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Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells

Yeh-Chuin Poh, Sergey P. Shevtsov, Farhan Chowdhury, Douglas C. Wu, Sungsoo Na, Miroslav Dundr & Ning Wang

Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Champaign, Illinois 61801, USA.

Introduction

Material & Methods

Results Results Results

Summary

Reference

Mechnotransduction has been implied as an important field relating physical environments with cellular physiology. It has been shown that mechanical force regulates a wide variety of cellular processes such as cell morphology, migration, differentiation and apoptosis, etc. . Recently, it was shown that force applied on the cell apical surface could display mitochondria within the cytoplasmic matrix of cells, and even the nucleolus within the nucleus.

• Can force directly alter nuclear function?

• Cajal body is nuclear protein complex consist of different proteins.

• Cajal body helps biogenesis of RNPs, maturation of telomerase and maintenance of telomere

HeLa cells were transfected with plasmids of fluorescent proteins of interest. Fluorescent levels of CFP- and YFP-proteins were recorded simultaneously before and after force application on cells. Force application was done by magnetic twist cytometry. CFP/YFP emission ratio was measured by a customized Matlab program. For substrate stiffness dependent experiments, cells were plated on glass and different stiffness polyacrylamide gels coated with type I collagen. For cellular traction experiments, fluorescent beads were embedded into gels, bead positions were recorded before and after cells were removed. The displacement field induced by each individual cell’s tractional forces was determined by comparing the fluorescent bead positions before and after removing cells.

CFP

YFP

FRET 433nm 476nm 433nm 527nm

Figure 1. FRET expreiment. Cells are co-transfected with YFP-Coilin and CFP-SMN. YFP and CFP are FRET pairs. Movements of SMN and Coilin were synchronized with the force appl icat ion through magnetic twist cytometry

CFP-SMNYFP-Coilin CFP-SMNYFP-Coilin

Figure 2. Dissociation of Cajal bodies was observed ~0.3s after force application.

-2 -1 0 1 2 3 4 5 12 15 18 21 240.900.951.001.051.101.151.201.251.30

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Dissociation of Cajal bodies in response to force

Dissociation is Magnitude dependent

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Figure 4. There is a threshold magnitude of force to trigger the dissociation of Cajal bodies in livinh cells between 14.0 Pa and 17.5 Pa.

Structural basis of force-induced dissociation

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Figure 5. By drug inhibition different force-transmitting components in cells, it is shown that F-actin, myosin II and Lamin A/C are necessary for force-induced dissociation of Cajal bodies.

Substrate stiffness dependent

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Figure 6. Force-induced dissociation of Cajal bodies was substrate stiffness dependent. Only on stiffer substrate, cells would exhibit dissociation of Cajal bodies in response to stress.

Figure 7. Actin bunles formation, cellular traction force and cell stiffness are proportional to substrate stiffness, implying prestress on cytoskeletons play a role in force propagation into nucleus

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1873

NATURE COMMUNICATIONS | 3:866 | DOI: 10.1038/ncomms1873 | www.nature.com/naturecommunications

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maximum stiffness (cell stiffness measured on a rigid dish) of ~1.51 kPa (Fig. 2e) and express ample actin bundles and high tractions on 2 and 8 kPa, but not on 0.6 kPa substrate (Fig. 2f,g). Together with the above data, this suggests that substrate rigidity regulates force-induced nuclear protein dissociation via controlling tension of the actin bundles in the cytoskeleton for long-distance force propagation.

Other protein pairs exhibit different force-induced changes. Next, we analysed how the local surface force affects interactions between the major interacting partners of coilin and SMN in CBs. Coilin and SMN are self-interacting proteins and their interaction is mediated by WRAP53 that is also able to self-interact27. Impor-tantly, both coilin and SMN interact with spliceosomal snRNPs28. In addition, coilin interacts with the nucleolar chaperone Nopp140 on its amino terminus29 and with the U4/U6 snRNP assembly fac-tor SART3 (ref. 30). In the absence of external stress, spliceosomal snRNP core SmE-SmG proteins had the lowest baseline CFP/YFP

emission ratios (~0.3), followed by coilin-coilin, coilin-SmE, coi-lin-Nopp140, WRAP53-WRAP53, coilin-WARP53, coilin-SART3, SMN-WRAP53, SMN-coilin, SMN-SMN, SMN-SmG and SMN-SART3 (~0.8) (Fig. 3a), suggesting that baseline distances between two proteins were closest for snRNP proteins of the Sm ring SmE-SmG and farthest for SMN-SART3 complexes. Interestingly, CFP-fibrillarin (a methylase that binds to SMN in the CB31) and YFP-coilin in the CB exhibited even higher baseline emission ratios than SMN-SmG, reaching almost 1.0 (Fig. 3a), consistent with previ-ously published data that fibrillarin and coilin do not directly inter-act with each other14 and that they might be far apart (~ > 10 nm) (a negative control). After a quick step-function force was applied, coilin-coilin self-interaction exhibited greatest FRET ratio increases from baseline values, followed by coilin-SmE, WRAP53-WRAP53, coilin-Nopp140, SMN-coilin, coilin-SART3, coilin-WARP53, SMN-SMN, SMN-SmG and SMN-WRAP53 (Fig. 3b; Supplemen-tary Figs S3 and S4). These results suggest that when these protein complexes were exposed to the same magnitudes of stress, their

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Figure 1 | A local surface force directly dissociates coilin from SMN in the CB in the nucleus. (a) Fluorescence images of a HeLa cell transfected with CFP-SMN and YFP-coilin (inset is the bright-field image of the cell; black dot shows the bead). Nucleus is outlined with dashed line. Scale bar, 10 m. (b) Displacements of the magnetic bead and of the CB proteins SMN and coilin as a function of cyclic forces (0.3 Hz). Displacements of the bead, coilin and SMN were all synchronized with the applied stress (peak magnitude = 24.5 Pa). (c) Displacement maps of coilin and SMN within the nucleus of the same cell. White arrows indicate direction of displacement, and the colour bar indicates the displacement magnitude. Pink arrow represents the direction of bead centre displacement (not drawn to scale). Scale bar, 10 m (d) FRET ratio map of force-induced dissociation of coilin and SMN. Inset shows an enlarged CB with FRET changes when stress is applied. (e) A representative time course plot of CFP-SMN and YFP-coilin anti-correlation in response to force. (f) FRET ratio change between coilin and SMN by mechanical is stress-magnitude-dependent and rapid ( < 0.35 s). Each load was applied to a cell only once. N = 5 CBs for stress of 17.5 Pa; 40 for 14 Pa; 95 for 1.8 Pa. (g) Stress-induced structural change to CB protein pairs is ‘plastic’. A step load of 24.5 Pa was applied. n = 13. All data points in (e), (f) and (g) were normalized to time zero when a step load was applied. (h) The dynamics of coilin and SMN were quantified before, during and after application of an oscillatory stress (24.5 Pa peak stress at 0.3 Hz). (P < 0.05 when t > 5.12 s). n = 106 CBs. Mean s.e.m.; data are pooled from > 4 independent experiments for each sub-figure. A two-tailed Student’s t-test was used to generate P values.

Figure 3. Cajal bodies did not reassembly after force application was turned off. Stress-induced structural change to CB protein pairs is ‘plastic’

• Mechanical Force is possible to directly altered nuclear functions

• Mechanotransduction in living cell nucleus depends on force magnitude, intact F-actin, cytoskeletal tension (prestress), Lamin A/C, or substrate rigidity

• Justified the tensegrity model of cells

• It is possible to manipulate cell growth, cell development by force-induced change in gene expression profile

Other protein pairs exhibit different changes

Figure 8. Other protein pairs in Cajal body has different sensitivity to force, implying that they have different interactions within the Cajal body.

Poh, Y.-., Na, S., Chowdhury, F., Ouyang, M., Wang, Y., and Wang, N. (2009) Rapid activation of Rac GTPase in living cells by force is independent of Src. PLoS ONE. 4(11)

Wang, N., Tytell, J.D., and Ingber, D.E. (2009) Mechanotransduction at a distance: Mechanically coupling the extracellular matrix with the nucleus. Nature Reviews Molecular Cell Biology. 10, 75-82

Wang, N., Butler, J., and Ingber, D. (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science. 260, 1124-1127