Supplementary Information Cofilin Regulates Nuclear Architecture through a Myosin-II Dependent Mechanotransduction Module O Neil Wiggan, Bryce Schroder, Diego Krapf, James R. Bamurg and Jennifer G. DeLuca
t = 6 s z z1 z2 z3 z4 t = 7 s z z1 z2 z3 z4 t = 8 s z z1 z2 z3 z4 t = 11 s z z1 z2 z3 z4 Mem. 1µm H2B Relative H2B intensity 1µm asal.2.16.12.8.4 z interval = 2.6 µm 4 8 12 z4 z3 z2 z1 z c Relative projected area 4 8 12 XYview e f g Time (s) apical COF+ADF 1.2 1.8.6.4.2 Time (s) d nucleus Normal Compressive Force lateral les projected area height nuclear displacement COF+ADF apical extensions lateral les projected area variale height XY YZ 3D-projection 1µm 2µm 2µm 1µm 6 4 2 : nuclear height (µm) ns n=116 n=1 n=1 COF+ADF COF+ADF excluding including apical apical extensions extensions nuclear projected area (µm 2 ) 5 4 3 2 1 : p <.1 COF+ADF Supplementary Figure S1
sal z z1 z2 z3 apical z4 H2B z-projection H2B H2B Memrane z-projection H2B Memrane Supplementary Figure S2
Nuclear envelope DAPI Supplementary Figure S3 COF+ADF+Myo2A COF+ADF COF+ADF+Myo2B COF+ADF COF+ADF+Myo2A COF+ADF+Myo2A+Myo2B c Myo2A F-actin F-actin 1 µm Merge 1 µm
LMNA #1 LMNA #2 LMNA #3 Sun1 #1 Sun1 #2 Sun2 #1 Sun2 #2 Sun2 #3 Sun2 #4 Sun1 LMNA Sun2 LMNA Sun1 Sun2 Sun2 LMNA LMNA c d e Nesprin1 #1 Nesprin1 #2 Nesprin1 #3 Nesprin2 #1 Nesprin2 #2 Nesprin2 #3 Nesp1 LMNA Nesp1 Nesp2 LMNA Nesp2 Supplementary Figure S4
F-actin Sun2 Merge single z Z-projection F-actin Sun2 Merge RPE-1 apical single z Z-projection HeLa apical Supplementary Figure S5
Uncropped lots for Figure 1f 12% gel - COF COF+ADF 37 kd GAPDH 25 kd 2 kd Cofilin/ADF Uncropped lots for Figure 2h 4-15% gel 2 kd 15 kd COF+ADF pmlc 2 kd 15 kd 1 kd 75 kd MLC lamin B Supplementary Figure S6
Supplementary Figure Legends Supplementary Figure S1. Induction of apical nuclear extensions in conjunction with plasma memrane le expansion in cofilin/adf depleted cells. (a) Confocal time lapse z-series depicting formation an apical nuclear extension in tandem with the apical expansion of a plasma memrane (Mem.) le. Images are of region and cell depicted y ox and arrows respectively in Fig. 3. () Quantification of H2B fluorescence for oxed region, relative to time, at each z-plane shows temporal asal to apical rise of nuclear intensity. (c) Formation of the apical nuclear extension depicted in (a) was not associated with any sustantial changes to projected area of that nucleus, as might e expected from episodic gloal nuclear compression. (d) Illustration of cell nuclear morphologies in response to different treatments. (e) Confocal XY (top), YZ (middle) and 3D-surface projections (ottom) of fixed DAPI stained HeLa cells, treated as indicated. Arrows illustrate the two different measurements made for nuclear height, quantified in (f). (g). Quantification of nuclear projected areas from fixed HeLa cells. Values are mean ± SD, n > 1/treatment. P values from Welch s t-test. Supplementary Figure S2. Apical nuclear extensions are emedded in plasma memrane les. Confocal z-series of live HeLa cells where DNA was laelled y RFP-H2B and the plasma memrane y GFP-CAAX. Bars, 1 µm. Arrows illustrate encapsulation of nuclear extensions in apical les. Note also the correlation of size and morphologies etween apical nuclear extensions and the memrane les that encapsulate them. Supplementary Figure S3. Co-silencing of myosin-ii isoforms rescues anormal nuclear morphologies in Cof/ADF depleted cells. (a, and c) Confocal fluorescence images of fixed Hela cells at 72 h post treatments as indicated. The nuclear envelope is laeled y lamin A/C immunostaining for the first three rows
nd y Sun2 for the last row of (a). Scale ars 1 µm, unless otherwise noted. Oserve F-actin accumulation at the cortex and in the central region of enlarged filopodia-like protrusions (oxed region of c, magnified in lower panel) for cells co-depleted of cofilin, ADF and oth myosin isoforms expressed in these HeLa cells. Supplementary Figure S4. Silencing of nuclear envelope LINC complex proteins. Immunofluorescence characterization of independent s for silencing of lamins A/C (a, LMNA), Sun1 (), Sun2 (c), Nesprin1 (d) and Nesprin2 (e). Bottom panels show overlay with a non-targeted nuclear envelope protein as indicated. Cells were laeled at 72 h post treatment of HeLa cells. Bars, 1 µm. Supplementary Figure S5. Examination of F-actin structures associated with the apical nuclear surface. (a and ) Confocal immunofluorescence images illustrating F-actin organization aove the nuclear surface, laeled y Sun2, of HeLa (a) and RPE1 cells (). Shown are z-projections of all apical slices aove the nucleus versus a single slice just aove the nucleus. Bars, 1 µm. Supplementary Figure S6. Full Western lots. Full lots related to figure 1f (a) and to figure 2h (). Cropped regions are oxed.