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

ABSTRACT: 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.

SUBMITTER: Ostlund C

PROVIDER: S-EPMC2776502 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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

Ostlund Cecilia C Folker Eric S ES Choi Jason C JC Gomes Edgar R ER Gundersen Gregg G GG Worman Howard J HJ

Journal of cell science 20091020 Pt 22

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-t ...[more]

PMID: 19843581

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Project description:BACKGROUND:Adipose-derived stem cells (ASCs) that show multidifferentiation and anti-immune rejection capacities have been widely used in plastic and reconstructive surgery. Previous studies have indicated that mechanical and biophysical interactions between cells and their surrounding environment regulate essential processes, such as growth, survival, and differentiation, and the cytoskeleton system plays an important role in the mechanotransduction. However, the role of mechanical force in the determination of lineage fate is still unclear. METHODS:Human ASCs (hASCs) were obtained from three different donors by liposuction. Adipogenesis and osteogenesis were determined by Oil Red O and Alizarin Red staining, respectively. The mRNA levels of the cytoskeleton system, PPARγ, and C/EBPα were determined by real-time polymerase chain reaction (RT-PCR). The level of cytoskeleton, PPARγ, and C/EBPα protein levels were measured by Western blotting. The morphology of the cytoskeleton system during adipogenesis was observed with confocal microscopy. hASCs were transfected with a SUN2-specific shRNA to knockdown sun2, and a nontargeting shRNA was used as a control. RESULTS:We found that disrupting the physiological balance between the cytoskeleton and the linker of the nucleoskeleton and cytoskeleton (LINC) complex (especially SUN2) could impact the adipogenesis of hASCs in vitro. Microtubule (MT) depolymerization with nocodazole (which interferes with the polymerization of MTs) increased the expression of SUN2 and PPARγ, while taxol (an inhibitor of MT disassembly) showed the opposite results. Meanwhile, hASCs with sun2 knockdown overexpressed MTs and decreased PPARγ expression, thereby inhibiting the adipogenesis. Furthermore, knockdown of sun2 changed the structure of perinuclear MTs. CONCLUSIONS:We demonstrated the presence of cross-talk between MT and SUN2, and this cross-talk plays a critical role in the rebalance of the mechanical environment and is involved in the regulation of PPARγ transport during adipogenic differentiation of hASCs.

Dataset Information

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

Publications

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

Similar Datasets

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