Identification of nesprin 4. (A) Amino acid sequence of mouse protein NP_705805, now designated nesprin 4 (Nesp4). Nesp4 contains a leucine zipper (L Zip; blue), a spectrin repeat (red), and a KASH domain (KASH4; blue highlight). The putative transmembrane sequence (TM) is underlined. (B) Alignment of the KASH domains (KASH1–4) from nesprins 1–4. (C) Nesp4 gene (eight exons, 4. 2 kb) and protein domain organization. (D) Immunofluorescence microscopy of HSG cells stably expressing HA–Nesp4. The cells were labeled with antibodies against HA and Sun2. DNA is visualized with Hoechst dye. (Bar: 10 μm.)

SUN- and KASH-dependent localization of Nesp4. (A) HeLa cells transiently transfected with either GFP-tagged full-length Nesp4 (GFP–Nesp4) or a variety of GFP-tagged deletion mutants (Nesp4ΔKASH–GFP, Nesp4ΔKASH–GST—GFP, and Nesp4ΔLum–GFP). Deletion of the KASH domain or the luminal portion of the KASH domain prevents localization to the NE. Conversely, GFP fused to the Nesp4 KASH domain (GFP–KASH4) localizes to the NE as revealed by colabeling with an antibody against the NPC protein Nup153. (B) Transfection of SS–HA–Sun1L–KDEL into HeLa cells stably expressing GFP–KASH4 displaces GFP–KASH4 from the NE. (C) Depletion of Sun1 and Sun2 by RNAi leads to loss of NE-associated GFP-KASH4. In the merged images, DNA (blue) was revealed by costaining with Hoechst dye. (Bars: 10 μm.)

Nesp4 is expressed in secretory epithelial cells. (A) Western blot analysis of HeLa cells using rabbit anti-Nesp4. The cells were either nontransfected (−) or expressed HA-Nesp4 (+). A single band (≈54 kDa) corresponding to HA–Nesp4 is evident only in the transfected sample. The relatively slow migration of HA–Nesp4 (its predicted molecular mass is 42 kDa) is likely caused by its high proline content (9%). (B) HeLa cells expressing HA–Nesp4 and labeled with rabbit anti-Nesp4 and mouse anti-HA. The rabbit anti-Nesp4 only decorates the NE of transfected cells (C) Immunofluorescence microscopy of mouse tissue cryosections using the rabbit anti-Nesp4 antibody. (D) Immunofluorescence microscopy of both undifferentiated and differentiated HC11 mouse mammary cells using the anti-Nesp4 antibody. In all positive cells Nesp4 is localized at the NE. All specimens were stained with Hoechst dye to reveal the DNA. (Bars: B and D, 10 μm; C, 40 μm.)

Nesp4 interacts with kinesin-I. (A) Anti-GFP IP analysis of ³⁵S-Met/Cys-labeled HEK293 cells expressing either Nesp4ΔKASH–GFP (large arrow) or GFP alone (small arrow). A faint ≈120-kDa band (*) present only in the Nesp4ΔKASH–GFP IPs was found, by mass spectrometry, to contain Kif5B, the kinesin-1 heavy chain. (B) Unlabeled HeLa cells expressing Nesp4ΔKASH–GFP, Nesp4ΔKASH–GST–GFP, or GFP were processed for IP using anti-GFP. IPs were analyzed by Western blot using either anti-GFP or anti-Kif5B. Kif5B (*) was clearly evident in IPs containing either Nesp4ΔKASH–GFP or Nesp4ΔKASH–GST–GFP. Ig heavy chains are indicated (arrowhead). (C) Similar analysis of HeLa cells expressing Nesp4ΔKASH–GFP, GFP–Nesp4(3–206), and Nesp4–GFP(207–307). Kinesin-1 coimmunoprecipitates only with Nesp4ΔKASH–GFP and Nesp4–GFP(207–307). (D) HeLa cells were transfected with various combinations of Nesp4ΔKASH–GFP and with KLC1 fused to a fluorescent protein and HA-tagged mCit-HA-KLC1. IP of Nesp4 followed by Western blot analysis using both anti-Nesp4 and anti-HA antibodies reveals association between Nesp4 and mCit–HA–KLC1. Immunofluorescence microscopy of HeLa cells expressing various combinations of nesprins and KLC1. (E) mCit–HA–KLC1 alone localizes to the cytoplasm. (F) HA–Nesp4 recruits mCit–HA–KLC1 to the NE. (G) Nesp3 has no effect on mCit–HA–KLC1. (H) An HA-tagged version of the KLC1 TPR domain [HA-KLC(TPR6)] is recruited to the NE by GFP–Nesp4. (I) The distribution of HA–KLC(1–176), which lacks the TPR domain, is unaffected by GFP–Nesp4. In the merged images, DNA, revealed by staining with Hoechst dye, is shown in blue. (Bar: 10 μm.) (J) In polarized epithelial cells microtubules are noncentrosomal. The bulk are arranged in lateral bundles with their plus (+) ends oriented toward the basal membrane. Kinesin-1 on the NE should drive the nucleus toward the base of the cell. (K) Nesp4-mediated recruitment of kinesin-1 to the NE in fibroblasts should induce separation of the nucleus and centrosome.

Nesp4 expression perturbs centrosome and Golgi positioning. (A) In ≈50% of HeLa cells, exogenous Nesp4 (GFP–N4) concentrates at a pole of the nucleus that is always distal to the centrosome (γ-tubulin). (B and C) Both A-type lamins (B) and NPCs (Nup153; C), copolarize with Nesp4. Nesp4 polarization is abolished by overexpression of KLC1 (A). Expression of Nesp4 causes separation of the nucleus from the centrosome. (D) In the scatter plot, the position of individual centrosomes (red dots) in multiple cells is displayed relative to the nucleus (in blue). (E) Centrosomes in nontransfected control cells or cells expressing GFP–KASH4 lie within 2 μm of the nucleus. Those in cells expressing GFP–Nesp4 (GFP–N4) are on average ≈13 μm from the nucleus (n = > 47; *, P = 0.0001). (F) This effect of Nesp4 can be abrogated by overexpression of KLC1. (G) Nesp4 has similar effects on the Golgi apparatus, causing it to depart from its normal perinuclear location (yellow arrows). In merged images, DNA is revealed by staining with Hoechst dye. In the right image in G, GFP–Nesp4 and the Golgi apparatus (in red) are overlaid on the same field visualized by using phase-contrast optics. (Bars: 10 μm.)