Herstellung eines polyklonalen Antikörpers ermöglichte die Verifizierung der subzellulären Lokalisation des NE81 durch Elektronenmikroskopie und gab Einblicke in das Verhalten des endogenen Proteins innerhalb des Zellzyklus. Mit der Generierung von NE81-Nullmutanten konnte demonstriert werden, dass NE81 eine wichtige Rolle bei der nukleären Integrität und der Chromatinorganisation von Zellen spielt. Des Weiteren führte die Expression von zwei CaaX-Box deletierten NE81 - Varianten dazu, den Einfluss des Proteins auf die mechanische Stabilität der Zellen nachweisen zu können. Auch die Bedeutung der hochkonservierten CaaX-Box für die Lokalisation des Proteins wurde durch die erhaltenen Ergebnisse deutlich. Mit der Durchführung von FRAP-Experimente konnte außerdem die strukturgebende Funktion von NE81 innerhalb des Zellkerns bekräftigt werden. Zusätzlich wurde im Rahmen dieser Arbeit damit begonnen, den Einfluss der Isoprenylcysteincarboxylmethyltransferase auf die Lokalisation des Proteins aufzuklären. Die Entdeckung eines laminähnlichen Proteins in einem einzelligen Organismus, der an der Schwelle zu den Metazoen steht, ist für die evolutionäre Betrachtung der Entwicklung der sozialen Amöbe und für die Erforschung der molekularen Basis von Laminopathien in einem einfachen Modellorganismus sehr interessant. Die Arbeit mit Dictyostelium discoideum könnte daher Wege aufzeigen, dass Studium der Laminopathien am Tiermodell drastisch zu reduzieren. In den letzten Jahren hat die Erforschung unbekannter Bestandteile des Centrosoms in Dictyostelium discoideum große Fortschritte gemacht. Eine zu diesem Zwecke von unserer Arbeitsgruppe durchgeführte Proteomstudie, führte zur Identifizierung weiterer, potentiell centrosomaler Kandidatenproteine. Der zweite Teil dieser Arbeit beschäftigt sich mit der Charakterisierung eines solchen Kandidatenproteins, dem CP75. Es konnte gezeigt werden, dass CP75 einen echten, centrosomalen Bestandteil darstellt, der mikrotubuli-unabhängig mit der Core Struktur des Zellorganells assoziiert ist. Weiterhin wurde deutlich, dass die Lokalisation am Centrosom in Abhängigkeit vom Zellzyklus erfolgt und CP75 vermutlich mit CP39, einem weiteren centrosomalen Core Protein, interagiert. / Lamins build the nuclear lamina together with lamin-associated proteins. The latter is required for mechanical stabilization of cells, chromatin organization, gene expression, cell cycle progression and cell migration. This became evident by the pathogenesis of laminopathies. Laminopathies are diseases which arise from mutations in genes encoding lamins, lamin-associated-or lamin-processing proteins. Prominent examples are the „Hutchinson-Gilford progeria syndrome“, the „Emery-Dreifuss“muscular dystrophy and dilated cardiomyopathy. Despite their universal importance, lamins have only been found in metazoans, but not in unicellular organisms so far. The amoeboid organism Dictyostelium discoideum is a haploid eukaryote widely used in different fields of cell biology. With the discovery of NE81, a protein associated with the inner nuclear membrane of Dictyostelium discoideum, for the first time a protein was identified, whose properties jutify denomination as a lamin-like protein in a lower eukaryote. This is based on the presence of lamin-typical sequences such as a CDK1 phosphorylation consensus sequence, followed by a central rod domain, a typical nuclear localization sequence and the highly conserved CaaX box. For the verification of NE81 as a primitive lamin, various different experiments were conducted in the frame of this work, which revealed structural and functional similarities to lamins of other organisms. Analysis of the behavior of the endogenous protein in cell cycle and the verification of the subcellular localization with electron microscopy was done with the generation of a polyclonal antibody. With a NE81 null mutant, it could be shown, that NE81 plays an important role in nuclear integrity and chromatin organization. The expression of two CaaX-box deleted protein variants confirmed the influence of NE81 on the mechanical stability of cells. These results furthermore underlined the importance of the presence of the highly conserved CaaX-box. FRAP-experiments further emphasized the structural function of NE81 in the nucleus. Furthermore, first steps were undertaken to determine the influence of the Isoprenylcysteinecarboxylmethyltransferase on the localization of NE81. In the light of evolution the discovery of a lamin-like protein in a unicellular organism is very interesting and could provide a simple experimental system for studies of the molecular basis of laminopathies. Hence, the study on laminopathies in animal models could be reduced dramatically. The identification of unknown centrosomal components in Dictyostelium discoideum has made significant proceedings in the last years. A proteomic approach which was accomplished for this purpose, yielded several potential centrosomal candidate proteins. The second part of this work focuses on the characterization of one of these proteins, CP75. It could be shown that CP75 is a genuine, centrosomal component, which is associated with the centrosomal core structure independently of microtubules. Furthermore, it could be demonstrated, that the localization of CP75 is cell cycle-dependent and that it presumably interacts with the core protein CP39.

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

The amino-terminal 33 amino acids of the baculovirus integral membrane protein, ODV-E66, are sufficient for localization of fusion proteins to viralinduced intranuclear microvesicles (MV) and occlusion derived virus envelopes during infection, and has been termed the sorting motif (SM). When abundantly expressed, SM-fusions are also detected in the inner nuclear membrane (INM), outer nuclear membrane and endoplasmic reticulum of infected cells, suggesting proteins with the SM use the same trafficking pathway as cellular INM proteins to traffic to nuclear membranes. This study identifies the essential characteristics required for sorting of the SM to the INM of uninfected cells, and the MV and ODV envelopes of infected cells. These features are an 18 amino acid transmembrane sequence that lacks polar and charged amino acids (a.a.) with a cluster of charged a.a. spaced 5-11 residues from the end of the transmembrane sequence. A comparison of the a.a. sequence of these SM features with cellular INM proteins shows the features are conserved. The model of INM protein sorting and localization predicts the only known sorting event during INM protein trafficking is immobilization/retention in the INM. This study uses confocal microscopy and fluorescence recovery after photobleaching to compare the localization and mobility of lamin B receptor (LBR) fusions (which contain SM-like sequences) to a viral SM fusion when expressed in either mammalian or insect cells. The results show that immobilization is not necessarily required for accumulation of proteins in the INM. Furthermore, the results from infected cells show that an active sorting event, likely independent of immobilization, can distinguish the viral SM from cellular sequences similar to the SM. The results of this study show that sorting of proteins to the INM can be mediated by the viral SM or INM protein SM-like sequences that can function either independent of, or in addition to, immobilization. These data combined with recent reports suggest that in addition to diffusion:retention a signal mediated mechanism for sorting and localization to the INM can occur.

The nuclear envelope forms the interface between the nucleus and the cytoplasm. The nuclear envelope consists of the two concentric lipid membranes, the nuclear pores and the nuclear lamina. The inner nuclear membrane contains hundreds of unique transmembrane proteins showing high tissue diversity. Mutations of some proteins in the nuclear envelope give rise to a broad spectrum of diseases called envelopathies or laminopathies. In this thesis, I aimed to study the functional organization of the nuclear envelope by identifying and characterizing interactions between the nuclear envelope proteins. For this, we developed a novel method called the Membrane Protein Crosslink Immuno-Precipitation, which enable identification of protein-protein interactions in the nuclear envelope in live cells. We identified several novel interactions of the inner nuclear membrane protein, Samp1, and studied the interaction between the Samp1 and the nuclear GTPase, Ran in detail. Samp1 can bind to Ran and is thus the first known transmembrane Ran binding protein and Samp1 might provide a local binding site for Ran in the inner nuclear membrane. We found that Samp1 also binds to the inner nuclear membrane protein, Emerin and Ran can regulate the Samp1-Emerin interaction in the nuclear envelope. During mitosis, Samp1 distributes in the mitotic spindle. Therefore, we investigated a possible functional role of Samp1 in the mitotic machinery. Samp1 depletion resulted in aneuploid phenotypes, metaphase prolongation and decreased distribution of γ-tubulin and β-tubulin in the mitotic spindle. We found that Samp1 can bind to γ-tubulin, which is essential for the microtubule nucleation and hence for the spindle stability. The new interesting features of Samp1 provide insights on the unforeseen functions of the nuclear envelope proteins. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.