Supplemental Data includes Supplemental Experimental Procedures, 4 Figures and 5 Movie Legends.

Transcription

1 SUPPLEMENTAL DATA Supplemental Data includes Supplemental Experimental Procedures, 4 Figures and 5 Movie Legends. Supplemental Experimental Procedures Dominant negative nesprin KASH (DN KASH) plasmid construction- To generate DN KASH fluorescently labeled with an amino-terminal mcherry domain, mcherry was amplified from pcdh-ef1- MCS1-puro-mCherry 2 (kindly provided by Dr. P. Patwari (1)) by PCR using the primers 5 - AATGGATCCTAACCATGGTGAGCAAGGGC-3 and 5 - CTTGTACAGCTCGTCCATGCCGCCGGT-3 and then subsequently subcloned into the BamHI and EcoRV (blunt end) sites of the pcdna4/his-max (Invitrogen) vector to create mcherry- pcdna4/his- Max. The KASH domain from GFP-mouse nesprin-1α (2) was amplified by PCR using primers 5 - ATAGCGGCCGCCGCGGCTTCCTGTTCAGAGTC-3 and 5 - TATTCTAGATCAGAGTGGAGGAGGGCCATT-3. The PCR product was digested with NotI and XbaI and inserted downstream of the mcherry sequence into the mcherry- pcdna4/his-max plasmid. mcherry-nesprin-1αkash was subcloned from pcdna4/his-max mcherry-nesprin-1αkash, into the BamHI and NotI (blunt end) sites of pcdh-ef1-mcs1-puro (System Biosciences) to create pcdh-ef1- MCS1-puro-mCherry-Nesprin-1αKASH. As a mock control (mcherry alone), pcdh-ef1-mcs1-puromcherry was used. All enzymes were obtained from New England Biolabs, Inc. The resulting plasmids are referred to as DN KASH (pcdh-ef1-mcs1-puro-mcherry-nesprin-1αkash) and mcherry control (pcdh-ef1-mcs1-puro-mcherry) in this manuscript. Dominant negative SUN1 luminal domain (DN SUN1L) plasmid construction- The 5 end of SS-HA- Sun1L-KDEL was amplified from pcdna3.1ss HA-Sun1L KDEL (3) by PCR using the pair of primers 5 - ATGCGGCCGCTTCGAACTATAGGGAGACCCAAGCTGG-3 and 3'- ATGAATTCATCGATAGTCGAGGCTGATCAGCGGTT-5. The SS-HA-Sun1L-KDEL sequence was then excised using NheI and EcoRI and the product was ligated into the NheI and EcoRI sites of the pcdh-cmv-mcs-ef1-copgfp-t2a-puro (System Biosciences) cut vector. The resulting plasmid was pcdh-cmv-mcs-ef1-copgfp-t2a-puross-ha-sun1l-kdel and is referred to as DN SUN1L in the manuscript. All enzymes were obtained from New England Biolabs, Inc. Primer sequences for strain-induced expression of mechanosensitive gene experiments- Gene expression of strain-induced cells was quantified by real-time PCR using probes for mechanosensitive genes Egr-1, Iex-1, Pai-I, tenascin C, talin and vinculin, using the following primers: mouse Egr CCTATGAGCACCTGACCACA-3 and 5 -TCGTTTGGCTGGGATAACTC-3 ; mouse Iex-1: 5 - GCCGAAGGGTGCTCTACC-3 and 5 -AAATCTGGCAGAAGATGATGG-3 ; mouse Pai-I 5 - GACTCTGGATGAGTGGAAAGC-3 and 5 -GGCGGCCTAAGTCTCCAAAAT-3 ; mouse Tenascin C 5 -ACGGCTACCACAGAAGCTG-3 and 5 -ATGGCTGTTGTTGCTATGGCA-3 ; mouse Talin 5 - CCTGCCGCATGATTCGTGA-3 and 5 - TCGGAGCATGTAGTAGTCCAAA-3 ; and mouse Vinculin 5 - GCACATCTGACCTACTGCTTAC-3 and 5 - TCTTAGTCATTCCTGGCCCAA-3. Expression was normalized to an endogenous control, TATA binding protein, using primers 5 - AGAACAATCCAGACTAGCAGCA-3 and 5 -GGGAACTTCACATCACAGCTC-3, and compared to unstrained controls and strained mcherry controls using the ΔΔC t method. 1

2 Supplemental Figures Figure S1. ER-targeted DN SUN1L localizes to the nuclear rim and ER. (A-B) Immunofluorescence images of fibroblasts transiently expressing DN SUN1L (containing a HA-tag; A) or GFP alone (B). Cells were stained for HA-tag (second panel) and DNA (Hoechst 33342, third panel). Fourth panel, merged image. The anti-ha-staining confirms localization of DN SUN1L to the nuclear envelope (perinuclear space) and endoplasmic reticulum. Scale bar, 10 µm. 2

3 Figure S2. Nesprin-2αext, mini-nesprin-2g, SS-GFP-KDEL and SUN1 control constructs do not displace endogenous nesprins. (A-D) Immunofluorescence images of fibroblasts transiently expressing EGFP-nesprin-2αext (A), GFP-mini-nesprin-2G (B), SS-GFP-KDEL (C) and GFP-SUN1 (D). Cells were stained for nesprin-2 (second panel) and DNA (Hoechst 33342, third panel). Fourth panel, merged image. The SS-GFP-KDEL construct is targeted to the perinuclear space and endoplasmic reticulum. The antinesprin-2 staining indicates that endogenous nesprin-2 remains at the nuclear envelope in cells expressing each of the control constructs. Scale bar, 10 µm. 3

4 Figure S3. LINC complex disruption does not alter mechanosensitive gene expression. (A) Mechanically-induced gene expression of Egr-1 and Iex-1 normalized to an endogenous control, TATA binding protein (TBP), and compared to unstrained controls and strained mcherry controls. Qualitative similar results were obtained for other mechanosensitive genes. Data are represented as mean ± s.e.m.; *, P <

5 Figure S4. Microtubule organization is normal in LINC disrupted fibroblasts. (A-B) Immunofluorescence analysis of fibroblasts expressing DN KASH domain fused to mcherry (A) or mcherry alone (B). First panel, mcherry signal. Cells were stained for β-tubulin (second panel) and DNA (third panel). Forth panel, merged image. Last panel, magnification of perinuclear area. Scale bar, 10 µm. 5

6 Movie Legends Movie 1. Microneedle manipulation assay in a mouse embryonic fibroblast. Phase contrast (left) and fluorescence (right) images of a cell labeled with Hoechst nuclear stain (blue) and MitroTracker Green mitochondrial stain (green). A microneedle was carefully inserted into the cytoskeleton 5 µm away from the nuclear periphery and moved 20 µm towards the cell periphery at 1 µm/second while collecting phase contrast and fluorescence images every 10 seconds, using a 60 objective (0.70 N.A.) on a inverted epifluorescence Olympus IX-70 microscope, with a digital CCDcamera (CoolSNAP EZ; Roper Scientific). Movie of the same cell shown in Fig. 2A-E. Movie 2. Induced nuclear and cytoskeletal deformation during microneedle manipulation in a mcherry expressing fibroblast. Phase contrast (left) and fluorescence (right) images of a cell expressing mcherry alone and labeled with nuclear stain (blue) and MitroTracker Green (green). A microneedle was carefully inserted into the cytoskeleton 5 µm away from the nuclear periphery and moved 10 µm towards the cell periphery at 1 µm/second while collecting phase contrast and fluorescence images every 10 seconds, using a 60 objective (0.70 N.A.) on a inverted epifluorescence Olympus IX-70 microscope, with a digital CCD-camera (CoolSNAP EZ; Roper Scientific). Movie 3. Induced nuclear and cytoskeletal deformation during microneedle manipulation in a dominant negative KASH expressing fibroblast. Phase contrast (left) and fluorescence (right) images of a cell expressing DN KASH and labeled with nuclear stain (blue) and MitroTracker Green (green). A microneedle was carefully inserted into the cytoskeleton 5 µm away from the nuclear periphery and moved 10 µm towards the cell periphery at 1 µm/second while collecting phase contrast and fluorescence images every 10 seconds, using a 60 objective (0.70 N.A.) on a inverted epifluorescence Olympus IX-70 microscope, with a digital CCD-camera (CoolSNAP EZ; Roper Scientific). Movie 4. Nuclear deformation in response to substrate strain application. Phase contrast images of mcherry (top) and DN KASH (bottom) expressing fibroblasts plated on fibronectin-coated silicone membranes and subjected to 20% uniaxial substrate strain. Membranes were placed on a custom-made strain device mounted on an inverted epifluorescence Olympus IX-70 microscope, using a 60 objective (0.70 N.A.) and a CoolSNAP EZ camera (Roper Scientific). One image was acquired before strain and a second image was acquired during strain. Movie of the same cells shown in Fig. 4C, D. Movie 5. Nuclear deformation in response to substrate strain application. Fluorescence images of the nuclei of mcherry (top) and DN KASH (bottom) expressing fibroblasts plated on fibronectin-coated silicone membranes and labeled with nuclear stain. The cells were subjected to 20% uniaxial substrate strain. Membranes were placed on a custom-made strain device mounted on an inverted epifluorescence Olympus IX-70 microscope, using a 60 objective (0.70 N.A.) and a CoolSNAP EZ camera (Roper Scientific). One image was acquired before strain and a second image was acquired during strain. Movies correspond to the same cells as shown in Movie 4. 6

7 References 1. Patwari, P., Chutkow, W. A., Cummings, K., Verstraeten, V. L., Lammerding, J., Schreiter, E. R., and Lee, R. T. (2009) J Biol Chem 284, Zhang, Q., Skepper, J. N., Yang, F., Davies, J. D., Hegyi, L., Roberts, R. G., Weissberg, P. L., Ellis, J. A., and Shanahan, C. M. (2001) J Cell Sci 114, Crisp, M., Liu, Q., Roux, K., Rattner, J. B., Shanahan, C., Burke, B., Stahl, P. D., and Hodzic, D. (2006) J Cell Biol 172,