Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner.

Pancreatic cancer is an aggressive and intractable malignancy with high mortality. This is due in part to a high resistance to chemotherapeutics and radiation treatment conferred by diverse regulatory mechanisms. Among these, constituents of the nuclear envelope play a significant role in regulating oncogenesis and pancreatic tumor biology, and this review focuses on three specific components and their roles in cancer. The LINC complex is a nuclear envelope component formed by proteins with SUN and KASH domains that interact in the periplasmic space of the nuclear envelope. These interactions functionally and structurally couple the cytoskeleton to chromatin and facilitates gene regulation informed by cytoplasmic activity. Furthermore, cancer cell invasiveness is impacted by LINC complex biology. The nuclear lamina is adjacent to the inner nuclear membrane of the nuclear envelope and can actively regulate chromatin in addition to providing structural integrity to the nucleus. A disrupted lamina can impart biophysical compromise to nuclear structure and function, as well as form dysfunctional micronuclei that may lead to genomic instability and chromothripsis. In close relationship to the nuclear lamina is the nuclear pore complex, a large megadalton structure that spans both outer and inner membranes of the nuclear envelope. The nuclear pore complex mediates bidirectional nucleocytoplasmic transport and is comprised of specialized proteins called nucleoporins that are overexpressed in many cancers and are diagnostic markers for oncogenesis. Furthermore, recent demonstration of gene regulatory functions for discrete nucleoporins independent of their nuclear trafficking function suggests that these proteins may contribute more to malignant phenotypes beyond serving as biomarkers. The nuclear envelope is thus a complex, intricate regulator of cell signaling, with roles in pancreatic tumorigenesis and general oncogenic transformation.

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of “nuclear envelopathies” is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

Connecting the nucleus to the cytoskeleton by SUN–KASH bridges across the nuclear envelope

Cellular migrations through constricted spaces are a crucial aspect of many developmental and disease processes including hematopoiesis, inflammation and metastasis. A limiting factor in these events is nuclear deformation. Here, we establish an in vivo model in which nuclei can be visualized while moving through constrictions and use it to elucidate mechanisms for nuclear migration. C. elegans hypodermal P-cell larval nuclei traverse a narrow space that is about 5% their width. This constriction is blocked by fibrous organelles, structures that pass through P cells to connect the muscles to cuticle. Fibrous organelles are removed just prior to nuclear migration, when nuclei and lamins undergo extreme morphological changes to squeeze through the space. Both actin and microtubule networks are organized to mediate nuclear migration. The LINC complex, consisting of the SUN protein UNC-84 and the KASH protein UNC-83, recruits dynein and kinesin-1 to the nuclear surface. Both motors function in P-cell nuclear migration, but dynein, functioning through UNC-83, plays a more central role as nuclei migrate towards minus ends of polarized microtubule networks. Thus, the nucleoskeleton and cytoskeleton are coordinated to move nuclei through constricted spaces.

The cardiomyocyte cytoskeleton, including the sarcomeric contractile apparatus, forms a cohesive network with cellular adhesions at the plasma membrane and nuclear–­cytoskeletal linkages (LINC complexes) at the nuclear envelope. Human cardiomyopathies are genetically linked to the LINC complex and A-type lamins, but a full understanding of disease etiology in these patients is lacking. Here we show that SUN2-null mice display cardiac hypertrophy coincident with enhanced AKT/MAPK signaling, as has been described previously for mice lacking A-type lamins. Surprisingly, in contrast to lamin A/C-null mice, SUN2-null mice fail to show coincident fibrosis or upregulation of pathological hypertrophy markers. Thus, cardiac hypertrophy is uncoupled from profibrotic signaling in this mouse model, which we tie to a requirement for the LINC complex in productive TGFβ signaling. In the absence of SUN2, we detect elevated levels of the integral inner nuclear membrane protein MAN1, an established negative regulator of TGFβ signaling, at the nuclear envelope. We suggest that A-type lamins and SUN2 play antagonistic roles in the modulation of profibrotic signaling through opposite effects on MAN1 levels at the nuclear lamina, suggesting a new perspective on disease etiology.

LINC (linker of nucleoskeleton and cytoskeleton) complexes play an essential role in nuclear migration by connecting the nucleus to the cytoskeleton and/or motor proteins. Plant LINC complexes have recently been identified in Arabidopsis thaliana, with the inner nuclear membrane SUN and outer nuclear membrane WIP proteins comprising the first identified complex. A recent study identified a nuclear movement defect in Arabidopsis pollen vegetative nuclei linked to the outer nuclear envelope WIP and WIT proteins. However, the role that SUN proteins may play in pollen nuclear migration has yet to be addressed. To explore this question, a SUN2 lumenal domain that was targeted to the ER specifically in pollen was over-expressed. It is shown that the ER-targeted SUN2 lumenal domain was able to displace WIP and WIT proteins from the pollen vegetative nuclear envelope. Expression of this dominant-negative transgene led to impaired VN mobility, impaired pollen tube guidance, and defective pollen tube reception. The observed pollen defects are similar to phenotypes observed in a wip1-1 wip2-1 wip3-1 wit1-1 wit2-1 mutant. It is also shown that these defects were dependent on the KASH-binding function of the SUN2 lumenal domain. These data support a model where LINC complexes formed by SUN, WIP, and WIT at the VNE are responsible for VN migration and suggest an important function of SUN, WIP, and WIT in pollen tube guidance and reception.

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

![Figure][1] Electron microscopy shows a large gap between the inner and outer nuclear membranes of a worm muscle cell lacking UNC-84. LINC complexes do not maintain the space between nuclear membranes, except in cells exposed to mechanical stress, [Cain et al.][2] reveal. SUN proteins in

Successful nuclear migration through constricted spaces between cells or in the extracellular matrix relies on the ability of the nucleus to deform. Little is known about how this takes place in vivo. We have studied confined nuclear migration in Caenorhabditis elegans larval P cells, which is mediated by the LINC complex to pull nuclei towards the minus ends of microtubules. Null mutations of the LINC component unc-84 lead to a temperature-dependent phenotype, suggesting a parallel pathway for P-cell nuclear migration. A forward genetic screen for enhancers of unc-84 identified cgef-1 (CDC-42 guanine nucleotide exchange factor). Knockdown of CDC-42 in the absence of the LINC complex led to a P-cell nuclear migration defect. Expression of constitutively active CDC-42 partially rescued nuclear migration in cgef-1; unc-84 double mutants, suggesting that CDC-42 functions downstream of CGEF-1. The Arp2/3 complex and non-muscle myosin II (NMY-2) were also found to function parallel to the LINC pathway. In our model, CGEF-1 activates CDC-42, which induces actin polymerization through the Arp2/3 complex to deform the nucleus during nuclear migration, and NMY-2 helps to push the nucleus through confined spaces.

The nuclear envelope (NE) is connected to the different types of cytoskeletal elements by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes exist from yeast to humans, and have preserved their general architecture throughout evolution. They are composed of SUN and KASH domain proteins of the inner and the outer nuclear membrane, respectively. These SUN–KASH bridges are used for the transmission of forces across the NE and support diverse biological processes. Here, we review the function of SUN and KASH domain proteins in various unicellular and multicellular species. Specifically, we discuss their influence on nuclear morphology and cytoskeletal organization. Further, emphasis is given on the role of LINC complexes in nuclear anchorage and migration as well as in genome organization.

Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

Intracellular nuclear migration is essential for many cellular events including fertilization, establishment of polarity, division and differentiation. How nuclei migrate is not understood at the molecular level. The C. elegans KASH protein UNC-83 is required for nuclear migration and localizes to the outer nuclear membrane. UNC-83 interacts with the inner nuclear membrane SUN protein UNC-84 and is proposed to connect the cytoskeleton to the nuclear lamina. Here, we show that UNC-83 also interacts with the kinesin-1 light chain KLC-2, as identified in a yeast two-hybrid screen and confirmed by in vitro assays. UNC-83 interacts with and recruits KLC-2 to the nuclear envelope in a heterologous tissue culture system. Additionally, analysis of mutant phenotypes demonstrated that both KLC-2 and the kinesin-1 heavy chain UNC-116 are required for nuclear migration. Finally, the requirement for UNC-83 in nuclear migration could be partially bypassed by expressing a synthetic outer nuclear membrane KLC-2::KASH fusion protein. Our data support a model in which UNC-83 plays a central role in nuclear migration by acting to bridge the nuclear envelope and as a kinesin-1 cargo-specific adaptor so that motor-generated forces specifically move the nucleus as a single unit.