Chapter 18 Sensing Cytoskeletal Mechanics by Ballistic Intracellular Nanorheology (BIN) Coupled with Cell Transfection

CHAPTER 18

Sensing Cytoskeletal Mechanics by Ballistic Intracellular Nanorheology (BIN) Coupled with Cell Transfection Melissa S. Thompson* and Denis Wirtz*,† *Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore, Maryland 21218 †

Howard Hughes Medical Institute graduate training program and Johns Hopkins Institute for NanoBioTechnology Johns Hopkins University Baltimore, Maryland 21218

Abstract I. Introduction A. Nanorheology as a Quantitative Tool for Molecular Cell Mechanics B. Transient Transfection to Induce Changes in the Cytoskeleton as Measured by Ballistic Intracellular Nanorheology (BIN) II. Materials and Instrumentation A. Cell Culture B. Nanoparticles and Macrocarriers C. Ballistic Injection D. Transfection and Drug Application E. Nanoparticle Imaging, Acquisition, and Analysis III. Procedures A. Preparation of Nanoparticles and Macrocarriers B. Ballistic Injection of Nanoparticles C. Transfection D. Multiple Particle Tracking Data Acquisition and Analysis IV. Pearls and Pitfalls V. Concluding Remarks References

METHODS IN CELL BIOLOGY, VOL. 89 Copyright 2008, Elsevier Inc. All rights reserved.

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0091-679X/08 $35.00 DOI: 10.1016/S0091-679X(08)00618-3

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Abstract Key processes in normal and diseased cells depend directly or indirectly on the viscoelastic properties of the cytoplasm. Particle-tracking microrheology is a highly versatile method that measures the viscoelastic properties of cytoplasm directly by tracking fluorescent nanoparticles embedded in the cytoskeleton with high spatial and temporal resolutions. Here we present a new method that combines cell transfection, ballistic injection, and particle-tracking microrheology to monitor changes in cytoplasmic micromechanics following controlled changes in protein expression. We demonstrate that cells transfected with GFP (green fluorescent protein) display viscoelastic properties identical to untransfected fibroblasts, that low levels of expression of GFP-a-actinin do not aVect cell microrheology, and that the transient transfection with GFP-C3 transferase reduces the elasticity of the cytoplasm of fibroblasts to a similar extent as C3 transferase toxin, which deactivates the GTPase Rho. Combining cell transfection with particle-tracking microrheology opens the way to quantitative, single live-cell mechanical studies where stable cell lines cannot be easily established, but where commonly used transfections can be exploited to manipulate cytoskeletal organization.

I. Introduction Key processes in normal and diseased cells depend directly or indirectly on the viscoelastic properties of the cytoplasm. For instance, cells migrating into a wound stiVen their leading edge to enable dendritic F-actin assemblies to produce net protruding forces (Kole et al., 2005; Lee et al., 2006; Pollard and Borisy, 2003). Cells adapt their intracellular physical properties to the physical properties of their extracellular milieu in order to grow, diVerentiate, and migrate (Pelham and Wang, 1997). The translocation of organelles (e.g., nucleus, mitochondria, ER) within the cytoplasm also depends on the local properties of the cytoplasm (Lee et al., 2005; Tseng et al., 2004b), which is stiVer (i.e., has a higher elasticity) in the cell periphery than in the perinuclear region of normal cells (Yamada et al., 2000). Transformed cells collected from cancer patients or cells harvested from mouse models for progeria (premature aging) can display significantly softer (i.e., less elastic) cytoplasm, which aVects their ability to adhere to substrata, to properly position their nuclei and centrosomes, to polarize under flow conditions, and to migrate at the edge of a wound (Lee et al., 2007).

A. Nanorheology as a Quantitative Tool for Molecular Cell Mechanics A major contributor to cytoplasmic stiVness is the cytoskeleton, which is composed of the filamentous proteins F-actin, microtubules, and intermediate filaments (Pollard and Borisy, 2003) and provides the cell’s cytoplasm with its

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structure and shape. In the cell, actin monomers assemble into semiflexible F-actin polymers that form highly entangled networks of filaments and bundles (Tseng and Wirtz, 2001; Tseng et al., 2004a), whose viscoelastic properties can be controlled by either altering the local density of assembled actin or the activity of F-actin crosslinking/bundling proteins such as a-actinin (Tseng et al., 2001, 2002a,c, 2005) or by subjecting these networks to external (Lee et al., 2006) or internal (Kole et al., 2004) mechanical stresses. Current experimental approaches to cell mechanics can be categorized into those measuring global and local cell properties, those indirectly measuring cytoplasmic mechanics through a required physical contact of the probe with the plasma membrane, and those measuring cytoplasmic mechanics directly. For instance, micropipette suction (Tsai et al., 1993)—well suited to probing suspension cells (e.g., red blood cells)—measures the global stiVness of a cell, which may include contributions from the plasma membrane, the nucleus, and the cytoplasm. Methods that measure local properties include atomic force microscopy (AFM) (Hoh and Schoenenberger, 1994) and magneto-cytometry (Valberg and Albertini, 1985), the latter probing apparent cell mechanics by subjecting large ECM-coated beads tethered to cell receptors to rotational movements. Because diVerent techniques measure diVerent (but often related) rheological quantities, intracellular mechanics is best characterized by multiple rheological parameters, including viscosity, elasticity, and creep compliance. Viscosity measures the propensity of cytoplasm to flow under mechanical forces. Elasticity (or elastic modulus) measures the stretchiness of a material, in this case, the pliability of the cytoplasm. A material that is more viscous than elastic is a viscoelastic liquid; a material that is more elastic than viscous is a viscoelastic solid. The cytoplasm of adherent cells is complex; it behaves as a viscoelastic liquid when sheared slowly, but as a viscoelastic solid when sheared rapidly (Kole et al., 2004; Tseng et al., 2002b). A similar behavior is observed in reconstituted actin filament networks in the presence of dynamic crosslinkers, such as filamin or a-actinin (Tseng et al., 2004a; Xu et al., 1998, 2000). When sheared more slowly than the lifetime of binding of the crosslinking protein to F-actin, the actin filament network flows and behaves largely as a liquid. When sheared at a rate faster than the inverse binding lifetime of the crosslinking protein, the actin filament network behaves as a stiV gel or solid. The creep compliance of the cytoplasm refers to its deformability. A high compliance indicates a low propensity to resist mechanical deformation following application of a shear stress; a low compliance indicates that a high propensity to resist such stress. In certain conditions, viscosity and elasticity can be directly computed from creep compliance measurements. We have introduced the method of particle tracking microrheology (Tseng et al., 2002b), which measures the viscosity, elasticity, and creep compliance of the cytoplasm. In this approach, fluorescent nanoparticles are injected directly in the cytoplasm of live cells are tracked with high spatial and temporal resolutions. The size of these nanoparticles is chosen to be larger than the average mesh size of the cytoskeleton, typically 50 nm, measured by probing the diVusion of fluorescent

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dextran using fluoresence recovery after photobleaching (Luby-Phelps et al., 1986). These nanoparticles are also smaller than
1 mm; therefore these nanoparticles undergo Brownian motion and inertial (gravity) forces are negligible. The small random forces produced by the bombardment of water molecules and the movements of cytoplasmic structures induce their random displacements (Tseng et al., 2002b). The amplitude of these Brownian displacements directly reflects the local viscoelastic properties of the cytoplasm in the vicinity of the nanoparticle (Gittes et al., 1997; Mason et al., 1997). Direct injection of the nanoparticles into the cytoplasm (Tseng et al., 2002b), as opposed to passive transfer, circumvents the endocytic pathway and, therefore, the engulfment of the nanoparticles in endocytic vesicles tethered via motor proteins to cytoskeleton filaments (Suh et al., 2003). The recent introduction of ballistic injection (Lee et al., 2006; Panorchan et al., 2006) has transformed the particle-tracking microrheology assay to highthroughput rather than using manual microinjection whereby thousands of cells are simultaneously injected with nanoparticles and, therefore, amenable to particle-tracking measurements (Fig. 1). Manual microinjection, and to a lesser extent ballistic injection, can cause trauma to cells, which may subsequently undergo necrosis or apoptosis in large numbers. Therefore, cell microrheology has been used almost exclusively to compare the mechanical response of cells stably expressing a mutant protein to that of their wild-type counterpart or has been restricted to comparing untreated cells to cells subjected to drug treatments. Cell transfection with genes encoding dominant negative proteins or constitutively active proteins, or with small interfering RNAs (siRNAs), in combination with particle-tracking microrheology, has not been demonstrated. This may be due in part to the fact that a fraction of cells do not seem to survive the double trauma of nanoparticle injection and cell transfection. Here we present a new method that allows one to combine cell transfection, ballistic injection, and particle-tracking microrheology. We demonstrate that cells transfected with GFP (green fluorescent protein) display viscoelastic properties identical to those of untransfected cells, that expression of GFP-a-actinin does not aVect cell microrheology, and that the transient transfection with GFP-C3 transferase reduces the elasticity of the cytoplasm of fibroblasts to a similar extent as C3 transferase toxin. Combining cell transfection with particle-tracking microrheology opens the way to quantitative, single live-cell nano-mechanical studies for cell lines that cannot be stably transformed, but are amenable to transfections commonly used to manipulate cytoskeleton organization. B. Transient Transfection to Induce Changes in the Cytoskeleton as Measured by Ballistic Intracellular Nanorheology (BIN) BIN represents a unique in vivo tool to measure global and local viscoelastic properties of cells by high-throughput injection of many beads/cell, thereby increasing population size and increasing the probability of probing typical biological scenarios. Previous experiments employed manual microinjection of

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Fig. 1 Ballistic injection of 100-nm fluorescent carboxylated-polystyrene nanoparticles yields a range of 3–20 measurable nanoparticles per cell, which are distributed throughout the cell’s cytoplasm. The diVraction-limited images of the nanoparticles are shown as red dots for ease of view. The displacements of the nanoparticles’ centroids are recorded for 20 s at a rate of 30 frames per second. (Left panels). Trajectories of the centroids are plotted to ensure that each bead is undergoing random, Brownian motion. (Right panel) MSDs of the nanoparticles are calculated, from which rheological parameters describing the viscoelastic behavior of cytoplasm—including viscous and elastic moduli, creep compliance, and shear viscosity of cytoplasm—can be derived. Scale bar for phase contrast image of the cell is 10 mm. Scale bar for trajectories is 0.05 mm.

beads into cells (Daniels et al., 2006), which also permitted the microinjection of purified proteins in the cytoplasm, to induce and quantify cytoskeletal changes (Tseng et al., 2002b). However, protein microinjection is cumbersome, and injected proteins are often degraded with time. Moreover, microinjection can cause major mechanical trauma to cells. In contrast, the genetic manipulation of protein expression in live cells is often more benign, providing a longer lasting eVect that is easier to implement. The use of transient transfection coupled with BIN provides a high-throughput means to probe the eVects on cytoplasmic mechanics caused by controlled changes in protein expression.

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Here we demonstrate how we use BIN to measure the rheological response of Swiss 3T3 cells to changes in protein expression. Swiss 3T3 fibroblasts exhibit strong stress fiber staining in serum (Fig. 2A). Numerous proteins regulate the formation and crosslinking/bundling of actin filaments into contractile stress fibers, including the small GTPase Rho and the F-actin-binding protein a-actinin, respectively. A

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Fig. 2 Combined BIN and cell transfection: Control experiments. (A) Swiss 3T3 fibroblasts were both ballistically injected with 100-nm fluorescent nanoparticles and transiently transfected with either GFP only (top panels) or GFP-a-actinin (bottom panels) expression constructs. Post-experiments, fluorescence microscopy was used to visualize GFP (left panels) and F-actin structure by phalloidin staining (right panels). Scale bar, 10 mm. (B) Ensemble-averaged MSDs were calculated from the 2D trajectories of the nanoparticles in untreated control cells (blue curve), GFP-transfected cells (red curve), and GFPa-actinin-transfected cells (green curve). The number of nanoparticles and cells used for each condition was: n ¼ 156 and 27 for control cells; n ¼ 33 and 20 for GFP-transfected cells; n ¼ 34 and 20 for GFP-aactinin cells. Error bars respresent standard error of the mean.

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In previous reports, microinjection of purified a-actinin significantly increased the cytoplasmic viscoelasticity of Swiss 3T3 fibroblasts (Yamada et al., 2000). We combined the BIN assay with transient transfection to induce expression of GFP-tagged a-actinin. This was accomplished by plating the cells on a culture dish and ballistically injecting the cells with 100-nm fluorescent polystyrene nanoparticles. Following immediate and extensive washing, the cells were replated on poly-llysine-coated dishes, and, after a 4–6 h incubation, were transfected using a traditional lipophilic transfection method (Fugene HD Transfection Reagent, Roche). This process yields 30–40% of cells containing beads in a range of 3–20 beads per cell used for measurements (Fig. 1). After a 16-h incubation, measurements of the displacements of injected nanoparticles in large numbers of untransfected and transfected cells were collected following standard methods of multiple particle tracking (Kole et al., 2004). Mean squared displacements (MSDs) of the nanoparticles were calculated, and rheological parameters that characterize the viscoelastic properties of the cytoplasm were computed, including creep compliance, viscoelastic moduli, and shear viscosities. The control vector, which expresses GFP only, induced no significant changes in the viscoelastic properties of the cytoplasm compared to untransfected cells (Fig. 2), as shown by the absence of changes in the averaged MSD of the nanoparticles and associated rheological parameters. Similarly, transfection of cells with GFP-a-actinin induced no extensive change in the stiVness of the cytoplasm (Fig. 2). This is because the GFP-a-actinin fusion protein expressed in the cells comprised only 1% of the total a-actinin concentration in the cell (Varker et al., 2003). Therefore, the additional crosslinking of F-actin induced by the expression of GFP-a-actinin was negligible and undetectable by our methods. C3 transferase, a cell permeable toxin used extensively to study Rho GTPases (Aktories and Hall, 1989), abolishes RhoA, RhoB, and RhoC activity in cells and therefore prevents the formation of stress fibers (Hall, 1992). We found that the expression of GFP-C3 transferase yielded a similarly robust phenotype as the C3 toxin, in which the cells contained little to no actin structure and exhibited a distinctly diVerent morphology than untreated cells (Fig. 3A–C). The altered cytoskeletal morphology leads to a more deformable cytoplasm than that of untreated cells, as measured by an increase in creep compliance, and softer cytoplasm, as shown by a reduced elasticity and viscosity (Fig. 3D–G). The measurements consisted of over a hundred MSD profiles, which were distributed similarly in the elastic regime but shifted significantly in the viscous regime (Fig. 4).

II. Materials and Instrumentation A. Cell Culture Swiss 3T3 cells (Cat. No. CRL-2658), Dulbecco’s Modified Eagle’s medium (Cat. No. 30-2002), and bovine calf serum (10% v/v with culture medium, Cat. No. 30-2030) are obtained from American Type Culture Collection (ATCC).

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Fig. 3 Combined BIN and cell transfection: Monitoring cell-mechanical changes following C3transferase transfection. Swiss 3T3 fibroblasts were grown in serum (control, A), transfected with the GFP-tagged C3 transferase construct (B), or treated with cell-permeable C3 transferase (C), then stained with phalloidin to visualize actin and DAPI to visualize the nucleus. Scale bar ¼ 10 mm. For each condition, nanoparticles were ballistically injected, and their centroid displacements were monitored by high-resolution time-lapsed fluorescence microscopy. The number of nanoparticles and cells used for each condition was: n ¼ 156 and 27 for control cells; n ¼ 55 and 24 for GFP-transfected cells; n ¼ 117 and 29 for GFP-C3-transfected cells; n ¼ 107 and 29 for C3 Toxin-treated cells. (D) These displacements were

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Fig. 4 Distributions of MSDs. (A and B) Over a hundred nanoparticles embedded in cytoplasm of Swiss 3T3 fibroblasts were tracked for each tested condition, cells transfected with GFP alone (A) or GFP-C3 transferase (B), which contributed to the ensemble-average MSDs (in red). (C and D) Distributions of MSD values for time lags of 0.1 s (C) and 1 s (D) are shown for control (blue) and GFP-C3-transfected cells (red).

averaged and converted to MSDs. (E) These averaged MSDs were converted to averaged creep compliances which measure the mean deformability of cytoplasm. (F) Mean frequency-dependent elastic modulus, G0 (o) (which measures the stretchiness of cytoplasm), and viscous modulus G00 (o) (which measures the propensity of cytoplasm to flow), were calculated from averaged MSDs, as well as (G) the mean shear viscosity. Error bars in each panel represent standard error of the mean. Significance was determined by student t-testing where * denotes statistical significance where P<0.05 compared with control. C3 Transferase, both in toxin and plasmid forms, reduces the elasticity of the cytoplasm.

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Cell culture medium is supplemented with 1:100 dilution of PenicillinStreptomycin solution (Cat. No. 15140-122, Invitrogen). Hank’s balanced salt solution (HBSS, Cat. No. 21-021-CV) is from Mediatech, Inc. and 0.25% trypsin—1 mM EDTA (Cat. No. 25200-056) is from Invitrogen. Cells are cultured on 100-mm polystyrene dishes (Cat. No. 353003, Falcon) prior to bombardment. For post-ballistic injection, chambered coverslips (Cat. No. 155383, Labtek, Nalge Nunc,) are coated with 0.01% poly-l-lysine (PLL, Cat. No. P8920, Sigma) for 1 h, washed, and used for plating. Cell cultures are maintained at 37  C in a humidified, 5% CO2 environment. B. Nanoparticles and Macrocarriers In all experiments, we use 0.1 mm carboxylated-polystyrene fluorescent spherical nanoparticles (Cat. No. F8801, Invitrogen). Nanoparticles are dialyzed by immersing 3 ml of stock nanoparticle in 300,000 MWCO dialysis tubing (Cat. No. LABDIS001, Spectrum Labs) in 100% ethanol (Cat. No. 1110002000PL05, Warner Graham Co.). Dialysis is performed at 4  C for 24 h with 1 change of ethanol. Nanoparticles are then stored in 500 ml aliquots at 4  C, protected from light. Dialyzed nanoparticles are coated on macrocarriers (see also below; Cat. No. 165-2335, Bio-Rad). C. Ballistic Injection Ballistic injection of the nanoparticles is performed by the PDS-1000/He Hepta System (Cat. No. 165-2258, Bio-Rad) equipped with a hepta adaptor (Cat No. 1652225) and a vacuum line produced by a pump (model 2560, Cat. No. 2560C-02, Welch Rietschle Thomas). The system is thoroughly cleaned with 70% ethanol prior to use, and an air canister is used to clean the hepta adaptor lines. System accessories used for injection include 1800 psi rupture discs (Cat. No. 165-2332), macrocarriers (Cat. No. 165-2335) and stopping screens (Cat. No 165-2336), all from Bio-Rad. Rupture discs require tweezers to pre-treat them with isopropanol (Cat. No I9516, Sigma). D. Transfection and Drug Application The plasmid expressing GFP-a-actinin is from AddGene (Cat. No. 11908, AddGene). The GFP vector was generated from the GFP-a-actinin by removing the a-actinin insert using restriction enzyme HindIII (Cat. No. R0104S, NEB Labs). The linear GFP-tagged DNA was ligated using T4 DNA Ligase (Cat. No. M0202S, NEB Labs), transformed into XL1-Blue competent cells (Cat. No. 200249, Stratagene) using Kanamycin plates (Cat. No. K0879, Sigma) and then midiprepped (Cat. No. A7640, Promega). The GFP-C3 transferase plasmid was kindly provided by Dr. Carol Williams, Medical College of Wisconsin. All plasmids were transfected into Swiss 3T3 cells using HD Transfection Reagent

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(Cat. No. 04709705001, Roche) and Opti-MEM (Cat. No. 31985, Invitrogen). The cell permeable toxin C3 transferase was obtained from Cytoskeleton (Cat. No. CT04).

E. Nanoparticle Imaging, Acquisition, and Analysis BIN experiments were imaged using a Nikon TE2000-E inverted microscope, which is equipped for epifluorescence and with a Nikon PlanFluor 60 oil immersion lens (N. A. 1.3). Movies were captured by a Cascade 1k camera (Roper Scientific). Data acquisition and analysis were executed by journal routines provided by the Metavue/Metamorph Imaging Series (MDS Analytical Technologies).

III. Procedures A. Preparation of Nanoparticles and Macrocarriers The nanoparticles obtained from Invitrogen are in a 2% w/v aqueous suspension. To coat the macrocarriers evenly with nanoparticles and to ensure that the solution remains sterile, the nanoparticle solution is dialyzed to replace the water with ethanol (Section II.B above). Ballistic injection involves accelerating nanoparticle-coated macrocarriers to a high-speed towards a wire-mesh stopping screen in a vacuum chamber. The macrocarriers are stopped by the screen, while the nanoparticles continue through the mesh at a high speed and lodged in the cytoplasm of the cells below. Success of the injection depends on the quality of the thin-film created on the macrocarrier prior to injection. a. Materials i. ii. iii. iv. v.

Macrocarriers Nanoparticles in ethanol Large Petri dish, sterile 20 ml micropipette and tip Ethanol

b. Steps i. Inside a laminar flow hood, make 7 small dabs (<5 ml each) of ethanol on the petri dish where the macrocarriers will be placed. Transfer the macrocarriers on top of these dabs. This will ensure that the macrocarriers lay flat on the dish. ii. Pipette 20 ml of nanoparticle solution to one macrocarrier and spread over the entire carrier with the micropipette tip. Continue to spread to keep beads from building at the edges of the layer as the ethanol dries. Stop spreading once solution layer becomes thin and continued spreading produces aggregation of the nanoparticles. Let dry.

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iii. Pipette an additional 20 ml of nanoparticle solution onto the same macrocarrier. Spread the nanoparticles very carefully with the pipette tip, attempting to only touch the liquid and not the macrocarrier. The beads should be maximally spread in area. Avoid scratching the carrier with the pipette tip to reduce the formation of aggregates. Let dry. iv. Repeat for each macrocarrier and let sit in laminar flow hood for at least 1 h or overnight for complete drying.

B. Ballistic Injection of Nanoparticles The PDS-1000/He Hepta system ballistically injects cells by using pressurized helium to accelerate the macrocarriers against a wire mesh. The nanoparticle beads are carried by momentum through the mesh and into the cells in the dish below. This process requires removal of cell media from the dish. Therefore, this injection must be performed quickly to reduce cellular stress and apoptosis. In addition, cells are likely to engulf these beads through endocytosis. After injection, great care must be taken to thoroughly wash the cells to remove nanoparticles that may have lodged on the surface of the cell. Following injection and subsequent wash, the cells are immediately incubated in cell culture media at 37  C and 5% CO2 to allow the holes in the membrane to close and reduce cell stress. The cells are then replated to an optimal confluency to remove any residual extracellular beads that may remain.

1. Preparation of PDS-1000/He Hepta System a. Materials i. ii. iii. iv. v.

70% Ethanol Helium Isopropanol Rupture disc, 1800 psi PDS-1000/He Hepta System and accessories including torque wrench and macrocarrier insertion tool vi. Tweezers vii. Vacuum pump viii. Coated macrocarriers b. Steps i. Thoroughly spray the inside of the injection chamber, the target tray and the macrocarrier holder set with 70% ethanol and wipe with a kimwipe. Place target tray on the third level.

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ii. Clean the wire mesh with 70% ethanol and a wire brush. Dry with a kimwipe. iii. Clean hepta adaptor lines with air canister to remove any remaining bits of previously ruptured discs. iv. Turn on system, helium tank, and vacuum pump. v. With the chamber empty, load helium lines with helium by pulling vacuum on the chamber and fire the mechanism to pull helium through the lines. vi. Using tweezers, transfer the macrocarriers to the holder. vii. Secure the macrocarriers in the well with the macrocarrier insertion tool provided with the system. Ensure that they are firmly in place by inverting the holder. Assemble with wire mesh and holder. viii. Immerse a single 1800-psi rupture disk in 100% isopropanol for 3 s. Dab on a kimwipe to remove excess isopropanol and place in the well inside the top part of the hepta adaptor. Screw onto flange inside the injection chamber. Tighten with the torque wrench tool as instructed. ix. Slide macrocarrier holder into the second position. x. Align pressure divider lines of the hepta adaptor with the holes in the macrocarrier holder.

2. Ballistic Injection a. Materials i. ii. iii. iv. v.

100-mm dish of cells Aspirator Laminar Flow Hood Hank’s Balanced Salt Solution (HBSS) Cell media

b. Steps i. Remove media from the 100-mm plate containing cells at approximately 80% confluency. Aspirate for an additional 30 s to remove residual fluid. ii. Quickly place the cells on the target tray in the third position and close chamber door. iii. Pull vacuum on the chamber until the vacuum gauge reaches 28 psi; press hold button on ballistic chamber. iv. Fire the mechanism until a rupture is attained and release the pressure inside the chamber. v. Immediately move the cells to the laminar flow hood and wash 2 times with 30 ml of HBSS by simultaneously adding media to one side of the dish and aspirating from the other side. Wash 2 times with 10 ml HBSS by applying directly

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on the cells, forcefully enough to dislodge surface beads without detaching cells from the dish. vi. Add 10 ml of pre-warmed media and let the cells incubate for 1 h in the cell culture incubator.

3. Replating Cells After 1-h incubation, the cells are replated to PLL-coated chambered coverslips. Chambered coverslips were incubated with 0.01% PLL for 1 h, washed three times with PBS and then used for plating. In this step, it is important to centrifuge the cells once they have been detached from the cell culture dish. Centrifugation will remove any remaining nanoparticles that were in the dish, exocytosed by cells, or freely detached from the cell surface during the hour incubation period. Centrifugation speed and duration depend on cell line and centrifuge; however a gentle speed should be selected to pellet cells only. The nanoparticles will stay in solution since their momentum due to centrifugation will not overcome the drag force on the sphere, thereby separating extracellular beads from the cells. The supernatant is aspirated and the cells are plated according to desired cell density. In the case of Swiss 3T3 cells that undergo growth inhibition through cell-cell contact, the cells should be plated at about
30% confluency. C. Transfection Transfection of the cells can be performed at the same time as plating or after cells have adhered to the dish, depending on what is being transfected and how it will aVect the adhesive properties of the cells. In our case, we transfect cells after they had begun to adhere to the dish. Transfection is performed using the protocol provided with the HD Transfection reagent with DNA:HD ratio of 2:8. Transfection solution is added to cells after plating, and the cells are incubated overnight. D. Multiple Particle Tracking Data Acquisition and Analysis Data acquisition for the BIN analysis requires collection of movies that capture the movement of nanoparticles embedded in the cytoplasm. Tracking software within Metamorph Imaging Series is utilized to extract the movement of the centroid of the spherical nanoparticles into x, y coordinates from which individual MSDs are calculated via: hDr2 ðtÞi ¼ h½xðt þ tÞ  xðtÞ2 þ ½yðt þ tÞ  yðtÞ2 i where t is time lag. For each condition, approximately 10 cells are tracked, each containing a range of 3–20 measurable beads. Experiments are performed in triplicate. Microscopy of these ballistically injected cells is performed using a 60 objective on a Nikon

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TE2000-E inverted microscope equipped with epifluorescence and a live cell incubator to maintain an 5% CO2, 37  C environment.

1. Sample Preparation prior to Microscopy a. Materials i. ii. iii. iv. v.

Cell culture media HBSS Drugs (C3 Toxin) Aspirator Laminar flow hood

b. Steps i. Remove cell culture media and wash cells gently with 1 volume of HBSS. ii. Add pre-warmed cell culture media or treatment. In the case of C3 Toxin, add serum-free media with 2 mg/ml of C3 Toxin to the cells 2 h prior to data acquisition.

2. Initial Data Acquisition of Raw Data a. Materials i. Metavue Imaging Series ii. TE-2000E equipped with epifluorescence, 60 objective iii. Live Cell incubator b. Steps i. Turn on live cell incubator and allow carbon dioxide and temperature to equilibrate for 5 min. ii. Scan sample for cells containing ballistically injected beads using a combination of fluorescence with phase contrast. For GFP transfection, the scan must be performed with the appropriate fluorescence filter set first and then the cells are checked for presence of nanoparticles. iii. Center the cell of interest. Adjust the focus to bring the beads into the brightest, clearest view. This may have to be repeated since beads may reside in diVerent focal planes within the cells. Acquire 20-s streams of video with a Cascade 1k camera at a frame rate of 30 frames per second. This frame rate is achieved by limiting the acquisition region to 500  300 pixels, with 3  3 binning. iv. Repeat for beads residing in diVerent focus planes within the cell.

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v. Acquire phase contrast and fluorescent still images for overlay purposes. vi. Save stack files and images files. vii. Repeat for each cell within the data set.

3. Analysis of Movie Streams a. Materials i. Metamorph Imaging Series ii. Microsoft Excel b. Steps i. Open stream file in Metamorph Imaging Series. ii. Calibrate the image using the pixel to distance calibration file. This must be obtained in advance using a stage micrometer. iii. Draw a square region around beads of interest. Zoom 400% and duplicate the entire stream with zoom. iv. Using the ‘‘track objects’’ command within Metamorph, create inner and outer regions around each nanoparticle in the first frame of the stream. Aggregated spheres are omitted from analysis since they violate assumptions made in subsequent calculations, as are spheres moved by cellular organelles. The inner region is created to fit just outside the perimeter of the nanoparticles. The outer region is adjusted to encompass the nanoparticle in the remaining frames. v. Duplicate this image with the regions and save. vi. Open a log to Excel and track the nanoparticles using the regions created. Log the bead number and the time-dependent coordinates for each frame. Save this data spreadsheet.

4. Conversion of Aquired Data to MSDs, Creep Compliance, and Viscoelastic Moduli a. Materials i. Matlab b. Steps The MSDs resulting from the thermally induced fluctuations of the nanoparticles are calculated from the time-dependent coordinates [x(t), y(t)] extracted from movie streams using the following equations: 2 2   N N xðti þ tÞ  xðti Þ yðti þ tÞ  yðti Þ X X MSDy ðtÞ ¼ ð1Þ MSDx ðtÞ ¼ ðN þ 1Þ ðN þ 1Þ i¼1 i¼1

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The MSD of the nanoparticle, hDr2 ðtÞi, is the sum of the x and y components: MSDðtÞ ¼ MSDx ðtÞ þ MSDy ðtÞ

ð2Þ

Trajectories are monitored as a function of time to ensure that all nanoparticles are undergoing random, Brownian motion. Nanoparticles with trajectories that exhibit directed motion due to being endocytosed, altered motion due to directed movement of other cytoplasmic bodies in the cell, or due to error by the tracking software (i.e., when beads change focal planes) are eliminated from analysis. As the nanoparticles are thermally excited and randomly fluctuate in the cytoplasm, force is exerted on the surrounding cytoplasm. The deformability of the surrounding cytoplasm due to these forces is described by the local creep compliance, G(t). The creep compliance is therefore proportional to the MSD: GðtÞ ¼

3pa hDr2 ðtÞi 2kB T

ð3Þ

where kB is Boltzmann’s constant, T is the absolute temperature of the cell (in degrees Kelvin), and a is the radius of the nanoparticle. The creep compliance is the inverse of pressure or modulus and is expressed in units of cm2/dyn. The method to obtain frequency-dependent viscoelastic moduli has been described in detail in (Kole et al., 2004). Briefly, the complex viscoelastic modulus, G*(o), is obtained using the following equation: G ðoÞ ¼

kB T paio Ju fhDr2 ðtÞig

ð4Þ

where o ¼ 1=t and Ju fhDr2 ðtÞig is the Fourier transform of hDr2 ðtÞi. The elastic modulus is the real part of Eq. (4) and the viscous modulus is the imaginary part of Eq. (4). While G*(o) may be calculated numerically, an analytical solution was obtained by (Mason et al., 1997) by approximating the Fourier transformation of hDr2 ðtÞi into the Fourier domain using a wedge assumption, which expanding hDr2 ðtÞi locally around the frequency of interest o using a power law, and retains the leading term (Mason et al., 1997). The Fourier transform of hDr2 ðtÞi then becomes: io Ju fhDr2 ðtÞig  hDr2 ð1=oÞiG½1 þ aðoÞiaðoÞ ln hDr2 ðtÞi=d

ð5Þ

ln tjt¼1=o is the local logarithmic slope of at where aðoÞ ¼ d the frequency of interest o ¼ 1=t and G is the gamma function. The frequencydependent elastic and viscous moduli, G0 and G00 , respectively, can then be calculated algebraically using the following relationships:   0 ð6Þ G ðoÞ ¼ jG∗ ðoÞjcos paðoÞ=2   00 G ðoÞ ¼ jG∗ ðoÞjsin paðoÞ=2

hDr2 ðtÞi

ð7Þ

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where jG∗ ðoÞj ¼

2kB T   3pahDr2 ð1=oÞiG 1 þ aðoÞ

ð8Þ

IV. Pearls and Pitfalls The process of ballistic injection of cells with nanoparticles contains many steps which present possible obstacles for success and must be optimized for each procedure. For instance, the appropriate quantity of nanoparticles to coat on the macrocarriers for bombardment can vary among cell lines and must be optimized empirically. Too few particles will not yield enough intracellular beads for tracking and too many will lead to overwhelming cell death, leaving only the cells without beads for tracking. Furthermore, the process of coating is key to success in that one cannot allow the formation of aggregates as the ethanol-based bead solution dries. Spreading technique to reduce aggregate formation can only be perfected with practice and may require coating more macrocarriers that necessary to ensure even coating for a ballistic run. The wash step immediately following injection is crucial to the success of the experiment. However, washing too harshly has the potential to wash away all the cells that remain after bombardment. Washing too lightly will leave beads that have nonspecifically bound to the cell surface, and can subsequently be endocytosed. Ballistic injection and transfection have the potential to endanger cell health and lead to cell death. Measures must be taken in both cases to increase the chances of cell survival. For example, using pre-warmed media each time cells are incubated or replated and minimizing the time cells are kept out of the incubator environment increase cell survival. For transfection, the use of antibiotics is detrimental to the health of cells and must be absent from cell media upon incubation with transfection reagents. Waiting until cells have mostly adhered to the cell culture substrate to add the transfection reagent is beneficial to cell survival. Finally, as is typical with any lipophilic transfection, the ratio of DNA:reagent as well as the total amount of reagent to add to the cells must be optimized. In general, enhancement of the percentage of cells that contain injected beads and of the percentage that exhibit protein expression will increase the probability of having cells with both beads and expression for experimental observations. Additionally, the use of cell culture dishes that are entirely glass-bottomed, as opposed to partially, increases the percentage of cells that can be tested and, therefore, the population size for each experiment. Finally, during microscopic data acquisition, beads must be finely focused to yield clear centriods for data analysis. Overlays should be created to ensure that the beads lie in the cells of interest (control, with toxin, or with GFP). In addition, movies should

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be monitored during data tracking to ensure that beads are undergoing random, Brownian motion and are tracked without error (i.e., changing focal planes).

V. Concluding Remarks We have introduced a new method that combines the key advantages of the BIN assay with the convenience of transient transfections to correlate changes in cytoskeleton organization with changes in local mechanical properties of the cytoplasm. This method retains all the advantages of BIN and adds those of transfections using standard transfection reagents. This approach should greatly expand the applications of BIN in cell and molecular biology.

References Aktories, K., and Hall, A. (1989). Botulinum ADP-ribosyltransferase C3: A new tool to study low molecular weight GTP-binding proteins. Trends Pharmacol. Sci. 10, 415–418. Daniels, B. R., Masi, B. C., and Wirtz, D. (2006). Probing single-cell micromechanics in vivo: The microrheology of C. elegans developing embryos. Biophys. J. 90, 4712–4719. Gittes, F., Schnurr, B., Olmsted, P. D., MacKintosh, F. C., and Schmidt, C. F. (1997). Microscopic viscoelasticity: Shear moduli of soft materials determined from thermal fluctuations. Phys. Rev. Lett. 79, 3286–3289. Hall, A. (1992). Ras-related GTPases and the cytoskeleton. Mol. Biol. Cell 3, 475–479. Hoh, J. H., and Schoenenberger, C. A. (1994). Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy. J. Cell Sci. 107(Pt. 5), 1105–1114. Kole, T. P., Tseng, Y., Huang, L., Katz, J. L., and Wirtz, D. (2004). Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation. Mol. Biol. Cell 15, 3475–3484. Kole, T. P., Tseng, Y., Jiang, I., Katz, J. L., and Wirtz, D. (2005). Intracellular mechanics of migrating fibroblasts. Mol. Biol. Cell 16, 328–338. Lee, J. S., Chang, M. I., Tseng, Y., and Wirtz, D. (2005). Cdc42 mediates nucleus movement and MTOC polarization in Swiss 3T3 fibroblasts under mechanical shear stress. Mol. Biol. Cell 16, 871–880. Lee, J. S., Panorchan, P., Hale, C. M., Khatau, S. B., Kole, T. P., Tseng, Y., and Wirtz, D. (2006). Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiVening response to fluid flow. J. Cell Sci. 119, 1760–1768. Lee, J. S., Hale, C. M., Panorchan, P., Khatau, S. B., George, J. P., Tseng, Y., Stewart, C. L., Hodzic, D., and Wirtz, D. (2007). Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration. Biophys. J. 93, 2542–2552. Luby-Phelps, K., Taylor, D. L., and Lanni, F. (1986). Probing the structure of cytoplasm. J. Cell Biol. 102, 2015–2022. Mason, T. G., Ganesan, K., van Zanten, J. V., Wirtz, D., and Kuo, S. C. (1997). Particle-tracking microrheology of complex fluids. Phys. Rev. Lett. 79, 3282–3285. Panorchan, P., Lee, J. S., Kole, T. P., Tseng, Y., and Wirtz, D. (2006). Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix. Biophys. J. 91, 3499–3507. Pelham, R. J., Jr., and Wang, Y. (1997). Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13661–13665. Pollard, T. D., and Borisy, G. G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465.

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Melissa S. Thompson and Denis Wirtz Suh, J., Wirtz, D., and Hanes, J. (2003). EYcient active transport of gene nanocarriers to the cell nucleus. Proc. Natl. Acad. Sci. USA 100, 3878–3882. Tsai, M. A., Frank, R. S., and Waugh, R. E. (1993). Passive mechanical behavior of human neutrophils: Power-law fluid. Biophys. J. 65, 2078–2088. Tseng, Y., An, K. M., Esue, O., and Wirtz, D. (2004a). The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks. J. Biol. Chem. 279, 1819–1826. Tseng, Y., An, K. M., and Wirtz, D. (2002a). Microheterogeneity controls the rate of gelation of actin filament networks. J. Biol. Chem. 277, 18143–18150. Tseng, Y., Fedorov, E., McCaVery, J. M., Almo, S. C., and Wirtz, D. (2001). Micromechanics and microstructure of actin filament networks in the presence of the actin-bundling protein human fascin: A comparison with a-actinin. J. Mol. Biol. 310, 351–366. Tseng, Y., Kole, T. P., and Wirtz, D. (2002b). Micromechanical mapping of live cells by multipleparticle-tracking microrheology. Biophys. J. 83, 3162–3176. Tseng, Y., Lee, J. S., Kole, T. P., Jiang, I., and Wirtz, D. (2004b). Micro-organization and viscoelasticity of the interphase nucleus revealed by particle nanotracking. J. Cell Sci. 117, 2159–2167. Tseng, Y., Schafer, B. W., Almo, S. C., and Wirtz, D. (2002c). Functional synergy of actin filament cross-linking proteins. J. Biol. Chem. 277, 25609–25616. Tseng, Y., and Wirtz, D. (2001). Mechanics and multiple-particle tracking microheterogeneity of alphaactinin-cross-linked actin filament networks. Biophys. J. 81, 1643–1656. Tseng, Y., Kole, T. P., Lee, J. S., Fedorov, E., Almo, S. C., Schafer, B. W., and Wirtz, D. (2005). How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response. Biochem. Biophys. Res. Commun. 334, 183–192. Valberg, P. A., and Albertini, D. F. (1985). Cytoplasmic motions, rheology, and structure probed by a novel magnetic particle method. J. Cell Biol. 101, 130–140. Varker, K., Phelps, S., King, M., and Williams, C. (2003). The small GTPase RhoA has greater expression in small cell lung carcinoma than in non-small cell lung carcinoma and contributes to their unique morphologies. Int. J. Oncol. 22, 671–681. Xu, J., Tseng, Y., and Wirtz, D. (2000). Strain hardening of actin filament networks: Regulation by the dynamic cross-linking protein a-actinin. J. Biol. Chem. 275, 35886–35892. Xu, J., Wirtz, D., and Pollard, T. D. (1998). Dynamic cross-linking by a-actinin determines the mechanical properties of actin filament networks. J. Biol. Chem. 273, 9570–9576. Yamada, S., Wirtz, D., and Kuo, S. C. (2000). Mechanics of living cells measured by laser tracking microrheology. Biophys. J. 78, 1736–1747.