The cytoskeleton : an introductory survey
And it is now apparent that the cytoskeleton has impact on other biological processes such as the control of gene expression, protein synthesis, cell cycle regulation, and development. This monograph outlines the basic properties of the major components of the polymeric filament networks and their interactions and associations. Wherever possible, emphasis is placed on more recent references. Any attempt to cover a research field this complex in an introductory mono graph is, by necessity, fragmentary, and oversights or omissions are inevitable.
I wish to apologize in advance to all those colleagues who feel that their work is not adequately represented. Selected pages Title Page. Table of Contents. Contents Prologue. Intermediate Filaments. Dynamic Aspects of Filament Assembly. Other Filament Types. The Cytoskeleton and the Cytoplasmic Matrix. MembraneCytoskeleton Interactions. Small experimentally induced extensions, as above, were observed to retract against even quite large tension loads imposed by a needle. In Fig. After achieving attachment, the needle was pulled with gradually increasing force producing a short extension some 46 min after initial placement of the needle.
By of this same experiment, the reference needle required to assess tension in the needle attached to the cell was at the very edge of the optical field. This was the maximum load we could measure at this magnification. As before, the cell spontaneously retracted induced lengthening of the cellular process data not shown. At this point, some 20 min after the sequence shown in Fig. The diameter of this cell process varied between 2. Forceful spontaneous retraction of experimentally induced cellular extension in GFP-MAP2c—transfected cell manipulated with a laminin-treated needle h:min.
Cell is shown before application of needle in fluorescent and phase images of Over the next 40 min, a cellular extension is induced and pulled by a laminin-treated needle, essentially by the same process shown in Figs. Subsequently, the extension was experimental towed to the right increasing in length to position 2 by The presence of an increasing force load on the cell extension can be seen by the change in the angle of the needle during the experiment. Note the high stability of the shape and position of the MT array in the cell.
The behaviors of the MTs during process extension and retraction was of some interest. First, a small number of MTs appear to be present in the experimentally induced cell process of Fig. More importantly, however, the images show very little change in the position or shape of the cell itself or of the nucleus. Further, there was little change in the prominent MT bundles seen within this cell. Particularly noteworthy is the bundle nearest the cone-shaped, mechanical anchor region of the experimentally induced extension.
This bundle of MTs deformed very little throughout this sequence despite the substantial forces that are being exerted on a neighboring local region. Untreated needles data not shown were applied to the surface of the cell but never formed detectable attachments, nor was GFP-labeled actin observed to concentrate at the application site of such needles or anywhere else. Needles were also treated with polylysine.
Essentially similar to Fig. As shown here, within 15 min of needle application, fluorescent actin has accumulated beneath the tip of the needle. With slow towing at modest forces, this actin accumulation was walked across the top surface of the cell to the cell margin over the next 40 min. However, increased tension at this time in an effort to elongate an extension caused the cell to release from the needle. Two fundamentals for a mechanical understanding of any complex structure, e. The development of GFP technology for cytoskeletal proteins Ludin and Matus, enabled us to make some direct observations of the actin and MT cytoskeletons in response to applied mechanical forces.
Indeed, these observations were most informative in assessing the time scale and spatial range over which the cytoskeleton changed or maintained form in response to forces that were of the same magnitude that these fibroblasts themselves exert. When a probe without attachment to the underlying cytoskeleton was used to apply force, we found that these attached cells behaved as predicted by the three-layer model of Dong et al.
The cell appears as a highly elastic nucleus that is surrounded by cytoplasmic MTs that behave like a viscoelastic fluid e. The third and outermost layer is an elastic cortical actin shell with a sustained tension pre-stress in the actin structures. The stiffness of this layer increased markedly when the experimental needle was treated with laminin to recruit the actin cytoskeleton to the surface.
By directly visualizing the actin recruitment, we confirmed a widely postulated model for mechanical connections between integrins and the actin cytoskeleton. Whether the probe applied simple deformations to the cell or interacted with the cytoskeleton, we found little evidence for strong connections between the actin cortex and linear elements of the cytoskeleton, either stress fibers or the underlying MT network. That is, we observed that experimentally applied forces produced unexpectedly local responses by the cytoskeleton.
In this regard, we found no evidence for a complementary force interaction between prestressed actin and compression-bearing MTs. In one experimental series Figs. The nucleus was highly compliant and highly elastic; poking with a needle caused the nucleus to undergo substantial local deformations that recovered very rapidly after release of the needle Fig. The contribution of the nucleus to the deformability of the cell has received very little attention, e. Our observations suggest that the properties of the nucleus are likely to play a significant role in the mechanical responses of cells, particularly the central region of attached cells.
As previously shown by Maniotis et al. Our observations did not allow us to determine whether this was by attachment or by steric entanglement, but movement of the nucleus clearly caused equivalent movements in the surrounding actin network, which also behaved elasticially Fig. Indeed, our results for the mechanical behavior of actin are entirely consistent with the well-established view of the cortex as being an elastic structure under sustained tension or prestress Lewis, ; Bray and White, ; Hochmuth, ; Ingber, Actin observed in our transfected REF cells recovered its shape and position after noninjurious deformation over the course of seconds indicating nearly pure elasticity Figs.
Sustained tension was clearly indicated by the behavior of actin to cutting, in which the severed actin bundle retracted strongly Fig. The sustained tension in the cortex is presumably balanced by positive fluid pressure in the cytoplasm, which also provides resistance to poking, but this appears not to be a large force in relation to cytoplasmic viscosity insofar as no cytoplasmic spillage followed any cutting intervention. However, the high degree of localization of the actin response to pushing, prodding, and cutting was surprising given the widespread view of an integrated cytoskeletal network Schliwa, ; Heidemann and Buxbaum, ; Forgacs, ; Ingber, By and large, only the actin filaments in the very immediate region of the intervention showed a response.
In contrast to the elastic behavior of the nucleus and actin network, MTs behaved as a viscoelastic fluid and we observed little evidence for tethering among MTs or between MTs and the overlying cortex. That is, rapid deformations produced elastic, solid behaviors while deformations on a longer time scale produced flow and permanent deformations. The most dramatic solid-like behavior of MTs occured when they buckled in response to rapid retractions of cellular regions where the MTs were arrayed axially to the direction of retraction Fig.
In all instances of buckling, no evidence for compression-induced MT disassembly was noted, although continued dynamic instability was observed. Because the buckling of MTs began almost immediately on cytoplasmic retraction, i. Nor did we ever observe a concerted or organized shift in the MT array suggestive of an integrated arrangement of MTs that distributed an increased compressive load throughout the array. Instead there was only random buckling and on even a slightly longer time scale min , MTs showed clear fluid behaviors.
In the relatively slow cellular retraction of Fig. When MT bundles were manipulated directly by the needle, the bundles moved differently indicating a lack of interconnection, and were deformed in shape and position over the time scale of 10 min, dramatically different from the elastic recovery within seconds for actin responses. In rounding cells at the beginning of the formation of retraction fibers Harris, , the MT cytoskeleton retracted independently of the overlying cortex, which remained attached at the same sites on the substratum Fig.
When large numbers of MTs were severed in a spread cell Fig. The cytoplasm at the cut edge of the living fragment behaved as if one had used a knife to cut through agar. We note that the rapid lysis of the severed fragment shown in Fig. Thus the responses of the cytoskeleton we observed cannot be ascribed to necrotic events.
Indeed, cells are known to survive mechanical insult rather well, due in part to the capacity of the plasma membrane to reseal rapidly McNeil and Steinhardt, Our results suggest that the localized mechanical responses of the cytoskeleton may also make an important contribution to injury resistance, e. Actin remained elastic in these experiments, but seemed more like a rigid, solid gel than like the relatively compliant cellular structure seen with untreated needles.
It is clear from Figs.
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We observed no integrated, wide-spread changes in the position of cytoskeletal or other cellular components. In the examples shown in Figs. Neither the actin nor the MTs in regions neighboring the extension were altered by changes in the length or position of the extension itself nor did the cytoskeletal filaments in these regions show significant responses to changes in the forces exerted nearby. Again, these behaviors and their time scale suggests the sort of viscoleastic behavior typical of rigid solids, i.
We presume that the contractions we observed by the experimentally induced cell processes Fig. The largest force we measured in this example was consistent with a recent measurement of the force generated by fibroblasts during locomotion Galbraith and Sheetz, and with contractile force exerted by essentially spherical fibroblasts Thoumine and Ott, The stress across the induced cellular process was similar to that of fibroblasts strongly stimulated to contract with thrombin Kolodny and Wysolmerski, In sharp contrast to cultured neurons that show a fluid-like growth response to tension and contract when slackened Heidemann and Buxbaum, ; Chada et al.
We think it likely that by experimentally applying forces with laminin-treated needles, we engaged adhesion, deformation-sensing, and tensile-response machinery normally engaged in the wound-closure function of fibroblasts Grinnell, We found the events of needle attachment interesting of themselves, although we can provide only a preliminary and incomplete interpretation.
First, we observed a local accumulation of actin in the cytoplasmic region corresponding directly with the extracellular site of the laminin, an important ligand for integrins. This lends support to a widely accepted model of integrin-mediated attachment: that ligand binding to integrins causes a mechanical connection to the underlying cytoplasmic actin network Ingber, ; Yamada and Miyamoto, ; Burridge et al. We were nevertheless surprised by the rapidity with which a visually dramatic accumulation of actin occurred and the strength of the connection between laminin and the cell surface.
The adhesion between the cell and the tip of the needle shown in Fig. There is no reason to assume that this represesents the upper limit of adhesive force, rather it was the largest force we could measure under the experimental circumstances. Intriguingly, the cortical actin accumulation at the needle could be dragged across the cell with only modest forces, again suggesting a lack of strong connections between the actin cortex and the underlying cytoplasm.
The ability to pull out cellular extensions with larger forces indicates that under some conditions the connections between the extracellular adhesion protein i. In this regard, Choquet et al. However, these tentative conclusions will require further study as the mechanical aspects of cellular attachment are currently less well understood than the underlying chemistry. In control experiments for laminin-treated needles, we found that polylysine-treated needles, but not untreated needles, also caused an accumulation of actin that was capable of being translocated beneath the cell surface.
However, the attachment was not nearly as strong as with laminin. Cytoskeletal involvement and the architecture of polylysine-mediated adhesion has not received much attention, presumably because polylysine is a nonphysiological, nonspecific adhesion protein. Our results suggest that polylysine also causes actin assembly beneath the surface site of adhesion. With our method of applying tension, however, polylysine-mediated adhesion was considerably weaker, we would estimate by an order of magnitude, than laminin-integrin adhesion.
Our results are difficult to reconcile with a tensegrity model of the cell in which sustained tension in the actin network is supported in part by compression of underlying MTs Heidemann and Buxbaum, ; Ingber, As implied by the origin of its name, tensegrity structures are those in which the tensional elements behave in an integral fashion to provide the large-scale shape of the structure; i. Classic tensegrity structures also involve intimate and distributed connections between the overlying tensile network and internal compressive struts so that changes in the network produce changes in the array of struts.
Yet we repeatedly observed that both the actin- and MT-based cytoskeleton responded only locally to either passive deformations or those in which the needle was attached to the actin cytoskeleton. In view of the direct evidence that some of the actin-based tension is borne by attachments to the dish e. For this reason, we manipulated both well-spread cells Figs. We observed no differences in the cytoskeletal behaviors of rounded cells or well-spread cells in response to manipulation.
Regardless of apparent degree of spreading and presumably attachment, the cytoskeletal responses to deformation were surprisingly local. In this regard, Thoumine and Ott conducted rheological measurements on essentially spherical chick embryo fibroblasts. Although no cytoskeletal inferences could be drawn, the overall behavior of their highly rounded cells were entirely similar to those reported here for fibroblasts of varying degrees of spreading: highly elastic responses over the time scale of seconds, viscoelastic responses over 5—15 min, and an active contractile response with adhesive conditions.
In aggregate, these results indicate that fibroblasts do not change their qualitative, and possibly quantitative, mechanical properties depending on their shape or degree of spreading, as would be predicted of a tensegrity structure. The use of GFP-technology to visualize the cytoskeleton of living cells in real time adds an additional dimension to cellular rheology and cytomechanics. This technology makes it possible to directly observe the behaviors and interconnections of cytoskeletal elements in response to changes in cell shape and activity.
We hope to exploit this technology in the future to better understand the cytoskeletal mechanics underlying cell crawling and the slower changes of cell shape change. We thank Phillip Lamoureux for expertly providing the large numbers of calibrated glass needles required for this study and David Mooney, Donald Ingber, and Fred Grinnell for stimulating discussions. Finally, S. Heidemann is most grateful to the members of the Matus lab for hospitality above and beyond the call of sanity. Heidemann , which made this collaboration possible.
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This work was also supported by the Friedrich Miescher Institute and by a grant S. Address correspondence to Dr. Fax: E-mail: ude. The first two authors contributed equally to this paper. National Center for Biotechnology Information , U. Journal List J Cell Biol v. J Cell Biol. Steven R. Robert E. Author information Article notes Copyright and License information Disclaimer. Received Oct 22; Revised Mar 3. Copyright notice. This article has been cited by other articles in PMC. Abstract Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape.
Keywords: cytoskeleton, cytomechanics, biorheology, integrins, cell shape. Mechanical Deformations and Force Measurements Cells were deformed by poking and prodding them with glass needles that had been calibrated to determine their bending constant, i. Results The Nuclear Region and Actin-rich Peripheral Regions Are Generally Elastic The nuclei of REF cells as well the actin cytoskeleton show almost purely elastic behavior in response to all manipulations that deformed their arrangement or shape.
Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Response of Fibroblasts to Towing Forces via Integrin-mediated Attachments Integrin-mediated cell attachment to a substratum also mediates mechanical attachments to the cytoplasmic actin cortex and has been shown to play a major role in regulating cytoplasmic architecture, cell shape, and motility Ingber, ; Hynes, ; Yamada and Miyamoto, ; Burridge et al.
Figure 8. Figure 9. Figure Discussion Two fundamentals for a mechanical understanding of any complex structure, e. Acknowledgments We thank Phillip Lamoureux for expertly providing the large numbers of calibrated glass needles required for this study and David Mooney, Donald Ingber, and Fred Grinnell for stimulating discussions. Footnotes Address correspondence to Dr. References Adams DS. Mechanisms of cell shape change: the cytomechanics of cellular respone to chemical environment and mechanical loading. Anderson, and W. Springer Verlag, Berlin.
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