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Comparative Study
. 2006 May;26(10):3738-51.
doi: 10.1128/MCB.26.10.3738-3751.2006.

SUN1 Interacts With Nuclear Lamin A and Cytoplasmic Nesprins to Provide a Physical Connection Between the Nuclear Lamina and the Cytoskeleton

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Free PMC article
Comparative Study

SUN1 Interacts With Nuclear Lamin A and Cytoplasmic Nesprins to Provide a Physical Connection Between the Nuclear Lamina and the Cytoskeleton

Farhana Haque et al. Mol Cell Biol. .
Free PMC article

Abstract

Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

Figures

FIG. 1.
FIG. 1.
SUN protein family sequence alignments and predicted structures. (A) Schematic representation of SUN protein structures highlighting the serine-rich domain (Ser), zinc (Zn) finger motif, transmembrane (TM) domains predicted by TMpred, coiled coils (CC), and conserved SUN domain (shaded box). Percent homology with mSUN1 over the relevant region (arrows) is shown for each homolog. (B) mSUN1 hydropathy plot obtained with TMpred software, predicting four transmembrane domains at amino acids 231 to 254, 358 to 383, 386 to 407, and 413 to 431. (C) Alignment of the C-terminal SUN domains of SUN protein family members. Identical residues and conservative substitutions are shown in black and gray, respectively.
FIG. 2.
FIG. 2.
The NTD of SUN1 interacts with lamin A and with itself. (A) Schematic representation of the SUN1 constructs used. TM, transmembrane. (B) Cell lysates were made from U2OS cells transfected with HA-SUN1 together with either GFP (lanes 1 and 3) or GFP-lamin A (lanes 2 and 4). HA-SUN1 was immunoprecipitated with anti-HA antibodies and bound to protein A-Sepharose beads. Initial lysates (lanes 1 and 2) and immunoprecipitates (IP; lanes 3 and 4) were immunoblotted with anti-GFP antibodies to detect coprecipitating proteins. Positions of molecular size markers (kilodaltons) are shown on the left. IgG, immunoglobulin G. (C) 35S-labeled lamin A, SUN1-N, and SUN1-C proteins were produced by in vitro translation (lanes 1). The proteins were each incubated with MBP (lanes 2), MBP-SUN1-N (lanes 3), or MBP-SUN1-C (lanes 4) previously bound to amylose resin. Following resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, bound proteins were detected by autoradiography. The Coomassie-stained gel shows equal expression of the MBP fusion proteins. (D) Lamin A (LA), lamin C (LC), lamin B1 (LB1), and lamin B2 (LB2) were produced by in vitro translation (top) and bound to MBP alone (middle) or to MBP-SUN1-N (bottom) previously immobilized on amylose beads. Bound proteins were detected by autoradiography.
FIG. 3.
FIG. 3.
SUN1 is an NE protein. (A) Nuclei were produced from untransfected NIH 3T3 cells, and total extracts were produced from NIH 3T3 cells transiently transfected with untagged or myc-tagged SUN1 constructs, as indicated. In vitro-translated (IVT) SUN1 was generated from pCI-mSUN1. Protein samples were immunoblotted and probed with either preimmune rabbit serum (lane 1) or affinity-purified anti-SUN1 antibodies (lanes 2 to 6). Arrows indicate the mSUN1 doublet. Positions of molecular size markers (kilodaltons) are shown on the left. (B) Total (T), cytoplasmic (Cyt), and nuclear (Nuc) fractions were produced from NIH 3T3 cells. The nuclear fraction was then subjected to further extraction with buffers containing Triton X-100, salt, and urea, as indicated. Following centrifugation, soluble (Sol) and insoluble (Ins) fractions were produced. All fractions were analyzed by immunoblotting with anti-SUN1 antibodies. (C to E) Immunofluorescence localization of SUN1 in interphase (C) and mitotic (D and E) cells. In panel C, cells were stained with anti-lamin A/C antibody XB10 (red) and either preimmune serum or affinity-purified SUN1 antibodies (green). In panel D, cells stained for SUN1 (green) are shown at different stages of mitosis: metaphase (Meta), anaphase A (AnaA), anaphase B (AnaB), and telophase (Telo). In panel E, a metaphase cell is shown costained with SUN1 and mouse anti-γ-tubulin antibodies. DNA is shown in blue. Scale bars in panels C to E, 10 μm.
FIG. 4.
FIG. 4.
The NTD of SUN1 is responsible for anchoring to the nuclear lamina. (A) Schematic representation of the myc-tagged SUN1 constructs. Labeling is as in Fig. 2A, except that hatched boxes indicate locations of myc tags. (B) NIH 3T3 cells were transiently transfected with myc-SUN1 constructs as indicated and fixed in methanol 24 or 72 h (c, f, h, j, and l) posttransfection. Cells were then costained with anti-myc 9E10 (left side) and 3262 anti-lamin A/C (right side) antibodies. In parts k and l, apparent colocalization of myc-SUN1(450-913) with lamin A/C at the NE is indicated by yellow areas in the merged color images [red, myc-SUN1(450-913); green, lamin A/C]. (C) Transfected NIH 3T3 cells were subjected to pre-extraction with 0.5% Triton X-100 in PBS for 5 min on ice, immediately fixed in methanol, and then subjected to immunofluorescence staining as for panel B. Scale bars, 10 μm. (D) NIH 3T3 cells were transfected with myc-tagged SUN1 constructs encoding the full-length (FL) protein or the NTD (amino acids 1 to 355). Nuclei (Nuc) were isolated and incubated in extraction buffer containing 7 M urea, separated into soluble (Sol) and insoluble (Ins) fractions, and immunoblotted with anti-myc 9E10 antibodies.
FIG. 5.
FIG. 5.
Lamin A/C is not required for localization of SUN1 to the NE. Subcellular localization of SUN1 in RNA interference-treated NIH 3T3 (A to C) or Lmna knockout MEF cells (D and E). NIH 3T3 cells were transfected with a mouse (A and B) or a control human (C) lamin A/C siRNA and processed for immunofluorescence microscopy 48 h after transfection. Cells were stained with anti-lamin A/C XB10 (red) and anti-SUN1 (green) antibodies. Hoechst staining of DNA is in blue. Arrows indicate disrupted SUN1 localization at the poles of nuclei. The arrowhead indicates apparent leakage of DNA from the nucleus. Scale bars, 10 μm.
FIG. 6.
FIG. 6.
The CTD of SUN1 is located in the NE lumen. NIH 3T3 cells left untransfected (A to D) or transfected with pCI-emerin-myc (E to H) were fixed with 4% paraformaldehyde and permeabilized with either 0.5% Triton X-100 (A, C, E, and G) or 40 μg/ml digitonin on ice for 5 min (B, D, and F) or 2 min (H). Immunofluorescence staining was performed with antibodies against SUN1, α-tubulin, lamin A/C (3262), and the myc epitope, as indicated. Scale bars, 10 μm.
FIG. 7.
FIG. 7.
Probing the topology of SUN1 using myc-tagged SUN1 constructs. (A) Schematic representation of the myc-tagged SUN1 constructs used. Positions of myc tags are shown as hatched boxes. (B to E) U2OS cells were transiently transfected with SUN1 constructs with a myc tag engineered at either the N terminus (B) or the C terminus (C) or internally following residue 355 (D) or 456 (E). The cells were fixed with 4% paraformaldehyde and permeabilized with either Triton X-100 at room temperature (top) or with digitonin at 4°C (bottom) and then costained with anti-SUN1 (left side) and anti-myc 9E10 (right side) antibodies. Scale bar, 10 μm.
FIG. 8.
FIG. 8.
The SUN1 CTD interacts with the KASH domain of nesprins 1 and 2. (A) Lysates of U2OS cells transiently transfected with myc-SUN1 and nesprin 2β were subjected to immunoprecipitation with either anti-SUN1 or nonspecific anti-HA tag antibodies. The initial lysate and immunoprecipitates were then immunoblotted with anti-nesprin N2 antibodies. The arrow and arrowhead indicate coprecipitated nesprin 2β and the antibody heavy chain, respectively. (B) U2OS cells were cotransfected with myc-SUN1 and GFP or GFP-nesprin fusions, as indicated. Expression of GFP- and myc-tagged proteins was confirmed by immunoblotting (upper and lower left parts, respectively). Anti-myc immunoprecipitates (IP) were probed with GFP antibodies to detect coprecipitating GFP fusion proteins (right side).
FIG. 9.
FIG. 9.
Model of SUN1 topology and interactions at the NE. SUN1 is anchored at the INM through interaction of the nucleoplasmic NTD with lamin A and other nuclear factors, most likely chromatin (indicated by the question mark). The CTD of SUN1 lies in the NE lumen, where it interacts with nesprins that contain a C-terminal membrane-spanning KASH domain, resulting in their anchoring at the ONM. The N terminus of long nesprin isoforms binds to actin. As a result, a direct physical connection between the nuclear interior and the actin cytoskeleton is created. NPC, nuclear pore complex; CH, calponin homology domain.

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