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

The nuclear lamina consists of a mesh-like network of lamin proteins anchored to the inner nuclear membrane by interactions with integral membrane proteins such as emerin. Emerin binding to lamin A/C is one of the interactions that connect the inner nuclear membrane to the lamina. Infection by herpesviruses results in changes in the organization of the nuclear lamina, perhaps in order to facilitate envelopment of capsids at the inner nuclear membrane. In HSV-1 infected cells, alterations to the lamin proteins have been shown to involve pUL34, pUL31, and pUS3 proteins, which are also required for normal nuclear envelopment. We tested hypotheses about the mechanism and significance of lamina disruption. This thesis presents the following data. Infection of multiple cell types induced emerin hyperphosphorylation that was dependent on the presence of pUL34 and kinase active pUS3 proteins. The pUL34-dependent component was also sensitive to Rottlerin treatment suggesting that cellular kinases sensitive to Rottlerin were involved in emerin modification. LAP2 (another lamin associated protein) was de-modified (perhaps de-phosphorylated) in a pUS3 and pUL34 independent manner. Emerin was not required for growth of HSV-1. Hyperphosphorylation of emerin was required for its disassociation from the lamina. PKC family members have been implicated in the disruption of the nuclear lamina during herpesvirus nuclear egress. To test the hypothesis that PKC activity was required for viral replication, PKC activity was blocked with PKC inhibitors and dominate negative PKC constructs. Chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta did not inhibit viral growth. In addition to lamin associated proteins, lamin localization is also disrupted during herpesvirus infections. Emerin and lamin A/C are binding partners and the localization of both proteins is disrupted by pUS3 and cellular kinase mediated phosphorylation. To test the hypothesis that HSV-1-induced lamin A/C disruption is mediated via a mechanism similar to emerin's, pUS3 and Rottlerin Sensitive Kinases were inhibited and lamin A/C localization was observed. Unlike emerin, HSV-1-induced disruption of lamin A/C was not altered by Rottlerin Sensitive Kinase inhibition suggesting that HSV has multiple mechanisms for disruption of the lamina. Phosphorylation of lamina components, by Rottlerin Sensitive Kinases, may be a required event prior to primary envelopment. To test this hypothesis, growth of HSV-1 was tested in Rottlerin treated infected cells. Although the inhibitor Rottlerin, did reduce viral growth, it was also was also associated with severe depression of viral late-gene expression. TEM analysis suggested that Rottlerin Sensitive Kinases(s) were required for: (i) nuclear egress and (ii) capsid accumulation or formation supporting the hypothesis that the capsids were made in the presence of Rottlerin were unable to leave the nucleus. pUS3 is a multi-functional protein in alpha-herpesviruses. It has been implicated in lamina disruption, protecting the infected cell from apoptosis, and de-envelopment at the outer nuclear membrane. In BT-549 cells, a breast cancer cell line with low PKC delta expression, the hypothesis was tested that in the absence of cellular lamina disrupting kinases, an US3-null virus would be blocked at the lamina disruption step. In BT-549 cells, the US3-null (vRR1202) virus was 10-fold decreased above the typical 10-fold decrease, compared to WT virus, to produce a 100-fold decrease in infectious PFU yet apoptosis was not increased. Lamin A/C disruption occurred via similar mechanism in both breast cancer cell lines: BT-549 and MCF-7. Interestingly, in the BT-549 cells, emerin was extensively hyperphosphorylated in an US3-null infection, yet was not redistributed along the NE. These data support a model that one or more specific residue(s) must be phosphorylated for emerin disconnection from lamina.

<p><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:HyphenationZone>21</w:HyphenationZone> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> </w:Compatibility> <w:BrowserLevel>MicrosoftInternetExplorer4</w:BrowserLevel> </w:WordDocument> </xml><![endif]--><!--[if gte mso 9]><xml> <w:LatentStyles DefLockedState="false" LatentStyleCount="156"> </w:LatentStyles> </xml><![endif]--> <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} p.Standardmedluft, li.Standardmedluft, div.Standardmedluft {mso-style-name:"Standard med luft"; margin-top:14.0pt; margin-right:0cm; margin-bottom:0cm; margin-left:0cm; margin-bottom:.0001pt; text-align:justify; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 2.0cm 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> <!--[if gte mso 10]><mce:style><! /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} --><!--[endif]--></p><p>Transvection is a widespread phenomenon affecting chromosomal and gene function. There are many examples of epigenetic machineries controlling gene regulation. Nuclear Lamin proteins could have this function. This project shows <em>zeste</em> dependent transvection<strong> </strong>in loss-of-function<strong> </strong><em>lamin</em> mutants in <em>Drosophila melanogaster</em>.<strong> </strong>The <em>zeste</em> locus<strong> </strong>encodes a regulatory gene product affecting the expression of other loci, e.g. <em>white</em>. No transvection effect in loss-of-function <em>lamin </em>mutants has so far been shown. The effect of homozygosity versus heterozygosity of <em>lamin</em> on <em>zeste</em>-dependent transvection at paired <em>white</em> loci was analysed by crossing fruit flies to get homozygous<em> </em><em>z¹</em>; <em>lam</em>^D395 individuals. Whether or not the <em>zeste (z¹</em>) transvection effect on <em>white</em> was affected by <em>lam</em> ^D395 loss-of-function mutation was determined by comparing the eye colour phenotypes of double mutant <em>z¹</em>; <em>lam</em>^D395 females to that of <em>z¹/Y</em>; <em>lam</em>^D395 males, which were used as an internal negative control since they are hemizygous for <em>zeste</em> that is located on the X chromosome. Females homozygous for <em>z¹</em> and <em>lam</em>^D395 displayed the <em>z¹</em>-characteristic yellow eye colour. The conclusion is that <em>zeste</em>-dependent transvection effect at <em>white</em> also occurs in <em>lamin</em> mutants. Future research on transvection is needed in order to understand the exact mechanisms of gene regulation. Even gene therapies for some human diseases can take advantage of <em>trans</em>-acting sequences to correct gene expression.</p><p> </p>

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The nuclear lamina is a filamentous network that underlies the nuclear envelope. Lamina components include the family of LEM domain (LEM-D) proteins, named for LAP2, emerin and MAN1. Mutations in genes encoding LEM-D proteins cause tissue-restricted human disease, even though these genes are globally expressed. To understand the contributions of the LEM-D proteins to nuclear lamina function, investigations of the Drosophila LEM-D proteins was undertaken. The Drosophila genome encodes four LEM-D proteins and this thesis describes work done on the Drosophila homologues of MAN1 and emerin, Drosophila MAN1 (dMAN1) and Otefin (Ote). Chapter 2 describes the generation and phenotypic analyses of dMAN1 mutants. These mutants display a range of tissue-specific defects associated with an increase in BMP/Dpp signaling. This suggests that dMAN1 downregulates BMP/Dpp signaling at the nuclear periphery. Chapter 3 describes the identification and phenotypic analyses of ote mutants. Loss of Ote is associated with a tissue-specific defect of the female germline where ote mutant females display defects in germline stem cell (GSC) maintenance. Loss of Ote causes defects in the germline cells, the cap cells of GSC niche and an increased sensitivity to Dpp signaling in both germline and somatic cells. These findings support models suggesting that laminopathies arise from dysfunction of the homeostasis in stem cell populations. Taken together, these studies suggest that the nuclear lamina may play tissue-specific roles through regulation of signal transduction pathways. Our data also support the use of Drosophila as a system to elucidate the mechanistic basis of diseases associated with defects in the nuclear lamina.

No description available.

The contents of nuclei are highly organized. Nuclear organization is facilitated by a dense protein network, called the nuclear lamina, which underlies the nuclear envelope. The nuclear lamina is composed of filamentous lamins and more than eighty lamin-associated proteins (LAPs). Among the first LAPs identified are LEM Domain (LEM-D) proteins, named after LAP2, emerin and MAN1. LEM-D proteins have many shared and unique functions that include providing structural support to the nucleus, regulating signal transduction pathways and gene expression, facilitating proper progression through the cell cycle and maintaining chromatin attachments at the nuclear periphery. Despite requirements for these processes in all cell types, loss of globally expressed LEM-D proteins causes tissue-restricted defects. Identification of the primary function in tissues susceptible to LEM-D protein loss is a persistent challenge in the nuclear lamina field. Research described here uses Drosophila as a model to understand LEM-D protein function. Loss of the Drosophila emerin homologue Otefin (Ote) causes a complex phenotype in the ovary wherein both somatic and germline cells are compromised. Using tissue-restricted expression experiments, it was determined that Ote function is only required in germline stem cells (GSCs) to maintain all cells in the ovary. Developmental, molecular and genetic analyses revealed that the primary defect in ote mutant ovaries is an early block in germline differentiation, followed by GSC death. Genetic rescue experiments revealed that both of these GSC defects are due to the activation of the DNA Damage Response (DDR) proteins ATR and Chk2. Interestingly, activation of ATR and Chk2 occurs independent of detectable canonical DDR triggers, including DNA damage. Immunohistochemical analyses suggest that Ote might be regulating chromatin condensation and/or heterochromatin organization in GSCs. Through studies of Ote, a rescue method was discovered that involves culturing animals at elevated temperatures. This novel rescue strategy, termed hyperthermia, acts independent of ATR or Chk2 inhibition. Interestingly, elevated temperatures leads to chromatin decondensation in Drosophila, suggesting that hyperthermia may rescue oogenesis by alleviating chromatin defects observed in ote mutant germ cells. Together, results from experiments discussed herein dissect a complex ovary phenotype to reveal the critical requirement for a nuclear lamina LEM-D protein. Investigations into Ote function have revealed several aspects of GSC biology. The ATR/Chk2 checkpoint activated in the absence of Ote uncovered a previously unidentified cause of female GSC death. Further, findings that ATR and Chk2 are activated in the absence of canonical triggers suggest that GSCs possess a system to monitor defects or changes in the nucleus that do not involve DNA damage. Therefore, studies of Ote function and ote mutant phenotypes have uncovered valuable insights into LEM-D protein function and revealed the existence of novel conditions required for GSC maintenance.

The nuclear matrix (NM) is proposed to be a permanent network of core filaments underlying thicker fibres, present regardless of transcriptional activity. It is found to be both RNA and protein rich; indeed, numerous important nuclear proteins are components of the structure. In addition to mediating the organisation of entire chromosomes, the NM has also been demonstrated to tether telomeres via their TTAGGG repeats. In order to examine telomeric interactions with the NM, a technique known as the DNA halo preparation has been employed. Regions of DNA that are tightly attached to the structure are found within a so-called residual nucleus, while those sequences forming lesser associations produce a halo of DNA. Coupled with various FISH methodologies, this technique allowed the anchorage of genomic regions by the NM, to be analysed. In normal fibroblasts, the majority of chromosomes and telomeres were extensively anchored to the NM. Such interactions did not vary significantly in proliferating and senescent nuclei. However, a decrease in NM-associated telomeres was detected in quiescence. Since lamin A is an integral component of the NM, it seemed pertinent to examine chromosome and telomere NM-anchorage in Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts, which contain mutant forms of lamin A. Indeed, genome tethering by the NM was perturbed in HGPS. In immortalised HGPS fibroblasts, this disrupted anchorage appeared to be rescued; the implications of this finding will be discussed. This study also suggested that telomere-NM interactions are aberrant in X-linked Emery-Dreifuss Muscular Dystrophy (X-EDMD), which is caused by mutant forms of emerin, another NM-associated protein. The positioning of selected genes in control and X-EDMD cell lines was examined in un-extracted nuclei using 2D and 3D FISH. Subtle shifts in the organisation of these genes were detected in diseased cells; however, their expression levels remained unaltered. Furthermore, in order to examine the architectural integrity of the nuclear lamina in lamin A and emerin mutant cell lines, scanning electron microscopy (SEM) was employed. This work revealed that such structures were indeed compromised in disease. The findings presented in this thesis highlight the importance of lamin A and emerin in mediating the organisation of the genome and taken together, promote the hypothesis that dysfunctional NM dynamics may well contribute to disease pathology.

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