The Nesprins Are Giant Actin-Binding Proteins, Orthologous to Drosophila melanogaster Muscle Protein MSP-300

Zhang, Qiuping; Ragnauth, Cassandra D.; Greener, Marc; Shanahan, Catherine M.; Roberts, Roland

doi:10.1006/geno.2002.6859

Cited by 178 publications

(169 citation statements)

References 30 publications

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“…This construct has recently been shown to displace actin-binding protein Nesprin-2 giant from the NE (14), which may connect the actin cytoskeleton to the nucleus (9,(19)(20)(21). We found that the fraction of EGFP-KASH2-transfected cells displaying an organized actin cap was dramatically decreased compared to WT cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

A perinuclear actin cap regulates nuclear shape

Khatau¹,

Hale²,

Stewart-Hutchinson

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

531

602

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Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap.LINC complexes ͉ nucleus I n 1921, Champy and Carleton suggested an apparent correlation between the shape of various types of animal cells and the shape of their respective nuclei (1). Moreover, defects in nuclear shape are routinely used in the lab and in clinical settings as markers of disease and differentiation in human cells and tissues (2). However, remarkably little is known about the factors that determine nuclear morphology in living cells. In particular, the molecular mechanisms that govern the shape of the interphase nucleus are unknown. Here we show that an actin filament structure that forms a cap or dome located above the apical surface of the nucleus tightly controls nuclear shape and identify key associated cytoskeletal regulators of its organization and nuclear shape-determining function. The organization and function of the perinuclear actin cap are deregulated in diseased cells with distorted nuclei. Results and DiscussionTo test the hypothesis of a correlation between the shape of the nucleus and the overall cell shape, mouse embryonic fibroblasts were dispersed on fibronectin (FN)-coated glass substrates. Using morphometric analysis, we found that nuclear shape and cellular shape correlated ( Fig. 1 A and B). Shape factor, defined as 4 A/P 2 (where A and P are the nuclear area and perimeter), approaches 1 for a rounded nucleus and approaches 0 for an elongated nucleus. Elongated cells typically showed an elongated nucleus of low shape factor; rounded cells showed a rounded nucleus of high shape factor (Fig. 1 A). To control cell shape and, therefore, be able to quantify nuclear shape as a function of cell shape, we developed adhesive FN-coated micropatterned stripes of width ranging between 10 and 50 m, which alternated with stripes covered with nonadhesive poly...

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Section: Resultsmentioning

confidence: 99%

A perinuclear actin cap regulates nuclear shape

Khatau¹,

Hale²,

Stewart-Hutchinson

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

531

602

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“…KASH domains are found in a variety of proteins that target to the nuclear envelope (Starr and Fischer, 2005). In all tested cases, the KASH domain is sufficient for nuclear envelope localization Zhang et al, 2002;Fischer et al, 2004). Moreover, a naturally occurring isoform of Klarsicht without the KASH domain does not localize to the nuclear envelope (Guo et al, 2005).…”

Section: The Function Of Kash Domainsmentioning

confidence: 96%

“…Moreover, a naturally occurring isoform of Klarsicht without the KASH domain does not localize to the nuclear envelope (Guo et al, 2005). KASH-domain proteins have been shown to play roles in positioning of nuclei in mammalian skeletal muscles, Drosophila melanogaster photoreceptor cells, and C. elegans syncytial cells Zhang et al, 2002;Patterson et al, 2004;Grady et al, 2005;Guo et al, 2005). These studies have implicated that KASH-domain containing proteins function at the outer nuclear membrane and likely represent the first group of proteins to localize specifically to the outer nuclear membrane but not the rest of the ER (reviewed in Starr and Fischer, 2005).…”

Section: The Function Of Kash Domainsmentioning

confidence: 99%

UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

McGee

Rillo

Anderson

et al. 2006

MBoC

127

180

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UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane. INTRODUCTIONA variety of cellular and developmental processes, including fertilization, cell division, cell migration, and establishment of polarity, depend on positioning the nucleus to a specific location within the cell. For example, in budding yeast the nucleus must migrate to the bud neck before the onset of mitosis. Also, nuclei actively follow the leading edge of migratory cells, such as those in the developing cerebral cortex. Nuclear migration defects in these two examples lead to the missegregation of chromosomes or the neurological disease lissencephaly, respectively (reviewed in Morris, 2000). The role of microtubules and associated dynein and kinesin motors in nuclear migration are well established (reviewed in Reinsch and Gonczy, 1998). Although less established, actin also plays an important role in many nuclear positioning events (reviewed in Starr and Han, 2003). Recently, a definitive role for actin networks has been described in nuclear migration during NIH 3T3 cell polarization (Gomes et al., 2005). It remains relatively unknown how the nucleus connects to the cytoplasmic cytoskeleton during nuclear migration. Furthermore, it is not clear how the forces involved in nuclear positioning are transferred across both membranes of the nuclear envelope from the cytoskeleton to the nuclear matrix.We have previously proposed that two C. elegans proteins, UNC-84 and UNC-83, function to control nuclear migration by bridging the nuclear envelope, connecting the cytoskeleton with the nuclear matrix (Starr et al., 2001;Lee et al., 2002;Starr and Han, 2003). Mutations in unc-83 or unc-84 disrupt nuclear migration in at least three cell types: embryonic hypodermal hyp7 precursors, larval hypodermal P-cells, and embryonic intestinal primordial cells (Horvitz and Sulst...

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“…Actin is physically linked to the nucleus via nesprin, which is a component of the LINC complex (49). To understand whether this physical link is necessary for nuclear deformability, cells were transfected with dominant negative (DN)-KASH fused with GFP, which displaces the endogenous nesprin from the nuclear envelope to the endoplasmic reticulum, thereby disrupting the LINC complex (50).…”

Section: Linc Complex and Microtubules Affect Amplitude Of Nuclear Areamentioning

confidence: 99%

Nuclear deformability and telomere dynamics are regulated by cell geometric constraints

Makhija

Jokhun

Shivashankar

2015

Proc. Natl. Acad. Sci. U.S.A.

189

186

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Forces generated by the cytoskeleton can be transmitted to the nucleus and chromatin via physical links on the nuclear envelope and the lamin meshwork. Although the role of these active forces in modulating prestressed nuclear morphology has been well studied, the effect on nuclear and chromatin dynamics remains to be explored. To understand the regulation of nuclear deformability by these active forces, we created different cytoskeletal states in mouse fibroblasts using micropatterned substrates. We observed that constrained and isotropic cells, which lack long actin stress fibers, have more deformable nuclei than elongated and polarized cells. This nuclear deformability altered in response to actin, myosin, formin perturbations, or a transcriptional down-regulation of lamin A/C levels in the constrained and isotropic geometry. Furthermore, to probe the effect of active cytoskeletal forces on chromatin dynamics, we tracked the spatiotemporal dynamics of heterochromatin foci and telomeres. We observed increased dynamics and decreased correlation of the heterochromatin foci and telomere trajectories in constrained and isotropic cell geometry. The observed enhanced dynamics upon treatment with actin depolymerizing reagents in elongated and polarized geometry were regained once the reagent was washed off, suggesting an inherent structural memory in chromatin organization. We conclude that active forces from the cytoskeleton and rigidity from lamin A/C nucleoskeleton can together regulate nuclear and chromatin dynamics. Because chromatin remodeling is a necessary step in transcription control and its memory, genome integrity, and cellular deformability during migration, our results highlight the importance of cell geometric constraints as critical regulators in cell behavior. mechanotransduction | cell geometry | actomyosin contractility | chromatin dynamics | telomere dynamics P hysical properties of the nucleus, such as its morphology and deformability, have been associated with important cellular functions like gene expression, genome integrity, and cell behavior (1-3). The major cellular components that regulate these physical properties are the cytoskeleton to nuclear links and the nuclear lamina (4-8). Lineage-specific physical properties of the nucleus emerge during cellular differentiation; although stem cell nuclei are highly deformable (9, 10) and have a dynamic chromatin with hyperdynamic chromatin proteins (11), with differentiation, nuclei lose their deformability and become less deformable (12). The nucleus in a differentiated cell is physically coupled to the cytoskeleton via lamins and the linker of nucleoskeleton and cytoskeleton (LINC) complex, which comprises transmembrane Sad1p, UNC-84 (SUN) and Klarsicht, ANC-1, Syne Homology (KASH) domain proteins (13-18). Any perturbation to these components is linked to changes in nuclear morphology and deformability (19). Therefore, the meshwork of actin stress fibers and lamin A/C serves as a critical physical intermediate in the maintenance of nuclear func...

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The Nesprins Are Giant Actin-Binding Proteins, Orthologous to Drosophila melanogaster Muscle Protein MSP-300

Cited by 178 publications

References 30 publications

A perinuclear actin cap regulates nuclear shape

A perinuclear actin cap regulates nuclear shape

UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

Nuclear deformability and telomere dynamics are regulated by cell geometric constraints

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