Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA replication and repair and development. Several recent proteomic studies uncovered a great variety of nuclear envelope transmembrane proteins (NETs). Diseases associated with several nuclear envelope proteins, mostly NETs, affect many tissues e.g. muscle, adipose tissue, skin, bones. Many NETs of the inner nuclear membrane have been shown to interact with chromatin, suggesting that their influencing gene expression might explain NET roles in disease. This work is focused on finding novel interactions of NETs with chromatin. First, SUN2 post-translational modifications were analysed and the effect of phosphomimetic and phospho-null mutants on heterochromatin and the cytoskeleton was tested by overexpression. However, no obvious changes were found. Second, several tissue-preferential NETs were tested in an adipocyte differentiation system. NET29 changed chromosome 6 position in pre-adipocytes. This matched changes in chromosome positioning that occur during adipocyte differentiation when NET29 is normally induced. Post-translational modifications of NET29 are likely to play a vital role in this process because a phospho-null mutant dominantly blocked chromosome repositioning. The effect of over-expression and down-regulation of NET29 on transcription was tested and results suggest that NET29 negatively regulates expression of myogenic genes during adipogenesis. This thesis is split into six chapters. Chapter I is an overview of the nuclear envelope, adipogenesis and chromatin remodelling, Chapter II is a detailed description of methods used in this study. Chapter III focuses on post-translational modifications of SUN2, as well as trials to identify novel partners of SUN2. Chapter IV and V deal with a novel nuclear envelope transmembrane protein and its role in adipogenesis. Finally, the last chapter includes a discussion and recommended future directions.

The nuclear envelope is a complex double membrane system that separates the activities of the nuclear and cytoplasmic compartments. A recent explosion in the number of proteins associated with this subnuclear organelle together with it now being linked to over 2 dozen diseases indicates the importance of better understanding its functional organisation. This thesis addresses two important questions for this: how do integral proteins of the nuclear envelope get to their sites of function and do any of these proteins direct genome organisation? To address the first question I used FRAP and photoactivation methods to find that different proteins use at least 4 distinct mechanisms to reach the inner nuclear membrane. Some appeared to be translocated by simple unaided lateral diffusion in the membrane while others needed Ran GTPase activity, others ATP, some others were aided by phenylalanine/glycines (FGs). Both Ran and FG mechanisms required the nucleoporin Nup35, albeit the mechanisms appeared to be completely independent of one another. To investigate the role of the nuclear envelope in genome organization, I screened for nuclear envelope proteins that reposition particular chromosomes to the nuclear periphery, finding five with this function. Interestingly, all of the proteins with this effect are tissuespecific. Depletion of two liver-specific nuclear envelope proteins reversed their effects on a specific chromosome for positioning with respect to the nuclear periphery. Finally, exogenous expression of these proteins in tissue culture cells caused induction of genes involved in differentiation pathways.

Microtubules are essential components of eukaryotic cytoskeleton and play a crucial role in variety of cell activities, such as cell mobility, intracellular transportation, cell division and organelle spatial organization. The initiation of microtubule nucleation is an important event to trigger the microtubule growth from different microtubule organizing centres (MTOCs) during the cell cycle in fission yeast. In interphase, the major MTOCs in the cells are nuclear envelope (NE) and microtubules existing in the cytoplasm. During mitosis, spindle pole body (SPB) that is the centrosome equivalent is the MTOC where astral and spindle microtubules initiate from. Once cells enter anaphase, the post-anaphase array (PAA) of microtubules will initiate from the equatorial MTOC (eMTOC) at the cell division site. To initiate microtubule nucleation, γ-tubulin small complex (γ-TuSC) will be recruited to the MTOC to form the “lock-washer” like γ-tubulin ring complex (γ- TuRC) as the scaffold of the microtubule. Fission yeast γ-TuSC is composed of two molecules of γ-tubulin and one each of GCP2 and GCP3 homologue, Alp4 and Alp6, respectively. Apart from Alp4 and Alp6, the homologue of human GCP4, GCP5 and GCP6, named Ghf1, Mod21 and Alp16 were identified as independent components of γ-TuRC in fission yeast. Protein Mto1 form a complex with its partner Mto2 and the complex directly interacts with γ-TuSC at all the MTOCs through the cell cycle. Mto1 is a large coiled-coil protein composed of 1115 amino acids. It has three main functional domains, including an N-terminal ~60 amino acids region termed CM1 motif that is required for the recruitment γ-TuSC to MTOCs, a central region that is required for the interaction with Mto2 at all cytoplasmic MTOCs and a ~44 amino acids region close to the C-terminus (named MASC) which is required for the binding of Mto1 to SPBs and eMTOC. The C-terminal truncation for Mto1 shows Mto1[1-549]-GFP mainly localizes on the NE and this Mto1 mutant is still functional for microtubule nucleation. It is referred as “Mto1[NE]”. In addition, truncation of 1-130 amino acids region for Mto1[1-549]-GFP creates the smallest Mto1 mutant that is able to initiate the microtubule nucleation in cytoplasm in a random manner. This Mto1[131-549]-GFP mutant fails to localize at any MTOCs, including NE. It is then referred as “Mto1[bonsai]”. To understand the mechanism that how is Mto1[NE] recruited to the NE, I performed two-step purification and mass spectrometry for both Mto1[1-9A1-549] and Mto1[131-9A1-549] strains, which form more significant puncta on both NE and cytoplasm respectively, to identify the potential receptor(s) of Mto1[1-9A1-549] on the nuclear envelope by comparing the different interactomes of Mto1[1-9A1-549] and Mto1[131-9A1-549]. Here, I show both exportin Crm1 and nucleoporin Nup146 are essential for the binding of Mto1 to the NE. I find the Localization of Mto1[1-9A1-549] GFP on the NE depends on binding of Mto1 to Crm1 via a nuclear export signal (NES)-like sequence within the N-terminus of Mto1. Further, I figure out that Spi1GTP (RanGTP in fission yeast) is involved in the formation of Crm1-Spi1GTP-Mto1 complex and required for the interaction of Mto1 to the NE. In addition, I also find that the FG repeats of Nup146 are essential for the binding of Mto1 to the nuclear envelope. It’s likely Nup146 anchors Crm1-Spi1GTP-Mto1 complex to the NE through the interaction between its FG repeats and Crm1. Last, my data are consistent with previous findings that the association of Mto1 to the NE is important for the microtubule nucleation from the NE.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

No description available.

Actin cytoskeletal regulation is of critical importance for a number of diverse cellular functions, including cell motility, endocytosis, cell division and transcription. Tight regulation of actin is critical for many aspects of cancer biology and in particular invasion and metastasis. ADF/cofilins are among the most important actin regulatory proteins. Mammals have three highly conserved members, ADF, CFL1 and CFL2, which regulate actin dynamics by severing and depolymerizing actin filaments. Despite a huge literature on the roles of ADF/cofilins in actin treadmilling and cell migration in vitro and in cancer cell behavior during invasion, very little is known about their collective roles in tissue homeostasis. By employing genetic knock-outs of ADF, in conjunction with conditional depletion of CFL1 using a Cre-LoxP system under the control of the keratin 14 promoter, we were able to study the effects of ADF/CFL1 loss in vivo in the mouse epidermis. Furthermore, by generating ADF-null squamous cell carcinoma (SCC) cell lines and by transiently downregulating CFL1 with RNAi, we were able to investigate further the cellular responses after ADF/CFL1 depletion in vitro. Co-depletion of ADF and CFL1 from the mouse epidermis triggered loss of tissue homeostasis characterized by abnormal thickening of the tissue, actin filament accumulation and nuclear deformation. Loss of ADF/CFL1 in cultured malignant keratinocytes also led to aberrant cell morphology accompanied by unrestrained accumulation of actin stress fibers tethered to enlarged focal adhesions. Enhanced SRF/MAL-mediated transcription fuels this uncontrolled actin polymerization which is also mediated by Arp3. Furthermore, these actin filaments are decorated with phospho-myosin light chain, which indicates their contractile nature. As a consequence, the increased intracellular acto-myosin tension results in nuclear deformation, which is promoted by the deregulated actin filaments tethered to the nuclear envelope via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Overall, we describe new conceptual insight into the cellular functions of ADF/cofilins. We show that their activities are essential for the dynamic regulation of contractile actin filaments that, if left unchecked, lead to loss of cellular homeostasis and cell death promoted by loss of nuclear integrity. Additionally, the critical roles of nuclear actin and actin-associated proteins have recently started being appreciated. Thus, for the first time we set out to investigate new functions of cofilins in the nucleus using proteomics, and identify new cofilin binding partners that implicate them in novel cellular pathways, expanding our knowledge on these small actin-binding proteins.

The nuclear envelope is a highly organised double membrane system that separates the activities of the nuclear and cytoplasmic compartments in eukaryotic systems. The wide range of functions recently associated with the NE and the identification of hundreds of proteins associated with this cellular structure indicates that it is a major signalling node for the cell. Recent work indicates NE functions in signalling innate immune responses to herpesviruses. The viruses, on the other hand, often target or usurp NE functions in different ways. The NE is also a physical barrier that must be overcome for viruses like the herpesviridae that assemble capsids in the nucleus. This thesis addresses two important questions: 1) How do herpesviruses cross the NE after new viral particles are produced in the nucleus? and 2) What is the nuclear envelope role of NET23/STING in the activation of immune factors upon herpesvirus infection? To address the first question, I followed two different approaches. The first used the isolation of microsomes from HSV-1 infected cells to identify possible host factors involved during herpesvirus exit through the NE on the prediction that such proteins would disperse into the ER during infection. I identified a group of vesicle fusion proteins that play a role in this herpesvirus exit through the NE. Depletion of three identified vesicle fusion proteins decreased the growth of HSV-1 in host cells, yielding accumulation of viral particles in the nucleus. The second approach was to follow the fate of nuclear envelope transmembrane proteins (NETs) during HSV-1 infection. To address the question of how NET23/STING is involved in innate immunity I tested the hypothesis that this NET acts as a transport receptor to carry signals through the peripheral channels of the NPC when central channel transport is blocked by pathogens. FRAP was used to quantify the mobility of NET23/STING upon the induction of the innate immune response, finding an increase of the mobility for this protein in the NE. To further elucidate its role within the NE I tested whether some NE-NET23/STING binding partners were being redistributed between the nucleus and cytoplasm during innate immune responses. This revealed two of these binding partners normally redistribute upon innate immune response activation and this is blocked in cells knocked down for NET23/STING. Finally, I confirmed that NET23/STING contributes to chromatin remodelling during infection involving an increase in the H3K9Me3 epigenetic mark. Collectively, these data argue the identification of novel host proteins involved in herpesvirus nuclear egress and the finding of a new role for NET23/STING within the NE.

Plusieurs publications, parues lors de ma thèse, ont révélé que les protéines de la membrane nucléaireinterne (INM) et plus particulièrement l’émerine, la lamine A, SUN1, l’actine et BAF, jouaient un rôleessentiel dans les propriétés mécaniques du noyau et de la cellule. L’assemblage de l’enveloppenucléaire et les interactions de ces protéines entre-elles sont régulées par des évènements dephosphorylation et d’oligomérisation. Mon objectif était de décrire les évènements moléculairesessentiels à l’assemblage de l’enveloppe nucléaire interne, afin de pouvoir par la suite comprendrecomment l’enveloppe nucléaire répond à un stress mécanique.J’ai dans un premier temps caractérisé les évènements d’oligomérisation et de phosphorylation de laprotéine émerine. J’ai montré que cette protéine était capable de former, in vitro et en cellules, de grosoligomères indispensables à son interaction avec la lamine A. J’ai également observé que desmutations dans l’émerine, aboutissant à la dystrophie musculaire d’Emery-Dreifuss, affectaient lespropriétés d’auto-association de cette protéine.En parallèle, j’ai étudié les interactions entre émerine, lamine, SUN1, actine et BAF in vitro. J’ai pumontrer des interactions directes entre le domaine C-terminal de la lamine A et les protéines émerine,actine et SUN1. Ces trois protéines lient la lamine A sur des surfaces différentes suggérant l’existencede complexes à 3 ou 4 protéines dans la cellule. L’analyse des modes de régulation des interactionsentre ces protéines doit être poursuivie afin de comprendre quels sont les évènements moléculairesessentiels au maintien de l'intégrité nucléaire et à la transmission d’un signal mécanique entre lecytosquelette et le nucléosquelette. / During my PhD, several papers revealed that the inner nuclear membrane (INM) proteins, andespecially emerin, lamin A, SUN1, actin and BAF, played an essential role in the mechanicalproperties of the nucleus and the cell. The nuclear envelope assembly and the interactions betweenthese proteins are regulated by phosphorylation and oligomerization events. My aim was to describemolecular events essential for inner nuclear envelope assembly as a first step to understand how thenuclear envelope responds to a mechanical stress.I first characterized the oligomerization and phosphorylation states of the protein emerin. I showedthat this protein is capable of forming, in vitro and in cells, large oligomers essential to its interactionwith lamin A. I also observed that several emerin mutations leading to Emery-Dreifuss musculardystrophy impaired the self-association properties of this protein.In parallel, I studied the interactions between emerin, lamin, SUN1, actin and BAF in vitro. I was ableto demonstrate direct interactions between the C-terminal domain of lamin A and the proteins emerin,actin and SUN1. These three proteins bind lamin A on different surfaces suggesting the existence ofcomplexes of 3 or 4 proteins in the cell. Analysis of the mechanisms regulating interactions betweenthese proteins should be pursued in order to understand what are the molecular events responsible forthe maintenance of nuclear integrity and the transmission of a mechanical signal between thecytoskeleton and the nucleoskeleton.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18496号 / 理博第4011号 / 新制||理||1578(附属図書館) / 31382 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 西村 いくこ, 教授 鹿内 利治, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM