Prestressed Nuclear Organization in Living Cells

Prestressed Nuclear Organization in Living Cells

CHAPTER 10 Prestressed Nuclear Organization in Living Cells Aprotim Mazumder*, T. Roopa*, Abhishek Kumar*,†, K. Venkatesan Iyer*,†, Nisha M. Ramdas*...

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CHAPTER 10

Prestressed Nuclear Organization in Living Cells Aprotim Mazumder*, T. Roopa*, Abhishek Kumar*,†, K. Venkatesan Iyer*,†, Nisha M. Ramdas*, and G.V. Shivashankar*,† * National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore 560065, India † Department of Biological Sciences and Research Center for Excellence in MechanoBiology, National University of Singapore, Singapore 117543

Abstract I. Introduction II. Mechanics of Isolated Nucleus A. Isolation of Nucleus from Living Cells and Study of Higher-Order Chromatin Organization B. Measurement of Entropic Force during Chromatin Decompaction C. Optical Trap: A Method to Probe Softening of Nucleus During Chromatin Decompaction D. Photo-Bleaching and Nuclear Swelling Techniques to Probe Chromatin Organization III. Nuclear Prestress in Cellular Context A. Chemical Inhibitors to Modulate Cytoskeletal Tension B. Laser Perturbation of Differentially Compacted Chromatin IV. Conclusions

Acknowledgments

References

Abstract The nucleus is maintained in a prestressed state within eukaryotic cells, stabilized mechanically by chromatin structure and other nuclear components on its inside, and cytoskeletal components on its outside. Nuclear architecture is emerging to be critical METHODS IN CELL BIOLOGY, VOL. 98 Copyright  2010 Elsevier Inc. All rights reserved.

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978-0-12-381009-0 DOI: 10.1016/S0091-679X(10)98010-2

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to the governance of chromatin assembly, regulation of genome function and cellular homeostasis. Elucidating the prestressed organization of the nucleus is thus important to understand how the nuclear architecture impinges on its function. In this chapter, various chemical and mechanical methods have been described to probe the pre­ stressed organization of the nucleus.

I. Introduction Chromatin, the primary constituent of nucleus, is a complex of nucleic acids and proteins packaging a meter-long DNA into a micron-scale nucleus. The interaction of histone and nonhistone proteins with DNA facilitates the condensation of DNA far beyond its radius of gyration of 220 µm defined by the entropic regime. In addition, DNA is condensed into euchromatin and heterochromatin regions enabling regulated access to genetic information. Maintenance of this state of the nucleus may require a prestressed nuclear organization with contribution from factors within the nucleus and from the cytoskeleton, but the principles of this organization are still unclear. Impor­ tantly, nuclear structure is emerging to be critical to the governance of chromatin assembly, regulation of genome function and cellular homeostasis. Elucidating the prestressed organization of the nucleus is thus essential to understand how the nuclear architecture impinges on its function. In eukaryotic cells, nucleus is the stiffest organelle (Dahl et al., 2008). In this context, tensegrity models have been proposed for cellular shape stability that depends on tension and compression in the cytoskeleton and physical integration of the nucleus with cytoplasm (Ingber, 1993; 2003a, b). A quantitative understanding of prestressed nucleus may also elucidate mechanisms underlying mechanotransduction of signals from extra-cellular matrix (ECM) that impinge on genome function. In this chapter, we outline some methods to probe the components involved in maintenance of nuclear shape and size.

II. Mechanics of Isolated Nucleus The prestressed organization of nucleus arises from both nuclear and cytoskeletal components. Hence it is pertinent to decouple these components and explore indivi­ dually their roles on nuclear prestress. Isolation of a nucleus out of its cytoplasmic milieu provides a mechanism to assess stresses on the nucleus directly. In this section, we present experimental methods for isolating nuclei from cells, and study the forces that work to maintain the prestressed state of the nucleus by enzymatic disruption of higher-order chromatin assembly. A. Isolation of Nucleus from Living Cells and Study of Higher-Order Chromatin Organization The nucleus of eukaryotic cell is 5- to 10-fold more rigid than the cytoplasm with variations depending on the cell type. Taking advantage of this property of the nucleus,

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mechanical and chemical methods have been employed to isolate individual nuclei from living cells (Caille et al., 2002; Dahl et al., 2005). In general, nuclear isolation techniques require the selective mechanical or chemical perturbation of the cell mem­ brane and loss of cytoplasmic contents. This method retains the integrity of nuclear membrane and chromatin organization inside the nucleus. Osmotic swelling and mechanical shearing techniques have been employed in our laboratory to isolate nucleus exploiting the considerable difference in the mechanical properties of cell and nucleus. A simple method for the isolation of nucleus involves the following steps: 1. Cultured cells are harvested and washed using 1 PBS (pH 7.4). 2. Cells are re-suspended in TM2 buffer (10 mM Tris–HCl, pH 7.4, 2 mM MgCl2, and 0.5 mM PMSF (added fresh before use)). 3. Cells are incubated for 5 min on ice and 5 min at room temperature. 4. To disintegrate the cytoplasm of cells, Triton X-100 is added and mixed well, before the cells are again incubated on ice for 5 min. 5. Cells are sheared by passing them through a syringe needle (22 gauge) 10 times and centrifuged at 12,000 rpm for 5 min. 6. Triton X-100 treatment is repeated if the nuclei were found to have cellular debris adhered to them. 7. Clean isolated nuclei are used for experiments. The integrity of nuclear membrane and chromatin assembly can be assessed by staining the nucleus with DNA dyes like Hoechst 33342 and DAPI. These nuclei can then be further used for studying either their mechanical properties or prestress involved in the isolated nucleus. The nucleus devoid of any cytoplasmic milieu is still a stable structure. In the entropic regime a meter-scale genome acquires a configuration which has a radius of gyration of 220 µm. But higher-order chromatin organization involving DNA–protein interactions in isolated nucleus forces it to stabilize and restrain from going to this entropic config­ uration (Krajewski and Ausio, 1996; Leuba et al., 1998). Perturbation of chromatin structure provides a mechanism to investigate forces involved within the prestressed nucleus. It has been known that histone tail–tail interactions are important for compac­ tion of the chromatin (Bertin et al., 2004; Placek and Gloss, 2002; Schalch et al., 2005). Probing the effect of perturbing proteins involved in higher-order chromatin assembly on integrity of the nucleus is crucial to understand mechanical stresses within the nucleus. Here, we describe proteolytic-cleavage-based methods (using the enzymes clostripain and trypsin) to probe the importance of higher-order chromatin structure in maintenance of nuclear prestress. Trypsin and clostripain are a class of proteases that cause cleavage of arginine residues starting at carboxyl terminal of the proteins (Dumuis-Kervabon et al., 1986); while trypsin also acts on lysine residues. We have used trypsin and clostripain to perturb higher-order chromatin compaction by digestion of histone tails and to observe responses of nucleus to such structural perturba­ tion (Mazumder et al., 2008). Isolated nuclei are adhered on coverslips using poly-D­ lysine (PDL) following which clostripain is added in concentrations ranging from 0.5 to 4 mU/ml. The enzyme needs to be activated by 2.5 mM DTT and calcium acetate at room

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(A)

0′′

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Clostripain

DNase

Nuclear area (μm2)

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0 0

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Fig. 1 Chemical perturbation of isolated nucleus to study higher-order chromatin compaction. (A) The panel of images shows the entropic swelling of an isolated H2B-EGFP nucleus digested with 0.5 mU/μl clostripain and 500 mU/μl DNase. No swelling is observed in nucleus for cleavage of nucleic acids. At high DNase concentrations (500 mU/μl), fragmented bits of chromatin are emitted from the nuclei though there is no expansion in size. The corresponding time-points are indicated. Scale bar = 10 μm. (B) Expansion kinetics for clostripain and DNase (filled circle is for clostripain and open square is for DNase).

temperature for 30 min. The action of the activated enzyme leads to an increase in isolated nuclear size which can be quantified by computing the cross-sectional area obtained from confocal images of nucleus. However, in contrast when chromatin is perturbed by nucleic acid targeted enzymes such as DNases, nucleus does not expand, instead fragmented chromatin is observed (Fig. 1). Quantification shows that the nuclear expansion increases significantly when protein interactions maintaining higher-order chromatin structure are violated resulting in an entropic swelling of chromatin. This assay can further be utilized to probe the role of nuclear scaffold proteins in the maintenance of chromatin assembly. Highly compacted heterochromatin regions of chromatin are organized along the nuclear periphery and are known to be anchored at distinct foci to the lamin matrix. The lamin scaffold via a host of proteins is involved in organizing the genome within the nucleus (Goldman et al., 2004; Gruenbaum et al., 2005; Mattout et al., 2006; Panorchan et al., 2004; Tzur et al., 2006). Perturbation of proteins that stabilize chromatin may also affect tethering of chromatin to the lamin scaffold. Cells are transiently transfected with EGFP-

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LaminB1 fusion plasmid and studied following the addition of trypsin. Dynamics of expansion of lamin scaffold studied by fluorescence time-lapse imaging shows an increase in the perimeter of nucleus leading to complete nuclear disintegration marked by a loss of lamin fluorescence intensity when the nucleus ruptures. Thus, these proteolytic-cleavage-based methods allow investigation of the role of higher-order chro­ matin structure in the maintenance of nuclear prestress.

B. Measurement of Entropic Force during Chromatin Decompaction Various experiments have been performed to understand the mechanical properties of nucleus (Guilak et al., 2000; Ofek et al., 2009; Tseng et al., 2004). Micropipette aspiration experiments have been employed to reveal elastic modulus and viscosity of the nucleus (Pajerowski et al., 2007; Rowat et al., 2006; Vaziri and Mofrad, 2007). Here, we describe a method to measure entropic force generated by chromatin when the nuclear prestress is removed by chemical perturbation of higher-order chromatin organization. Atomic Force Microscope (AFM) has been effectively used to study forces stabilizing nanoscale structures and force fluctuations (Bao and Suresh, 2003; Milani et al., 2009). AFM cantilever sensitivity to small deflections enables them to be used for measuring minute displacements generated by the mechanics of nuclear expansion. In order to measure the entropic expansion of nucleus, we used an AFM cantilever (Veeco Instru­ ments Inc., NY) of stiffness kcant = 0.02 N/m which is mounted on -SNOM Microscope (WiTEC, Germany). The cantilever is employed to measure deflections on a nucleus adhered on a coverslip dish coated with PDL. A saturating concentration of trypsin can be used to hasten the process of nuclear swelling. Deflection of the cantilever is constantly monitored using a quadrant photodiode (QPD). A change in the position of the cantilever is manifested as a differential change in the voltages of four quadrants of the QPD, where voltages are precalibrated against defined displacements of the cantilever. When the enzyme starts to act, the cantilever shows an upward movement, indicat­ ing swelling of the nucleus. The pressure on the cantilever can be tracked as a function of time (Fig. 2A). Quantification reveals a large increase in outward pressure 3 kPa or force of 300 nN before the nuclear membrane and lamin scaffold disintegrates. This is the force that the entropic chromatin exerts on the nuclear membrane upon digestion of protein interactions. After the rupture of nuclear membrane, the cantilever recoils back to its initial position indicating a decrease in pressure owing to the absence of nuclear membrane to confine the expanding chromatin (Fig. 2A). This assay demonstrates the use of AFM to probe chromatin structure.

C. Optical Trap: A Method to Probe Softening of Nucleus During Chromatin Decompaction Perturbation to the structural components of nucleus, like deletion of Lamin A/C results in a decrease in the rigidity of nucleus as observed in micropipette aspiration experiments (Pajerowski et al., 2007). Chromatin and lamin scaffold are known to be key determinants of the structural integrity of the nucleus. In this section, we describe a method using an

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Pressure (kPa)

(A) 3

2

Quadrant photodetector

Initial direction of cantilever deflection on trypain digestion of the nucleus

920 nm laser

k = 0.02 N/m

1

0 102

105

20

Change in δx (nm)

(B)

10

0 0

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Time (min)

Fig. 2 Quantification of mechanical property of an isolated nuclei. (A) Pressure exerted by nucleus on the cantilever is estimated from deflection and stiffness of the cantilever. Upon trypsin digestion the pressure due to prestress is released and a large increase in the pressure on cantilever is observed followed by a dip due to rupture of the nuclear envelope. Inset shows schematic of the experimental set up. (B) Fluctuation of a bead adhered on the nuclear membrane increases with time upon trypsin digestion (open circles) suggesting softening of the nucleus. No increase in fluctuation is observed when the nucleus is fixed with paraformaldehyde (open squares).

optical trap to measure the change in mechanical properties of isolated nucleus upon perturbation of higher-order chromatin organization by enzymatic digestion. Optical traps have been used to trap dielectric beads and thereby study various biological processes (Sheetz, 1998). Fluctuations of beads trapped in an optical trap can be interpreted to elucidate the structural properties of underlying substrate (Hodges et al., 2009; Soni et al., 2003). Our laboratory has quantified the fluctuations of bead in a trap to study the stiffness of chromatin fiber and additionally the nucleus. The optical trap we used is built on an inverted fluorescence microscope (Model: IX70, Olympus, Tokyo, Japan) using a current controlled diode laser (wavelength 830 nm, GaAlAs;

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SDL Inc, San Jose, CA) and controller ILX Lightwave, Bozeman, MT). In order to track position of the bead in the trap, a 5 mW, 635 nm diode laser (Coherent Inc., Santa Clara, CA) is employed to image the back-scattered light onto a photodetector. The output voltage of the photodetector is amplified using two low noise current pream­ plifier (Model SR570; Stanford Research Systems, Sunnyvale, CA). The difference in the two voltages provides the position of the bead from the center of the optical trap. Data acquisition and analysis are done using DAQ (PCI-MIO-16XE-10; National Instruments, Austin, TX) and LabVIEW (National Instruments, TX). In vitro experiments have been employed to study the mechanical properties of single chromatin fibers (Claudet and Bednar, 2006; Cui and Bustamante, 2000; Dame, 2008). To understand the mechanistic effect of the loss of compaction by enzymatic digestion of histone tails on stiffness of chromatin and to estimate local chromatin fluidity, we have used the optical trap assay in combination with micromanipulation methods (Roopa and Shivashankar, 2006). The quantification of chromatin stiffness rests on the assumption of treating the chromatin-trap as a system of springs in parallel. For the system used, the effective position standard deviation (PSD) of the bead can be calculated by the following equation: 1 2 trap

¼

1 2trap

þ

1 2Chr

where, Chr is PSD of the bead due to chromatin and trap is the PSD of the bead due to the trap. A PDL-coated micropipette kept at a fixed tension is used to pull a chromatin fiber from purified chromatin adhered onto a coverslip by PDL. The fluctuations of a bead which is adhered onto the chromatin fiber are monitored as a function of time, and studied in comparison to fluctuations that result after chromatin structure is modulated via the addition of trypsin. A histogram of fluctuations after enzymatic digestion of histone tails plotted as a function of time can be used to probe the rigidity of the underlying substrate quantified by the width of the histogram or the dispersion of fluctuations. Softer substrates show a larger dispersion than rigid substrates. This method can further be used to estimate the fluidity of the cell nucleus upon perturbation of the chromatin structure (Mazumder et al., 2008). A 2 µm bead is adhered on the membrane of isolated nucleus and the rigidity of the nucleus is estimated from fluctuations of the bead, i.e., standard deviation of its fluctuations (). The structural response of the nucleus to enzymatic perturbation is obtained by following  of the bead on treatment with the enzyme. The bead shows   10 nm before the nucleus is digested. Upon trypsin digestion of the nucleus,  increased to 30 nm which is comparable to the   50 nm of an unbound bead in the optical trap suggesting that trypsin digestion indeed leads to softening of nuclear material (Fig. 2B).

D. Photo-Bleaching and Nuclear Swelling Techniques to Probe Chromatin Organization Apart from the inward force on chromatin due to histone tail–tail interactions, as described in earlier section, there exists a repulsive electrostatic force between DNA segments owing to the presence of a net negative charge (Tkachenko, 2006). DNA inside

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the nucleus is in a bath of positively charged ions that screen the repulsions between the negatively charged DNA segments (Fenley et al., 1994; Taubes et al., 2005). Removal of these screening charges by altering the buffer conditions leads to an expansion of the nucleus, owing to repulsion between DNA segments. Such an expansion of the nucleus is reversible upon restoration of the buffer conditions. This phenomenon of reversibility of the nucleus can be employed as a method to probe the structural integrity of chromatin organization. Isolated nuclei are adhered onto PDL-coated coverslip dishes, stained with a DNA binding dye Hoechst or DAPI and imaged in 1 Phosphate Buffered Saline (PBS) which represents the physiological condition for the isolated nucleus. Nuclear size is then cyclically modulated by replacement of PBS buffer with water and restoration of PBS. Nuclear area increases when PBS is replaced by water and recovers completely upon restoration of buffer conditions (Fig. 3). Merging the images obtained before and after restoration of PBS buffer shows pixel-wise colocalization suggesting structural integrity of chromatin anchored within the nucleus. Further, this technique can be used to probe the effect of screening charges on the chromosome organization by titration of the buffer concentration, providing a handle on understanding the dynamics involved in the structural maintenance of chromatin assembly.

III. Nuclear Prestress in Cellular Context Within the cellular context, the eukaryotic nucleus is maintained in a prestressed condition by balance of both nuclear and cytoplasmic forces (Mazumder and Shivashankar, 2007). Apart from the forces in an isolated nucleus which stabilizes the prestressed state, cytoplasmic forces play an important role in establishing prestress in adherent cells as well as governing various functions (Janmey, 1998; Stossel, 1993). The physical links between cytoplasm and focal adhesion (FA) on one side and cytoskeleton and the nucleus on the other side are beginning to be elucidated (Ingber, 1993; 2008; Ingber et al., 1994, 1995). FAs or focal contacts are the subcellular sites where the cell contacts the ECM. FAs are points of cross-talk between trans-membrane integrin receptors and cytoplasmic filaments. Additionally, at these loci a large number of receptors are present, thus making FA a key site for various biochemical and mechan­ otransduction pathways (Vogel and Sheetz, 2006; Wang et al., 1993). FAs are major mechano-sensors present at the plasma membrane of the eukaryotic, mainly adherent cells (Geiger et al., 2001; Gillespie and Walker, 2001; Hamill and Martinac, 2001). These are dynamic structures and change their size and morphology in response to physical forces (Liu et al., 2010). The heterodimeric (–) trans-membrane integrin receptors interact with various anchor proteins like talin, -actinin, and tensin which either directly make connections with cytoplasmic filament actin or are mediated through other adaptor proteins like vinculin (Calderwood et al., 2003; Garcia-Alvarez et al., 2003; Geiger and Bershadsky, 2001; Jamora and Fuchs, 2002; Liu et al., 2000). By interacting with the ECM and cytoplasmic filaments, these provide a rigid structural support to the cellular structure. In cytoplasmic milieu, microtubule applies compressive load on the nucleus while actomyosin complex applies tensile force on the nuclear

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Before

H2O

After

5 μm

5 μm

Fig. 3

Reversible swelling of isolated nuclei in water. Isolated H2B-EGFP HeLa nuclei in PBS buffer is washed four times in deionized water, to show large swelling. The swelling is reversed on restoring back PBS, to regain the same configuration as before. No loss of histones is apparent. Individual nuclei are imaged with photobleaching marks to discern local length scales of swelling. Every node comes back to the original configuration on restoration of physiological buffer conditions, as is apparent from the merge image (bottom).

membrane. These filaments make connections with the nuclear envelope mediated by various anchor proteins mainly SUN and KASH domain proteins (Crisp et al., 2006; Haque et al., 2006; Padmakumar et al., 2005; Tzur et al., 2006). It has now been shown that apart from diverse function in nuclear positioning and centrosome localization, SUN

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(Sad1 and UNC-84 homology domain) along with its partner KASH (Klarsicht, ANC-1 and SYNE1 homology) may play an important role as mechanical couplers (Haque et al., 2006; Tzur et al., 2006). These act like bridges connecting most of the cytoplasmic filaments to nucleus (Stewart et al., 2007). The nucleus which consists mainly of chromatin and other nuclear proteins is held together by nuclear envelope (Gruenbaum et al., 2005). Further, inner and outer nuclear membranes join at nuclear pore complexes, the entry-exit site for various proteins. Also, lamins, an intermediate filament, which is connected to nuclear envelope via various anchor proteins and to chromatin mainly at heterochromatin sites via heterochromatin binding proteins like HP1a, provide a struc­ tural integrity to the nucleus (Georgatos and Blobel, 1987; Haithcock et al., 2005; Houben et al., 2007; Makatsori et al., 2004; Mattout et al., 2006; Nelson et al., 1986; Pajerowski et al., 2007; Panorchan et al., 2004; Shumaker et al., 2003). The size of cell nucleus reflects both total chromatin content and the balance of nuclear and cytoplasmic forces at the nuclear envelope. In addition, the size of eukaryotic nucleus is extraordi­ narily variable not only during different cell cycle stage but also between different cell types derived from various tissues of the same organism. In the following sections, we shall briefly describe chemical and physical methods to probe the cytoplasmic contribu­ tion toward a prestressed nuclear organization. A. Chemical Inhibitors to Modulate Cytoskeletal Tension In order to estimate contribution of the cytoskeleton toward prestress of nucleus, comparative measurements of size of the nucleus can be made in presence and absence of cytoskeletal filaments. Studies have explored the use of chemical reagents to perturb nucleo-skeletal and cytoskeletal balance of forces that define cellular integrity and thereby modulate nuclear prestress. A number of drugs are known to chemically perturb the cytoskeleton. Cytochalasin D is known to inhibit actin polymerization (Nair et al., 2008), while Latrunculin depolymerizes actin and Nocodozole has been shown to perturb micro­ tubule organization. Chemical disruption of these cytoskeletal filaments illustrate the force balance that exists within the cytoskeleton meshwork with resulting altered change to nuclear size. While different concentrations of these drugs have been utilized, a protocol for their concentrations to aid chemical perturbation of cytoskeleton is described as follows: 1. 2. 3. 4.

For For For For

de-polymerizing microtubules, use nocodazole at 1 µg/ml for 16 h. de-polymerizing actin, use cytochalasin D at 1 µM for 2.5 h. inhibiting myosin II, use blebbistatin at 5 µM for 2.5 h. inhibiting kinesin Eg5, use monastrol at 125 µM for 16 h.

The area of the nucleus is measured upon treatment with cytoskeletal inhibitors to estimate the contribution of each component to maintain the prestressed state (Fig. 4A and B). An increase in the nuclear area upon Nocodazole treatment suggests the role of microtubule as a compressive load bearer, whereas a decrease in the nuclear area upon Cytochalasin D or Blebbistatin treatment indicates the role of actin and myosin in providing outward tensile force on nucleus to maintain the prestressed state. Mild perturbation of actin cytoskeleton at low concentration of Cytochalasin D has revealed the role of a structural

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(A) Control

(B)

Noc

CytoD

Monst

Blebb

140000

Area (μm2)

120000

* 300

**

** 200

**

il)

G

en

om

e

(e

nt

ro

pi

c

co

ab l et

st

eb b

H

Bl

on M

oD yt

oc C

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Fig. 4 Chemical deploymerization of cytoplasmic filaments or inhibition of associated motor proteins cause a variation in nuclear size in PMEF cells. (A) Representative images of drug-treated and fixed nuclei with the DNAstained with Hoechst. Scale bar = 20 mm. (B) Statistics for 100 nuclei each. The error-bars are standard errors. Also shown are the size that nuclei shrink under a heterochromatin ablation (17 cells), and the estimated hydrodynamic radius of the genome in these cells. ‘*’ implies p < 0.05 and ‘**’ implies p < 0.001.

component of the actin cytoskeleton that forms a cap above the apical surface of the nucleus that modulates prestress. Disruption of this actin cap results in rounding up of nuclei (Khatau et al., 2009). The structural correlation of cell shape with nuclear shape and dependence of nuclear size on adherence is illustrated by the interplay between anchorage with cell and nuclear shape, with loss of anchorage resulting in nuclear retraction and rounding. Intermediate filaments form a continuous mesh from attachment points at the cell surface to the nuclear envelope (Herrmann et al., 2007), and the role of intermediate filaments as mechanical integrators and tensile stiffeners has been suggested more than a decade ago (Hollenbeck et al., 1989). However, their role on cellular strength and integrity requires more investigation.

B. Laser Perturbation of Differentially Compacted Chromatin Though pharmacological perturbation of cytoskeletal filaments reveals their roles in maintaining cellular and nuclear prestress, this method lacks specificity in structural

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perturbations. Further this does not indicate the regions in the nucleus and cytoplasm which are critical structural regions of the cell. To probe this aspect of the cellular structure, the technique of laser induced ablation is described in this section. The ablation of physical structure via laser by local heating can efficiently be employed to perturb structural components and thereby explore their contribution to cellular pres­ tress of the nucleus (Berns, 2007; Quinto-Su and Venugopalan, 2007). The incorporation of fluorescently tagged histone protein stably expressed in the cells of interest can be used as a method to address structures in distinct chromatin regions. The identification and comparative study of loosely packaged euchromatin and tightly com­ pacted heterochromatin is obtained by the differential fluorescence intensity of the core histone-tagged nucleus. Local ablation of cellular and subcellular structure within the cell is obtained by gold-nanoparticle-mediated laser perturbation. Incorporation of these particles into cells can be accomplished via methods of microinjection or hypotonic shock to cells (Mazumder and Shivashankar, 2007). Earlier studies have addressed issues such as the biocompatibility, low cytotoxicity, and method of endocytic incorporation of gold nano­ particles into cells (Shukla et al., 2005). Thus gold-nanoparticle incorporation can be employed as a mechanism to result in localized heating and disruption to structure. A brief protocol for incorporation of gold nanoparticle into living cells is provided below: 1. Cells pregrown on dishes are incubated in media supplemented with gold particles for 1 h to ensure the presence of the particle in the endocytosed fluid. 2. Cells are washed with PBS pH 7.4 prior to being subjected to a hypotonic shock for 3 min at 37°C to allow for incorporation of gold nanoparticles. 3. Cells are gently washed and allowed to recover for 3–4 h before experimental use. 4. Near-infrared radiation focused at specific location within the cell is employed with diffraction resolution. 5. Gold-nanoparticle-incorporated cells suffer local heating by exposure to pulsed Ti-sapphire laser mode locked at 835 nm employing laser power of 56 mW for a period of 3 s. Alternatively, since gold particles serve merely to ensure efficient local absorption of near infrared radiation, ablation without use of gold particles can achieve similar results but by employing instead higher laser powers (120–140 mW). Laser heating at powers smaller than those used results only in local photo-bleaching. Selective laser ablation of differentially compacted chromatin regions within the nucleus viz. euchromatin and heterochromatin has revealed their contributive roles toward the maintenance of nuclear prestress (Fig. 5A). Perturbation of dense heterochromatin regions results in decrease in nuclear area and volume, while perturbation of comparatively less dense euchromatin regions result only in marginal shrinkage (Fig. 5B). Analysis of shrinkage dynamics of heterochromatin ablated nucleus show mean decrease in nuclear volume by 35 ± 11% in comparison to unperturbed nuclei. This observed nuclear shrink­ age upon ablation reveals the role of heterochromatin as structural nodes for cytoskeleton attachment to the nucleus. The decrease in anisotropy of nuclear shape illustrates the role of outward cytoskeletal tension, as well as loss of force balance between nucleoskeletal

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(A) 0′′

Before Het

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Eu

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Het

Eu

(C) Normalized area

Fractional Δarea

(B)

1.0 τ ~ 152 s

Het Eu

0.8 0.6 0

200 Time (s)

400

Fig. 5 Representative images of the differential response of PMEFs to heterochromatin (Het) and euchromatin (Eu) perturbations. H1e-EGFP transfected PMEF cells are ablated at the heterochromatin at indicated time-points, using a 6.8 s exposure of a 1.5 µm diameter region to a pulsed titanium sapphire laser (80 mW at a fixed spot). Experiments are done 24 h posttransfection. The ablation points are shown by the white arrows, and time-points in seconds from the ablation are indicated above the images. Nuclei showed a fall in size in response to heterochromatin ablation, while such an effect was not present upon euchromatin ablation. (B) Fractional change in area (n = 17 each) and (C) average time-trace upon heterochromatin ablation (Het) and euchromatin ablation (Eu) in PMEF cells transfected with H1e-EGFP (n = 9 each). Scale bar = 5 µm.

and cytoskeletal components. Dynamics of nuclear collapse on heterochromatin ablation reveals an initial lag phase followed by a sudden decay in nuclear volume (Fig. 5C). The reason for this lag may probably be due to the time required for the disruption of cytoskeleton filaments. Figure 6 shows the disruption of various cytoskeletal structures upon laser ablation as visualized using immunostaining methods. During mitosis majority of chromatin is highly condensed. This condensation of the nucleus is reflected at the scale of chromatin organization. Drosophila SR2þ cells have a small number of chromosomes and hence such condensation can be more tractably visualized. To test the effect of release of nuclear prestress on chromosome condensa­ tion, laser ablation of the nuclear envelope was performed in Drosophila SR2þ cells (Fig. 7A). These experiments resulted in a condensation of the chromatin into indivi­ dual chromosome territories, as envisaged by a decrease in nuclear volume and a concomitant rise in average pixel intensity (Fig. 7B). This is reminiscent of an important natural state of cell division, where there is a large condensation of nuclear volume, immediately following nuclear envelope breakdown. In this case, nuclear envelope breakdown is caused by invaginating microtubules at prophase (Beaudouin

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(A) EGFP­ LaminB1

BA

PA

(E) Tau-EGFP

(B) H1e-mRFP

BA

PA

(F) Anti-α-tubulin

BA

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Normal (C) Actin-EGFP

PA

BA

Ablated

(G) Anti-vimentin

Normal

(D) H2B-EGFP

Actin

(H) Paxillin

Ablated

Merge

Fig. 6 State of the various nucleo-cytoskeletal components on heterochromatin perturbation. HeLa cells are cotransfected with (A) EGFP-Lamin B1 and (B) H1e-mRFP. Images before (BA) and after (PA) perturbation are presented for the same cell. (C) Images of HeLa cells transiently transfected with ActinEGFP before and after heterochromatin perturbation. Heterochromatin is identified by Hoechst staining of the DNA in these cases (images not shown). (D) H2B-EGFP expressing HeLa cells are perturbed at the heterochromatin, fixed and stained with an antibody against paxillin (visualized by a Cy5-labled secondary antibody), and filamentous actin bound by an Alexa-568-labeled phalloidin. Individual and merged images are presented. (E) Images of HeLa cells transiently transfected with Tau-EGFP as a microtubule-marker before and after heterochromatin perturbation. As in (C), heterochromatin is identified by Hoechst staining of the DNA. Images of H2B-EGFP expressing HeLa cells perturbed at the heterochromatin, and stained with primary antibodies against (F) -tubulin and (G) vimentin on separate plates. A Cy3-labeled secondary antibody is used. Normal and perturbed cells are imaged on the same plate. Green indicates H2B-EGFP fluorescence, while red shows the respective cytoplasmic filaments. Scale bars = 5 µm in (A), (B), (C), and (E). Scale bars = 10 µm in (D), (F), and (G). (See Plate no. 5 in the Color Plate Section.)

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(A) 0′′

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120

160 140 Area (μm2)

308′′

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120 80

100 80

Intensity

Before

60

60 40

40 0

50

100 150 200 250 300 Time (s)

Fig. 7 Condensation of chromatin upon envelope ablation. (A) Representative images of condensation of individual chromatin territories in SR2þ cells. Time in seconds is indicated on top of the panel. Scale bar = 5 μm. (B) Fall in area of the confocal slice (filled squares), and corresponding rise in fluorescence intensity (open circles), both indicating condensation.

et al., 2002; Panorchan et al., 2004). The methods described in this section provide mechanical and chemical tools to probe the role of the cytoskeletal filament structure on nuclear prestress.

IV. Conclusions The prestressed state of nucleus has been illustrated by probing their structural organization across a variety of cellular systems. Employing both mechanical and chemical perturbation of cytoskeletal filament and nuclear structure, a prestressed state of the cell nucleus is studied. In Fig. 8 a schematic of this prestress is shown. On one scale, histone tail–tail interactions results in condensing DNA into a micrometer size while on the other regime entropic forces drives this assembly to several hundred microns given its polymeric nature. Balancing these two scale results in an equilibrium size of a few micron-sized nucleus. As depicted in the schematic, in interphase cell the nucleus is further prestressed by variety of nucleo-cytoplasmic skeleton. This pre­ stressed dynamic equilibrium can be modulated by mechanical perturbations to hetero­ chromatin nodes, as well as chemical alterations to nuclear histone tail–tail interactions and cytoskeletal assemblies. The nucleus is thus a part of an integrated network that spans across cytoskeletal filament structure to the plasma membrane which emerges

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Entropic configuration

Isolated nucleus

Condensation due to histone tail−tail Interactions

~10 μm

~1 μm

~200 μm

Nucleus in cellular context (~20 μm)

Fig. 8 Schematic of the force balance that stabilizes chromatin assembly in an intact isolated nucleus and in cellular context. The outward entropic force due to confinement of DNA polymer to a small volume is countered by an inward condensing force due to histones and other nonhistone proteins of nucleus. These forces act even in an isolated nucleus. In a cellular context, equilibrium of forces is further modified by compressive forces due to microtubules and an outward tension due to actomyosin cytoskeleton.

through the process of cellular differentiation and development. Further, as cells are subject to various physical cues from their environment, mechanotransduction of such signals to the nucleus requires an understanding of the prestressed organization of the nucleus and its modulation (Wang et al., 2009). Cells potentially can exploit this to regulate expression level of genes responding to such mechanotransduction. Acknowledgements We thank the Nanoscience Initiative of Department of Science and Technology (DST) for funding and the NCBS Common Imaging and Flow Facility (CIFF). AK and KVI thank Council for Scientific and Industrial Research (CSIR) for their graduate research fellowships.

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