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Molecular mechanisms of centrosome and cytoskeleton anchorage at the nuclear envelope

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Abstract

Cell polarization is a fundamental process underpinning organismal development, and tissue homeostasis, which requires an orchestrated interplay of nuclear, cytoskeletal, and centrosomal structures. The underlying molecular mechanisms, however, still remain elusive. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies, spanning the entire nuclear envelope (NE), function in cell polarization by anchoring cytoskeletal structures to the nuclear lamina. Nesprin-2 forms complexes with the kinesin-1 motor protein apparatus by associating with and recruiting kinesin light chain1 (KLC1) to the outer nuclear membrane. Similar to nesprin-2, KLC1 requires lamin A/C for proper NE localization. The depletion of nesprin-2 or KLC1, or the uncoupling of nesprin-2/SUN-domain protein associations impairs cell polarization during wounding and dislodges the centrosome from the NE. In addition nesprin-2 loss has profound effects on KLC1 levels, the cytoskeleton, and Golgi apparatus organization. Collectively these data show that NE-associated proteins are pivotal determinants of cell architecture and polarization.

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Fig. 1a, b
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Fig. 4a–d
Fig. 5a–c
Fig. 6a–e
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Fig. 9a–g
Fig. 10a, b

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Abbreviations

AD:

Gal4-activating domain

BD:

Gal4-DNA-binding domain

β-gal:

β-Galactosidase

CT:

C-terminus

DN:

Dominant negative

ER:

Endoplasmic reticulum

G:

Giant

GST:

Glutathione-S-transferase

GFP:

Green fluorescent protein

IF:

Intermediate filament

KHC:

Kinesin heavy chain

KLC:

Kinesin light chain

MT:

Microtubule

NE:

Nuclear envelope

N2-SR:

Nesprin-2 SR

NT:

N-terminus

PDI:

Protein disulfide isomerase

Pc:

Pericentrin

shRNA:

Small hairpin RNA

SP:

Signal peptide

SR:

Spectrin repeat

TPR:

Tetratricopeptide repeat

WB:

Western blot

WT:

Wild type

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Acknowledgments

We thank Drs. S.T. Brady, R.D. Vale, W.T. Dauer, C.J. Hutchinson, and U. Euteneuer for providing reagents. We thank Dr. Martin Goldberg for critically reading the manuscript and Drs. R. Foisner, P.J. Hussey, R. Quinlan, and A. Smertenko for valuable discussions, as well as M. Munck and R. Blau-Wasser for technical advice. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KA 2778/1-1), the St. Moritz-Foundation, and the Wellcome Trust.

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Authors and Affiliations

  1. School of Biological and Biomedical Sciences, University of Durham, Durham, DH1 3LE, UK

    Maria Schneider, Wenshu Lu & Iakowos Karakesisoglou

  2. Center for Biochemistry, Center for Molecular Medicine Cologne (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Medical Faculty, University of Cologne, 50931, Cologne, Germany

    Maria Schneider, Sascha Neumann & Angelika A. Noegel

  3. Max F. Perutz Laboratories, Medical University of Vienna, 1030, Vienna, Austria

    Andreas Brachner & Josef Gotzmann

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  1. Maria Schneider
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Correspondence to Iakowos Karakesisoglou.

Electronic supplementary material

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18_2010_535_MOESM1_ESM.doc

Supplemental Table 1: Vectors, inserts, amino acids, primers and comments regarding plasmid construction of molecular reagents used in the present manuscript. (DOC 64 kb)

18_2010_535_MOESM2_ESM.jpeg

Supplementary Fig. S1 KASH-domain protein associations with the inner nuclear membrane determine cellular polarization. Monolayers of WT (A, C) and two independent, stably transfected DN-SUNL HaCaT cell clones (A’-A’’, C’-C’’) were wounded and subjected after 6 hours to indirect immunofluorescence using antibodies against pericentrin (Pc, centrosome, A-A’’) and Gm130 (Golgi, C-C’’). Nuclei were visualized with DAPI. The dashed lines indicate the migration front. Note that centrosomes and Golgi structures are not positioned towards the leading edge in DN-SUNL expressing clones (arrows). (B and D) Statistical evaluation of the representative experiments shown in A-A’’ and C-C’’ indicates a defective organelle polarization in DN-SUNL expressing cells. Centrosomes and Golgi positioned within a 120° sector facing the wound were assessed as polarized. Results are the mean ± SD Scale bars 10 µm. (JPG 325 kb)

18_2010_535_MOESM3_ESM.jpeg

Supplementary Fig. S2 KLC1 and MTs colocalize at the NE in various cell lines. Indirect immunofluorescence was performed on primary mouse fibroblasts (A-A’’) and COS7 cells (B-B’’) using KLC1 (pAb H-75; panels A, B) and tubulin (mAb WA3; panels A’, B’) specific antibodies. Scale bars: 10 µm. (JPG 421 kb)

18_2010_535_MOESM4_ESM.jpeg

Supplementary Fig. S3 Nesprin-2 silencing affects KLC expression levels. Equal amounts of untransfected HaCaT cell lysates as well as protein homogenates from control and nesprin-2 C-terminal RNAi transfected HaCaT cells were separated through gradient SDS-PAGE and subjected to Western blot analysis. (JPG 85 kb)

18_2010_535_MOESM5_ESM.jpeg

Supplementary Fig. S4 Nesprin-2 loss affects F-actin organization. Indirect immunofluorescence analysis using nesprin-2 (pAb Nes2CT) antibodies and F-actin counterstaining (FITC-Phalloidin: Phall) were performed on transiently transfected control (A) and nesprin-2 knock-down (B) HaCaT cells. DAPI was used to visualise the nuclei. Insets (A’, B’) are higher magnifications of denoted areas in panels A and B. While in nesprin-2 positive control cells (A’, arrowheads) Phalloidin staining is preferentially accumulated at cell-cell junctions, or the cell cortex (A’, arrows), in nesprin-2 silenced cells (B’, arrowheads) the Phalloidin staining reveals pronounced arrays of stress fibers (B’, arrows). (JPG 304 kb)

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Schneider, M., Lu, W., Neumann, S. et al. Molecular mechanisms of centrosome and cytoskeleton anchorage at the nuclear envelope. Cell. Mol. Life Sci. 68, 1593–1610 (2011). https://doi.org/10.1007/s00018-010-0535-z

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