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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
1Orthopaedic Biomechanics Laboratory, Department of Mechanical Engineering, University of California, Berkeley; and 2UCSF/UCB Joint Graduate Program in Bioengineering, University of California, San Francisco-University of California, Berkeley, California
Submitted 27 October 2007 ; accepted in final form 11 August 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-actinin, filamin, and talin in the cytoskeleton was measured using Western blot analyses. After mechanical loading, there was no change in the amount of actin monomers in the cytoskeleton, but the cross-linking proteins -actinin and filamin that cofractionated with the cytoskeleton increased by 29% (P < 0.01) and 18% (P < 0.02), respectively. Localization of the cross-linking proteins by fluorescent microscopy revealed that they were more widely distributed throughout the cell after exposure to fluid shear. The amount of vimentin in the cytoskeleton also increased by 15% (P < 0.01). These results indicate that osteoblasts responded to mechanical loading by altering the cytoskeletal composition, which included an increase in specific proteins that would likely enhance the mechanical resistance of the cytoskeleton.
MC3T3-E1; human fetal osteoblasts; -actinin; filamin; cytoskeleton
Cells protect themselves from mechanical loads by rearranging and reinforcing the cytoskeletal network. Of the three types of filaments in the cytoskeleton, the actin microfilaments are the most sensitive to mechanical stimulus (30). Microfilaments have also been shown to make a substantial contribution to whole cell mechanical behavior (6, 53, 61), although there is evidence that intermediate filaments may also contribute to cytoskeletal toughness (32) and interact synergistically with actin (13). Cross-linking proteins, in particular -actinin and filamin, organize the cytoskeleton and alter the mechanical properties of microfilament networks (57, 58, 60, 64). -Actinin can organize the actin microfilaments in various orientations but usually forms parallel bundles (4). Filamin cross-links the filaments perpendicularly to form orthogonal networks (56). These cross-linking proteins are present throughout the cell (38) and play an important role in determining the structure of the actin cytoskeleton (32, 63).
Few studies have considered how the cytoskeletal composition changes in response to mechanical loads. Network polymer models of the cytoskeleton predict that recruitment of cross-linking proteins to the filament network could be an important mechanism of controlling cell stiffness (29, 47), and this has been supported by several studies. -Actinin is involved in the cellular mechanoprotective response (52), and increasing the amount of -actinin in the cytoskeleton is sufficient to increase the whole cell resistance to deformation (27). Filamin has also been implicated in the rearrangement and stiffening of cytoskeletal structures in regions that are exposed to localized mechanical stimulus (20). These studies suggest that cells alter the cytoskeletal composition to increase their whole cell mechanical resistance in response to loading, but it remains unknown how these cytoskeletal proteins contribute to changes in the cytoskeletal composition at the whole cell level following exposure to externally applied loads. Therefore, the overall hypothesis of this study was that osteoblasts would respond to fluid shear stress by altering the amount of specific cross-linking proteins in the composition of the cytoskeleton. The specific objectives were to determine the effect of fluid shear stress on 1) the amount of polymerized actin microfilaments and vimentin intermediate filaments; 2) the amount of actin cross-linking proteins -actinin and filamin associated with cytoskeleton; and 3) to observe how the localization of these cross-linking proteins in the cytoskeleton changes in response to fluid shear relative to the actin microfilaments.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-actinin monoclonal IgM (clone BM-75.2), mouse anti-vimentin monoclonal IgM (clone VIM 13.2), mouse anti-talin monoclonal IgG (clone 8D4), and mouse anti-GAPDH monoclonal IgM (clone 17.1) were obtained from Sigma (St. Louis, MO). Mouse anti-filamin A monoclonal IgG (clone FLMN01 [PM6/317]) was obtained from ABCam (Cambridge, UK). Rabbit anti-ERK polyclonal IgG (clone C-14) was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). All antibodies were reactive against human antigens, and all antibodies except anti-filamin were cross-reactive with mouse antigens. Donkey IgG raised against mouse IgG and IgM were obtained from Jackson ImmunoResearch (West Grove, PA) and conjugated to either horseradish peroxidase or FITC. Donkey IgG raised against rabbit IgG and conjugated with horseradish peroxidase was obtained from Santa Cruz Biotechnologies. Rhodamine Phalloidin, Alexa Fluor 488 conjugated DNase and Hoestch were obtained from Molecular Probes (Eugene, OR).
Cell culture and mechanical loading with fluid shear stress.
Human fetal osteoblasts 1.19 (hFOBs) and murine osteoblasts (MC3T3-E1s) were obtained from ATCC (Manassas, VA). The hFOBs were cultured with a 1:1 mixture of dMEM without phenol red and Ham's F12 medium (GIBCO, Herndon, VA) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 0.3 mg/ml G814 (Sigma) in an humidified incubator at 34°C and 5% CO2 (hFOBs have a temperature-sensitive promoter of osteogenic lineage commitment that is activated at 37°C). The MC3T3-E1s were cultured with -MEM (Cellgro, Gathersburg, MD) supplemented with 10% FBS and 1% penicillin-streptomycin cocktail (GIBCO) in a humidified incubator at 37oC and 5% CO2. The cells were grown to confluency on either glass coverslips or polycarbonate sheets treated with O2 plasma. All surfaces were coated with 10 µg/cm2 of Type I collagen (BD Biosciences, Bedford, MA) before cell seeding.
Fluid shear was applied to the cells in a parallel plate flow chamber (45, 51) maintained at 37°C for MC3T3-E1s and 34°C for hFOBs. The chamber was placed in a closed loop that was perfused with CO2 independent media (GIBCO) supplemented with 10% FBS and 4 mM L-glutamine (GIBCO). A digitally controlled peristaltic pump (Cole-Palmer, Vernon Hills, IL) maintained a constant hydrostatic pressure head to drive media through the chamber at the required flow rate to expose the cells to the prescribed magnitude of fluid shear stress (40). In all experiments, cells were exposed to 2 h of steady fluid shear stress at 20 dyn/cm2 (2 Pa), which is a physiological magnitude of loading (62), and compared with a control group in static media.
Cell lysis and fractionation. For Western blot analysis, cells were cultured on polycarbonate plates. After exposure to flow, the cells were washed briefly with PBS and fractionated by detergent extraction or differential centrifugation. Two methods of fractionation were compared to ensure the cross-linking proteins were fractionated with the cytoskeleton. Cells fractionated with detergent extraction were incubated with 1 mM dithiobis(succinimidyl propionate) (DSP, Pierce, Rockford, Il) in Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.5) for 5 min to stabilize the interaction of cytoskeletal proteins. Triton-X 100 insoluble cytoskeletons were then generated by incubation with 0.5% Triton-X 100 in Tris buffer for 7.5 min. The proteins that were soluble in the 0.5% Triton-X 100 were collected as the soluble fraction, and the Triton-X insoluble cytoskeletons were treated with 5 mM EDTA and 0.5% Triton-X 100 in Tris buffer, scraped off the surface, and collected as the cytoskeleton fraction. Differential centrifugation was performed by scraping cells off the surface in lysis buffer (0.5% Trion-X 100, 0.25% deoxychloric acid, and 5 mM EDTA in Tris buffer). The lysates were centrifuged at 10,000 g for 30 min, and the supernatant was removed as the soluble fraction. The pellet was dissolved in the lysis buffer with 0.1% SDS to make the cytoskeletal fraction. All the lysates were brought to 0.1% SDS and 5 mM EDTA, mixed vigorously, and sheared through a 23-gauge needle. All lysis buffers contained protease inhibitor cocktail for mammalian tissue (Sigma) and protease inhibitor cocktail 2 (Sigma). The concentration of protein in each fraction was quantified using the BCA kit (Pierce), and each sample was normalized to the sum of the concentrations for its soluble and cytoskeletal fraction.
Western blot analysis. The protein fractions were mixed with Laemmli sample buffer (37), separated by SDS-PAGE in a 7.5% gel, and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked in 4% milk and 1% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) and then incubated with primary antibodies for 90 min at room temperature in TBST with whole donkey IgG diluted 1:1,000. After washing in TBST was completed, the secondary antibody was applied for 30 min at room temperature in TBST with 0.5% milk. The blots were visualized with ECL chemiluminescence (GE Biosciences, Piscataway, NJ) on BioMax Light X-ray film (Kodak, Rochester, NY), and densitometric analysis was performed in ImageJ (NIH, Bethesda, MD).
Immunostaining. For immunostaining experiments, cell were grown on glass coverslips and washed once before fixing. The cytoskeletons were extracted by incubating for 5 min with 1 mM DSP (Pierce) in Tris buffer and then for 5 min with 0.5% Triton-X 100 in cytoskeleton buffer [in mM: 20 2-(N-morpholino)ethanesulfonic acid, 276 KCl, 6 MgCl, 6 EGTA, and 320 sucrose, pH 6.1]. The Triton-X insoluble cytoskeletons were fixed with 1 mM 3-3'-dithiobis-(sulfosuccinimidyl propionate) (DTSSP, Pierce), a water-soluble analog of DSP, in cytoskeleton buffer for 15 min, and the unbound cross-linkers were quenched with 100 mM glycine (Sigma) in PBS. Cells were fixed without extraction in 3% paraformaldehyde in cytoskeleton buffer for 20 min and permeablized with 0.4% Triton-X 100 in cytoskeleton buffer for 10 min. Fixed cells were blocked in 2% BSA (Sigma) for 30 min. Primary antibodies were incubated in PBS with whole donkey IgG diluted 1:100 for 2 h at room temperature or overnight at 4°C. The FITC conjugated secondary antibodies incubated in PBS with rhodamine phalloidin and Hoesch for 30 min. The coverslips were then mounted to slides with VectaShield Hardset (Vector Labs, Burlingame, CA) and visualized at x400 with a TE300 inverted florescence microscope (Nikon, Melville, NY). Images were captured in Simple PCI (Compix, Cranberry Township, CA) using an ORCA 100 cooled CCD camera (Hamamatsu, Japan).
Statistics.
Each flow sample was matched with a static control, and all experimental procedures for each pair were performed simultaneously. The experiment was repeated 12 times with a single pair of samples in each experiment. The blots from the soluble and cytoskeletal fractions generated in each pair were analyzed using matched pair t-tests in JMP (Version 5, SAS Institute, Cary, NC). Statistical significance was assigned to = 0.03 adjusted for six independent tests using the Simes procedure of controlling the false discovery rate (3). The mean percent increases were determined by dividing the mean difference in blot densities between the static and flow sample for each protein by the mean density of the static blot for that protein. Confidence intervals of 95% were calculated for the percent increase of each protein following exposure to flow to demonstrate whether the confidence interval contained zero.
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RESULTS |
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-actinin (P < 0.01), filamin (P < 0.02), and vimentin (P < 0.01) in the cytoskeletal fractions was significantly different in hFOBs exposed to fluid shear compared with the static controls (n = 12, Fig. 2, A–C). There was no significant difference detected for any of the proteins in the soluble fraction or for β-actin and talin in the cytoskeletal fractions. The mean percent increase of -actinin, filamin, and vimentin in the cytoskeleton was 29%, 18%, and 15%, respectively. There was also a significant change in the stoichiometric relationship between the cross-linking proteins and the actin filaments after exposure to fluid shear (Fig. 2D). In the cytoskeletal fractions, the ratio of -actinin to β-actin increased 26% (P < 0.001), and the ratio of filamin to β-actin increased 16% (P < 0.05). There was no significant difference in the ratios of the cross-linking proteins to β-actin in the soluble fractions.
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-actinin. There were punctuate regions of intense staining for -actinin that did not appear to localize with the cytoskeleton and may have been cellular debris or vesicle bound -actinin. -Actinin also appeared to colocalize with the actin microfilaments wherever they were not completely depolymerized. However, the -actinin staining was weak and diffuse throughout most of the cell. In cells that were treated with cytochalasin D and fixed without extraction, the -actinin staining in the body of the cell remained intense, but there were fewer regions where the -actinin appeared to colocalize with the cytoskeleton.
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-Actinin and filamin were widely distributed throughout hFOBs and MC3T3-E1s after exposure to fluid shear. Filamin colocalized with f-actin bundles in the cells exposed to static cultures, and these structures are more highly concentrated at the periphery of the cells (Fig. 5). Additional distinct stress fibers formed throughout the cell in response to the fluid flow (Fig. 5, B vs. E), and filamin was also colocalized with these newly formed structures (Fig. 5, C vs. F). In static control cells, the -actinin was primarily localized to punctate regions (Fig. 6), which were often near the end of actin bundles (Fig. 6A). After exposure to fluid shear, stress fibers formed, and -actinin was more evenly distributed throughout the cell and along the stress fibers (i.e., it is no longer confined to the punctate regions, Fig. 6F). Although the stress fibers were dense, there were regions of diffuse -actinin staining that remained in the cytoskeletal residue following extraction but were not clearly colocalized with actin stress fibers (Fig. 6D).
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DISCUSSION |
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-actinin and filamin that was available to interact with the cytoskeleton throughout the cell. Since there was no significant change in the amount of these proteins in the soluble fraction, the cells may have upregulated the synthesis of these cross-linking proteins in response to mechanical loading to account for the observed increase in the cytoskeletal fraction. Furthermore, there was no change in the amount of monomeric g-actin in either the cytoskeletal or soluble fraction. Taken together, these results suggest that the change in cytoskeletal structure and whole cell mechanical behavior in response to fluid shear may be driven by the recruitment of cross-linking proteins to the cytoskeleton rather than by increasing the amount of actin filaments. There was also a significant increase in the amount of vimentin intermediate filaments in the cytoskeleton, which is likely a mechanoprotective response by the cells to minimize the risk of mechanical failure (32). These findings provide an improved understanding of how the composition of the cytoskeleton changes in response to the extracellular mechanical environment.
There are several features of this study that support the validity of our findings. First, we stabilized the interaction of the cytoskeleton and adjacent proteins before cytoskeletal extraction (2, 41). -Actinin and filamin are highly dynamic cross-linking proteins and disassociate easily from the cytoskeleton (56, 60), and their availability to interact with the cytoskeleton is tightly regulated by a variety of mechanisms (16, 56). We performed a rigorous set of control experiments to ensure that soluble cellular components were extracted from the cell after detergent extraction (Figs. 1 and 3), and the proteins that remained in the cytoskeletal residue after extraction were in close proximity to the cytoskeleton (Figs. 1 and 4). This method made it possible to observe differences in the amount of cross-linking proteins that were available to interact with the cytoskeletal fraction in response to a particular treatment. Second, the cells were mechanically stimulated using a magnitude of fluid shear that is within the predicted range of in vivo mechanical loads (1, 35, 62) and sufficient to increase the whole cell stiffness in osteoblasts (26). Therefore, the changes that were observed in the cytoskeleton may represent a part of the physiological response to loading. Finally, to verify that the increase in cross-linkers could be considered a whole cell response, the extracted cytoskeletons were immunostained for -actinin and filamin after exposure to fluid shear. Both cross-linking proteins were localized throughout the cytoskeletal structure, which confirmed that they had not been sequestered to specific regions of the cell. Talin, used to control for focal adhesion proteins (19), was not significantly increased in the cytoskeletal fraction after exposure to fluid shear. Therefore, it is unlikely that the concentration of -actinin and filamin was increased in the cytoskeleton as a result of focal adhesion contact formation.
Despite these strengths, two caveats should be noted. First, the cells were exposed to a magnitude of fluid shear that was consistent with other studies, but the type of flow in this study was limited to steady fluid shear stress. The physiological responses of osteoblasts to fluid shear stress, such as the expression of prostaglandin E2, cyclooxygenase-2, and osteopontin are induced by both steady and oscillatory flow (43, 46, 50), although oscillatory flow might more closely emulate in vivo loading of osteoblasts by pericellular fluid flow (28). In contrast, the arrangement of stress fibers (43, 50, 65), viscoelastic response (36), and role of the cytoskeleton in mechanotranstuction (43) is different after application of steady flow compared with oscillatory flow. Therefore, the optimal cytoskeletal composition also likely depends on the type load being applied, and these effects should be investigated in future studies. Second, Western blot densities were normalized to the total protein concentration in each sample rather than to an internal control in each fraction, as was necessary to avoid a priori assumptions about which proteins would remain constant in the cytoskeletal composition between the static and flow samples (14). The measurement of protein concentration may not be completely reliable, but a large sample size was included in this investigation to offset any errors associated with this measurement. A more conservative analysis was also performed by normalizing the amount cross-linking protein to the amount of actin in each fraction (Fig. 2D). There was a significant change in the ratio of -actinin to actin filaments (P < 0.01) after exposure to fluid shear, and this alteration to the the stoichiometry of the cross-linker/filament interaction is sufficient to indicate a change in the cytoskeletal composition (58).
Though morphological changes occur to the structure of the actin cytoskeleton in response to mechanical loading, the amount of polymerized actin remained unchanged. This has been previously observed using histomorphometry on fluorescently stained f-actin in insoluble MC3T3-E1 osteoblast cytoskeletons before and after exposure to fluid shear stress (46). In addition, the total amount of β-actin monomer did not increase in osteoblasts exposed to biaxial stretch (44) or in 3T3 fibroblasts that were exposed to tensile loads through focal adhesion contacts (10, 11). Though neither study investigated whether there was a change in the amount of polymerized versus soluble actin, they corroborate the present study since the total amount of β-actin in both the soluble and cytoskeletal fractions did not change. These results imply that any changes in the mechanical behavior of the cell that occur in response to loading are due to other changes in the cytoskeletal composition, such as the cross-linking proteins that determine the microstructure of the cytoskeleton.
The present study does not address the mechanism used by the cells to increase the total concentration of cross-linking proteins in the cytoskeleton in response to fluid shear stress. However, several previous studies that corroborate these observations indicate that mechanical loading can stimulate a redistribution of existing cross-linking proteins in the cell as well as gene expression of these proteins. It has been demonstrated that -actinin is recruited to stress fibers in MC3T3-E1 osteoblasts after exposure to 1 Pa of steady fluid shear for 1 h (49), though an increase in the total amount of -actinin could not be detected under these conditions (7), possibly because there was insufficient time for protein synthesis and the change in -actinin was undetectable. Since -actinin is constitutively expressed in eukaryotic cells (12), translation of -actinin could also have been upregulated rapidly after the onset of loading. Other investigators have observed an increase in filamin mRNA within 15 min in primary fibroblasts that were exposed to tensile loads through integrins (20). Furthermore, there was a substantial increase in the amount of total filamin in the fibroblasts after 4 h, which suggest that the cells may also rapidly upregulate the synthesis of filamin (10, 11). Additional investigation in needed to determine the extent to which de novo synthesis of cross-linking proteins is necessary to reinforce the cytoskeleton at the whole cell level.
Based on the mechanical function of the proteins studied in this investigation, increasing their concentration the in cytoskeleton will likely increase the whole cell resistance to deformation. The concentration of -actinin and filamin has a substantial effect on the mechanical behavior of gels made from reconstituted actin microfilaments (57, 58, 60, 64) and of whole cells (27, 58). There is additional evidence that cross-linking proteins interact synergistically to enhance the mechanical properties of the cytoskeletal network (18, 58, 59). Intermediate filaments also protect the cell against mechanical overloads (22) because they form networks that are tough and become very stiff at high magnitudes of strain (31). Vimentin and actin may also have a synergistic effect on the stiffness of the cytoskeletal network (48). Further investigation is needed to determine whether the observed change in cytoskeletal composition occurs to increase the stiffness of the cell at physiological load levels or to provide protection against mechanical rupture from pathological overloads.
In summary, this study demonstrated that osteoblasts respond to fluid shear by increasing the amount of -actinin, filamin, and vimentin that is present in the cytoskeleton. Based on the mechanical function of these proteins, it is likely that this response would be sufficient to increase the whole cell stiffness, which has been observed to occur in osteoblasts exposed to identical mechanical loads (26). As such, this study adds to the wealth of evidence suggesting that -actinin and filamin contribute to the whole cell response to mechanical loading. It also describes methods to facilitate further investigation into proteins that are redistributed in the cell by recruitment to or from the cytoskeleton in response to loading or other stimuli. Finally, this study indicates specific changes to the cytoskeletal composition that may be significant to mechanical and signaling models of the cytoskeleton and further characterizes the whole cell responses to mechanical loading.
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ACKNOWLEDGMENTS |
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Current address of W. M. Jackson, The National Institutes of Health-NIAMS, 50 South Dr., Room 1531, MSC 8022, Bethesda, MD 20892 (E-mail: jacksonw2@mail.nih.gov).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Bell PB Jr. The application of scanning electron microscopy to the study of the cytoskeleton of cells in culture. Scan Electron Microsc: 139–157, 1981.
3. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Royal Stat Soc Series B 57: 290–300, 1995.
4. Blanchard A, Ohanian V, Critchley D. The structure and function of alpha-actinin. J Muscle Res Cell Motil 10: 280–289, 1989.[CrossRef][Web of Science][Medline]
5. Cao F, Yanagihara N, Burke JM. Progressive association of a "soluble" glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells. Cell Motil Cytoskeleton 44: 133–142, 1999.[CrossRef][Web of Science][Medline]
6. Charras GT, Horton MA. Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys J 82: 2970–2981, 2002.[Web of Science][Medline]
7. Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, Duncan RL. Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol 278: C989–C997, 2000.
8. Christerson LB, Vanderbilt CA, Cobb MH. MEKK1 interacts with alpha-actinin and localizes to stress fibers and focal adhesions. Cell Motil Cytoskeleton 43: 186–198, 1999.[CrossRef][Web of Science][Medline]
9. Collins NT, Cummins PM, Colgan OC, Ferguson G, Birney YA, Murphy RP, Meade G, Cahill PA. Cyclic strain-mediated regulation of vascular endothelial occludin and ZO-1: influence on intercellular tight junction assembly and function. Arterioscler Thromb Vasc Biol 26: 62–68, 2006.[CrossRef][Web of Science][Medline]
10. D'Addario M, Arora PD, Ellen RP, McCulloch CAG. Interaction of p38 and Sp1 in a mechanical force-induced, beta 1 integrin-mediated transcriptional circuit that regulates the actin-binding protein filamin-A. J Biol Chem 277: 47541–47550, 2002.
11. D'Addario M, Arora PD, Fan J, Ganss B, Ellen RP, McCulloch CAG. Cytoprotection against mechanical forces delivered through beta 1 integrins requires induction of filamin A. J Biol Chem 276: 31969–31977, 2001.
12. de Jonge HJ, Fehrmann RS, de Bont ES, Hofstra RM, Gerbens F, Kamps WA, de Vries EG, van der Zee AG, te Meerman GJ, ter Elst A. Evidence based selection of housekeeping genes. PLoS ONE 2: e898, 2007.[CrossRef][Medline]
13. Esue O, Carson AA, Tseng Y, Wirtz D. A Direct Interaction between actin and vimentin filaments mediated by the tail domain of vimentin. J Biol Chem 281: 30393–30399, 2006.
14. Ferguson RE, Carroll HP, Harris A, Maher ER, Selby PJ, Banks RE. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics 5: 566–571, 2005.[CrossRef][Web of Science][Medline]
15. Forgacs G. On the possible role of cytoskeletal filamentous networks in intracellular signaling: an approach based on percolation. J Cell Sci 108: 2131–2143, 1995.[Web of Science][Medline]
16. Fraley TS, Pereira CB, Tran TC, Singleton C, Greenwood JA. Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling. J Biol Chem 280: 15479–15482, 2005.
17. Galbraith CG, Yamada KM, Sheetz MP. The relationship between force and focal complex development. J Cell Biol 159: 695–705, 2002.
18. Gardel ML, Shin JH, MacKintosh FC, Mahadevan L, Matsudaira P, Weitz DA. Elastic behavior of cross-linked and bundled actin networks. Science 304: 1301–1305, 2004.
19. Giannone G, Jiang G, Sutton DH, Critchley DR, Sheetz MP. Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation. J Cell Biol 163: 409–419, 2003.
20. Glogauer M, Arora P, Chou D, Janmey PA, Downey GP, McCulloch CAG. The Role of Actin-binding Protein 280 in Integrin-dependent Mechanoprotection. J Biol Chem 273: 1689–1698, 1998.
21. Glogauer M, Arora P, Yao G, Sokholov I, Ferrier J, McCulloch C. Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching. J Cell Sci 110: 11–21, 1997.[Abstract]
22. Goldmann WH, Galneder R, Ludwig M, Xu W, Adamson ED, Wang N, Ezzell RM. Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy. Exp Cell Res 239: 235–242, 1998.[CrossRef][Web of Science][Medline]
23. Harrison RE, Sikorski BA, Jongstra J. Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton. J Cell Sci 117: 2151–2157, 2004.
24. Hartwig JH, Kwiatkowski DJ. Actin-binding proteins. Curr Opin Cell Biol 3: 87–97, 1991.[CrossRef][Medline]
25. Hayakawa K, Tatsumi H, Sokabe M. Actin stress fibers transmit and focus force to activate mechanosensitive channels. J Cell Sci 121: 496–503, 2008.
26. Jaasma MJ, Jackson WM, Tang RY, Keaveny TM. Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells. J Biomech 40: 1938–1945, 2007.[CrossRef][Web of Science][Medline]
27. Jackson WM, Jaasma MJ, Baik AB, Keaveny TM. Over-expression of alpha-actinin with a GFP fusion protein is sufficient to increase whole-cell stiffness in human osteoblasts. Annals Biomed Eng In press.
28. Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, Donahue HJ. Differential effect of steady versus oscillating flow on bone cells. J Biomech 31: 969–976, 1998.[CrossRef][Web of Science][Medline]
29. Janmey PA. The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol Rev 78: 763–781, 1998.
30. Janmey PA. Mechanical properties of cytoskeletal polymers. Curr Opin Cell Biol 3: 4–11, 1991.[CrossRef][Medline]
31. Janmey PA, Euteneuer U, Traub P, Schliwa M. Viscoelastic properties of vimentin compared with other filamentous biopolymer networks. J Cell Biol 113: 155–160, 1991.
32. Janmey PA, Hvidt S, Lamb J, Stossel TP. Resemblance of actin-binding protein/actin gels to covalently cross-linked networks. Nature 345: 89–92, 1990.[CrossRef][Web of Science][Medline]
33. Ko KS, McCulloch CA. Partners in protection: interdependence of cytoskeleton and plasma membrane in adaptations to applied forces. J Membr Biol 174: 85–95, 2000.[CrossRef][Web of Science][Medline]
34. Ko KS, McCulloch CAG. Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading. Biochem Biophys Res Commun 285: 1077–1083, 2001.[CrossRef][Web of Science][Medline]
35. Kreke MR, Huckle WR, Goldstein AS. Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner. Bone 36: 1047–1055, 2005.[CrossRef][Web of Science][Medline]
36. Kwon RY, Jacobs CR. Time-dependent deformations in bone cells exposed to fluid flow in vitro: investigating the role of cellular deformation in fluid flow-induced signaling. J Biomech 40: 3162–3168, 2007.[CrossRef][Web of Science][Medline]
37. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[CrossRef][Web of Science][Medline]
38. Langanger G, de Mey J, Moeremans M, Daneels G, de Brabander M, Small JV. Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method. J Cell Biol 99: 1324–1334, 1984.
39. Lee JS, Panorchan P, Hale CM, Khatau SB, Kole TP, Tseng Y, Wirtz D. Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow. J Cell Sci 119: 1760–1768, 2006.
40. Levesque MJ, Nerem RM. The elongation and orientation of cultured endothelial cells in response to shear stress. J Biomech Eng 107: 341–347, 1985.[Web of Science][Medline]
41. Lindroth M, Bell PB Jr, Fredriksson BA, and Liu XD. Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells. Microsc Res Tech 22: 130–150, 1992.[CrossRef][Web of Science][Medline]
42. Maciver SK. Microfilament organization and actin-binding proteins. In: The Cytoskeleton, edited by Hesketh JE and Pryme IF. Greenwich: JAI, 1995, p. 1–45.
43. Malone AM, Batra NN, Shivaram G, Kwon RY, You L, Kim CH, Rodriguez J, Jair K, Jacobs CR. The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3–E1 osteoblasts. Am J Physiol Cell Physiol 292: C1830–C1836, 2007.
44. Meazzini MC, Toma CD, Schaffer JL, Gray ML, Gerstenfeld LC. Osteoblast cytoskeletal modulation in response to mechanical strain in vitro. J Orthop Res 16: 170–180, 1998.[CrossRef][Web of Science][Medline]
45. Nauman EA, Satcher RL, Keaveny TM, Halloran BP, Bikle DD. Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE2 but no change in mineralization. J Appl Physiol 90: 1849–1854, 2001.
46. Norvell SM, Ponik SM, Bowen DK, Gerard R, Pavalko FM. Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3–E1 osteoblasts does not require intact microfilaments or microtubules. J Appl Physiol 96: 957–966, 2004.
47. Nossal R. On the elasticity of cytoskeletal networks. Biophys J 53: 349–359, 1988.[Web of Science][Medline]
48. Osue E, Carson A, Tseng Y, Wirtz D. Synergy Between Actin and Vimentin. Baltimore, MD: Biomedical Engineering Soc, 2005.
49. Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh YF, Qiu J, Duncan RL. Fluid shear-induced mechanical signaling in MC3T3–E1 osteoblasts requires cytoskeleton-integrin interactions. Am J Physiol Cell Physiol 275: C1591–C1601, 1998.
50. Ponik SM, Triplett JW, Pavalko FM. Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles. J Cell Biochem 100: 794–807, 2007.[CrossRef][Web of Science][Medline]
51. Reich KM, Frangos JA. Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts. Am J Physiol Cell Physiol 261: C428–C432, 1991.
52. Rivero F, Koppel B, Peracino B, Bozzaro S, Siegert F, Weijer CJ, Schleicher M, Albrecht R, Noegel AA. The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development. J Cell Sci 109: 2679–2691, 1996.[Abstract]
53. Rotsch C, Radmacher M. Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys J 78: 520–535, 2000.[Web of Science][Medline]
54. Schmitz HD, Bereiter-Hahn J. Glyceraldehyde-3-phosphate dehydrogenase associates with actin filaments in serum deprived NIH 3T3 cells only. Cell Biol Int 26: 155–164, 2002.[CrossRef][Web of Science][Medline]
55. Smith PG, Garcia R, Kogerman L. Strain reorganizes focal adhesions and cytoskeleton in cultured airway smooth muscle cells. Exp Cell Res 232: 127–136, 1997.[CrossRef][Web of Science][Medline]
56. Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M, Shapiro SS. Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol 2: 138–145, 2001.[CrossRef][Web of Science][Medline]
57. Tseng Y, Fedorov E, McCaffery JM, Almo SC, Wirtz D. Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with alpha-actinin. J Mol Biol 310: 351–366, 2001.[CrossRef][Web of Science][Medline]
58. Tseng Y, Kole TP, Lee JS, Fedorov E, Almo SC, Schafer BW, Wirtz D. How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response. Biochem Biophys Res Commun 334: 183–192, 2005.[CrossRef][Web of Science][Medline]
59. Tseng Y, Schafer BW, Almo SC, Wirtz D. Functional synergy of actin filament cross-linking proteins. J Biol Chem 277: 25609–25616, 2002.
60. Wachsstock DH, Schwarz WH, Pollard TD. Cross-linker dynamics determine the mechanical properties of actin gels. Biophys J 66: 801–809, 1994.[Web of Science][Medline]
61. Wang N. Mechanical interactions among cytoskeletal filaments. Hypertension 32: 162–165, 1998.
62. Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 27: 339–360, 1994.[CrossRef][Web of Science][Medline]
63. Xu J, Tseng Y, Wirtz D. Strain hardening of actin filament networks. Regulation by the dynamic cross-linking protein alpha-actinin. J Biol Chem 275: 35886–35892, 2000.
64. Xu J, Wirtz D, Pollard TD. Dynamic cross-linking by alpha-actinin determines the mechanical properties of actin filament networks. J Biol Chem 273: 9570–9576, 1998.
65. You J, Yellowley CE, Donahue HJ, Zhang Y, Chen Q, Jacobs CR. Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. J Biomech Eng 122: 387–393, 2000.[CrossRef][Web of Science][Medline]
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15% (P < 0.02). C: there was a significant increase of 29% and 18% of the cross-linking proteins 
