Supplementry informtion Verticl uniformity of cells nd nuclei in epithelil monolyers Srujn Neelm b, Peter Hyes, Qio Zhng, Richrd B. Dickinson nd Tnmy P. Lele, Figure S1. Histogrm plots compre the frequency distribution of spect rtios in x-y view of cells in monolyers nd isolted cells. Vrince in x-y spect rtio is not different. Figure S2. x-y nd corresponding x-z views of cells of monolyers in control, cytochlsin-d treted, E- cdherin inhibited, ML-7 treted nd blebbisttin treted conditions. Tretment with cytochlsin-d, blebbisttin nd E-cdherin ntibody disrupted the cell-cell linkges nd cused rounded nucler pexes. Sttisticl dt is vilble in Tbles 1 nd 2 for these conditions. Figure S3. Flt nucleus in GFP control cells. (A) x-y nd x-z view of cells in monolyers trnsfected with GFP. Nucleus is GFP expressing cell, b is non-trnsfected cell djcent to GFP expressing cell nd, c is cell frther wy from the GFP expressing cell. The corresponding x-z views of the nuclei show no effect of GFP expression. (B) The plot compres the nucler heights of cells expressing GFP nd GFP-KASH4. The height of the nucleus in GFP-KASH4 cells is significntly higher. P <.5, n =2. Vlues re the men ± SEM. Figure S4. (A) x-y view of isolted MCF 1A cells seeded nd fixed t 3 mins, 6 mins, 6 hours nd 24 hours. The nuclei re stined with lmin A/C (green) nd show cler folds/ wrinkles t erlier time points nd t 24 hours the surfce of the nucleus is completely smooth. (B) The plot shows the rtio of nuclei with grooves to the totl number of nuclei t ech time point. As the seeding time incresed, less nuclei with grooves were found. N=3 replictes from n> 12 nuclei per time point. (C) The plot shows the nucler height t ech time point. The nuclei re flt fter 6 hours of seeding. N=3 replictes from n> 48 nuclei per time point. (B) nd (C) Vlues re the men ± SEM. P <.5, compring to 24 hours of seeding. Figure S5. (A) MCF 1A cells stbly expressing scrmbled shrna (control) nd were cultured in monolyers. Top pnel show the x-y view of nuclei immunostined for lmin A/C nd the bottom pnel shows the monolyers immunostined for E-cdherin nd nucleus. (B) x-z spect rtio nd nucler height of the cells expressing re significntly smller thn the scrmbled. P <.5, n > 3, # P<.5, n >3 for the indicted comprisons. Vlues re the men ± SEM. (C) Plot shows tht the cell spreding re of cells stbly expressing is significntly lrger. Figure S6. Vlidtion of ML-7 tretment. Similr to the results of (Shewn et l., 25), phosphomyosin stining ws observed in cell-cell contcts in control monolyers, while this stining ws lost upon ML-7 tretment. Plot shows quntittive comprisons of verged corrected totl cell fluorescence using the method of (McCloy et l., 214); imges were cptured under constnt microscope settings for control nd ML-7 tretment. corresponds to p<.1. Geometricl explntion for decresed height of the nucleus in monolyer compred to isolted cells. Here we show tht nucleus with flt picl surfce will hve smller height H thn one with rounded picl surfce, for the sme surfce re, A, nd volume V. We represent the nucleus s short cylinder of rdius nd height b, with sphericl cp of height h, s shown in the cross-section below. Since A nd V re ssumed fixed, the dimensions nd b depend on h.
The totl height of the nucleus is. When curved cp region increses in height, some of nucler volume is contined in the cp, nd the cylindricl volume nd height must reduce correspondingly. The totl height increses with h when (1) where The totl re nd volume re (2) (3) respectively. We look t the cse where the picl surfce is nerly flt nd sk whether the inequlity in Eq. 1 holds. Setting the derivtives of constnt A nd V with respect to zero, (4) yields nd b t h =, (5) (6) Plugging b into the inequlity Eq 1, provides the condition where the totl height increses with h: Hence, for nucleus with rdius greter thn 1.5X its height (typiclly the cse for cells), it is expected to hve lower height when the picl nucler surfce is flt in monolyer (h = ) rther thn rounded (in isolted cells). REFERENCES McCloy, R.A., Rogers, S., Cldon, C.E., Lorc, T., Cstro, A., nd Burgess, A. (214). Prtil inhibition of Cdk1 in G 2 phse overrides the SAC nd decouples mitotic events. Cell Cycle 13, 14-1412. Shewn, A.M., Mddugod, M., Kremer, A., Stehbens, S.J., Verm, S., Kovcs, E.M., nd Yp, A.S. (25). Myosin 2 is key Rho kinse trget necessry for the locl concentrtion of E-cdherin t cell-cell contcts. Mol Biol Cell 16, 4531-4542.
Frequency Figure S1 12 1 Monolyer Isolted 8 6 4 2.4.5.6.7.8.9 1. Aspect rtio in x-y view
Figure S2
Figure S3 Nucler height (µm) A Nucleus GFP E-cdherin b b c c B 7 6 5 4 3 2 1 GFP 1 GFP-KASH4 2 d
Nuclei with grooves/ Totl number of nuclei Nucler Height (µm) 6 hours fter seeding 24 hours fter seeding 3 mins fter seeding 6 mins fter seeding A Figure S4 Lmin A+C B 1.9.8.7.6.5.4.3.2.1 3 mins 6 mins 6 hours 24 hours C 1 9 8 7 6 5 4 3 2 1 3 mins 6 mins 6 hours 24 hours
Nucler height Cell Spreding re y Figure S5 A Scrmbled shrna x Lmin A+C b Nucleus E-cdherin Actin c d c b d z b c d b c d x B 6 5 4 3 2 1 Aspect rtio # Scrmbled MCF1A non shrna silenced Height MCF1A.3.25.2.15.1.5 C Aspect rtio 25 2 15 1 5 MCF1A Scrmbled non silenced shrna MCF1A
Figure S6 Intensity/µm² DMSO ML-7 12 1 8 6 4 2 DMSO ML7