Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells

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

Östlund

Luxton

et al. 2010

166

202

Mutations in LMNA, which encodes A-type lamins, result in disparate diseases, known collectively as laminopathies, that affect distinct tissues, including striated muscle and adipose tissue. Lamins provide structural support for the nucleus and sites of attachment for chromatin, and defects in these functions may contribute to disease pathogenesis. Recent studies suggest that A-type lamins may facilitate connections between the nucleus and the cytoskeleton mediated by nuclear envelope nesprin and SUN proteins. In mammalian cells, however, interfering with A-type lamins does not affect the localization of these proteins. Here, we used centrosome orientation in fibroblasts, which requires separate nuclear and centrosome positioning pathways, as a model system to understand how LMNA mutations affect nucleus-cytoskeletal connections. We find that LMNA mutations causing striated muscle diseases block actin-dependent nuclear movement, whereas most that affect adipose tissue inhibit microtubule-dependent centrosome positioning. Genetic deletion or transient depletion of A-type lamins also blocked nuclear movement, showing that mutations affecting muscle exhibit the null phenotype. Lack of A-type lamins, or expression of variants that cause striated muscle disease, did not affect assembly of nesprin-2G and SUN2 into transmembrane actinassociated nuclear (TAN) lines that attach the nucleus to retrogradely moving actin cables. Nesprin-2G TAN lines were less stable, however, and slipped over the nucleus rather than moving with it, indicating that they were not anchored. Nesprin-2G TAN lines also slipped in SUN2-depleted cells. Our results establish A-type lamins as anchors for nesprin-2G-SUN2 TAN lines to allow productive movement and proper positioning of the nucleus by actin.nesprin SUN | linker of nucleoskeleton and cytoskeleton complex | muscular dystrophy L amin A and lamin C, the predominant A-type lamins, are expressed in most differentiated somatic cells. Yet, different mutations in the LMNA gene encoding these proteins result in a variety of diseases that affect specific tissues. LMNA mutations cause autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) and related diseases with dilated cardiomyopathy (DCM) that affect cardiac muscle and skeletal muscle to variable degrees (1). Other LMNA mutations cause Dunnigan-type familial partial lipodystrophy (FPLD) that affects adipose tissue (2), Charcot-Marie-Tooth type 2B1 disease that affects peripheral neurons (3), and progeroid syndromes with features of accelerated aging (4, 5). The mechanism whereby mutations in a single gene that is widely expressed cause such diverse diseases remains a puzzle.There are two prevailing hypotheses to explain the pathogenesis of laminopathies. The mechanical stress hypothesis proposes that alterations in A-type lamins compromise nuclear integrity in tissues susceptible to stress, such as striated muscle. This model is supported by findings that A-type lamin deficiency disrupts nuclear integrity in model systems (6, 7). The secon...

Section: Resultssupporting

confidence: 81%

“…Loss of lamin A results in impaired cell migration (18,24). We tested whether disease-causing lamin A variants affected cell migration by expressing the variants in wound edge cells and stimulating migration with serum.…”

Section: Resultsmentioning

confidence: 99%

Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement

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

Östlund

Luxton

et al. 2010

166

202

“…Disruption of the LINC complex leads to a loss of mechanical stiffness in Swiss3T3 cells, similar to that seen in Lmna -/-fibroblasts (Broers et al, 2004;Lammerding et al, 2004;Lee et al, 2007;Stewart-Hutchinson et al, 2008). Lmna -/-fibroblasts also have defects in migration and polarization (Houben et al, 2009;Lee et al, 2007). These findings all point to a role for the LINC complex as a linker between the nucleoskeleton Journal of Cell Science 122 (22) (C)Recovery rates are expressed in terms of t 1/2 (see Materials and Methods) for FRAP of GFP-mini-nesprin-2G, GFP-KASH or GFP-KASH DL in the nuclear envelope region of wild-type MEFs (blue columns), Lmna -/-MEFs (red columns) or Lmna -/-cells co-transfected with exogenous RFP-lamin A (yellow columns).…”

Section: Discussionmentioning

confidence: 84%

Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins

Östlund

Journal of Cell Science

Choi

et al. 2009

168

166

The linker of nucleoskeleton and cytoskeleton (LINC) complex is situated in the nuclear envelope and forms a connection between the lamina and cytoskeletal elements. Sun1, Sun2 and nesprin-2 are important components of the LINC complex. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts and studied their diffusional mobilities using fluorescence recovery after photobleaching. We show that they all are more mobile in embryonic fibroblasts from mice lacking A-type lamins than in cells from wild-type mice. Knockdown of Sun2 also increased the mobility of a short, chimeric form of nesprin-2 giant (mini-nesprin-2G), whereas the lack of emerin did not affect the mobility of Sun1, Sun2 or mini-nesprin-2G. Fluorescence resonance energy transfer experiments showed Sun1 to be more closely associated with lamin A than is Sun2. Sun1 and Sun2 had similar affinity for the nesprin-2 KASH domain in plasmon surface resonance (Biacore) experiments. This affinity was ten times higher than that previously reported between nesprin-2 and actin. Deletion of the actin-binding domain had no effect on mini-nesprin-2G mobility. Our data support a model in which A-type lamins and Sun2 anchor nesprin-2 in the outer nuclear membrane, whereas emerin, Sun1 and actin are dispensable for this anchoring.

“…19,40,41 A type lamins have been implicated in nuclear-cytoplasmic interactions because lamin A/C deficient fibroblasts fail to orient their centrosome toward the leading edge, although it was unclear whether this was due to an effect on the centrosome or on the nucleus (or both). 42,43 To explore the role of lamins in nuclear movement, we first tested whether lamins might be found in TAN lines and did not detect accumulation of either endogenous lamin A/C or lamin B1 in TAN lines. Nonetheless, in fibroblasts derived from lamin A/C knockout mice or NIH3T3 fibroblasts acutely depleted of lamin A/C, we observed that the lack of centrosome orientation was due to a defect in nuclear movement.…”

confidence: 99%

TAN lines

Luxton

Gomes

et al. 2011

Nucleus

111

N uclear position is actively controlled and can be adjusted according to the needs of a cell by nuclear movement. Microtubules mediate the majority of nuclear movements studied to date, although examples of nuclear movements mediated by the actin cytoskeleton have been described. One such actin-dependent nuclear movement occurs during centrosome orientation in fibroblasts polarizing for migration. Here, the centrosome is maintained at the cell center while the nucleus is moved to the cell rear by actin retrograde flow thus positioning the centrosome between the nucleus and the leading edge of the cell. We have explored the molecular mechanism for actin dependent movement of the nucleus during centrosome orientation. We found that a novel linear array of nuclear envelope membrane proteins composed of nesprin-2G and SUN2, called transmembrane actin-associated nuclear (TAN) lines, couple the nucleus to moving actin cables resulting in the nucleus being positioned at the cell rear. TAN lines are anchored by A-type lamins and this allows the forces generated by the actin cytoskeleton to be transmitted across the nuclear envelope to move the nucleus. Here we review the data supporting this mechanism for nuclear movement, discuss questions remaining to be addressed and consider how this new mechanism of nuclear movement may shed light on human disease.