Topographically induced direct cell mechanotransduction

Medical Engineering & Physics 27 (2005) 730–742

Topographically induced direct cell mechanotransduction Matthew J. Dalby ∗ Centre for Cell Engineering, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK Received 7 February 2005; accepted 11 April 2005

Abstract This review is designed to introduce the cytoskeleton and then discuss how mechanical forces may be transduced to the cell nucleus. In addition to this, it also tries to explain current thinking as to how the nucleus turns these mechanical cues into gene changes and is especially interested in mechanotransduction arising from topographically induced morphological changes, specifically nanotopography. Thus, this review also describes cell responses to topography. © 2005 IPEM. Published by Elsevier Ltd. All rights reserved. Keywords: Cytoskeleton; Mechanotransduction; Tensegrity; Percolation; Topography; Nanotopography

1. General mechanotransduction

2. The cytoskeleton

It is becoming clear that mechanical forces are important in tissue and cell function. Mechanotransduction has obvious roles in blood pressure regulation, vascular response to fluid shear stress, remodelling of bone, maintenance of muscle and perception of touch and sound [1]. It is likely that cell growth, migration and gene expression are influenced by mechanotransductive events in most, if not all, cell types [2]. Mechanotransduction can be roughly divided into two categories: direct and indirect. Direct utilizes conformational changes in the cytoskeleton to pass information about the extracellular environment (shape, flow, strain, etc.) to the nucleus as mechanical signals. Indirect mechanisms use chemical messaging (G-proteins, kinases, ion channels) to relay information to the nucleus through signalling cascades. These mechanisms are highly entwined and thus complex. Here, this review tries to simplify the direct mechanotransductive pathways i.e. study signalling using changes in focal adhesion and cytoskeletal conformation and then to relate these to cell response to topography.

2.1. Introduction to the cytoskeleton

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First, as the cytoskeleton is critical to direct mechanotransduction, the reader must have a basic understanding – thus, if you are unfamiliar, then two good reference textbooks – Alberts et al. ‘Molecular Biology of the Cell’ [3] and Cooper ‘Cell’ [4] are recommended. Basically, The cytoskeleton is a network of protein filaments extending through the cell cytoplasm within eukaryotic cells. The cytoskeleton is of fundamental importance in the control of many aspects of cell behaviour, movement and metabolism including proliferation, intracellular signalling, movement and cell attachment. The three main cytoskeletal components are microfilaments (actin), microtubules (tubulin), and intermediate filaments (e.g. vimentin, cytokeratin, desmin depending on cell type). 2.1.1. Microfilaments (actin) There are two types of actin bundles, involving different bundling proteins. One type of bundle, containing closely spaced actin filaments aligned in parallel, supports projections in the cell membrane (filopodia or microspikes involved in cell sensing, lamellipodia involved in cell crawling). In these bundles, all the filaments have the same polarity,

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M.J. Dalby / Medical Engineering & Physics 27 (2005) 730–742

with their positive ends adjacent to the plasma membrane. The second type is composed of more loosely spaced filaments, and is capable of contraction; these are stress fibres (Fig. 1A). Most cell surfaces have protrusions involved in movement, phagocytosis, or absorption of nutrients. Endothelial cells have finger-like projections called microvilli. The microvilli form a layer on the apical surface, the brush border (approx. 100 microvilli per cell), increasing surface area for absorption. Stereocilia are specialised microvilli found on auditory hair cells, and detect sound vibrations. The ability of cells to crawl across substrate surfaces is a function of the actin cytoskeleton (by pulling (see Section 2.1.2) against focal adhesions (see Section 3.5)). 2.1.2. Myosin Whilst myosin is not generally considered as a cytoskeletal element, it is important for actin function and thus should be considered here. Myosin II is always present in cells where actin filaments form contractile bundles in the cytoplasm. The best example of actin and myosin II interaction is in muscle cells. Globular myosin heads bind actin filaments and hydrolyse the ATP. Each actin molecule in an actin filament is capable of binding one myosin II head to form a complex. The myosin II heads thus ’walk up’ the actin filaments in a single direction, towards the positive end resulting in a contractile action [3].

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2.1.3. Microtubules (tubulin) The microtubules provide a system by which vesicles and other membrane bound organelles may travel. They also help regulate cell shape, movement and the plane of cell division. Microtubules, composed of tubulin, exist as single filaments that radiate outward through the cytoplasm from the centrosome, near the nucleus [5]. Microtubules emanating from the centrosome (microtubule organising centre (Fig. 1B)) act as a surveying device that is able to find the centre of the cell. 2.1.4. Intermediate filaments Intermediate filaments are tough protein fibres in the cell cytoplasm. They are constructed like woven ropes around the nucleus, extending out in gently curving arrays to the cell periphery (Fig. 1C), and they are particularly dominant when the cells are subject to mechanical stress. The intermediate filaments are classified into four broad bands: type I are keratin based proteins, type II includes vimentin and desmin, type III includes neurofilament proteins and type IV are nuclear lamins. In cells of mesenchymal origin vimentin is the main intermediate filament protein. The filaments provide mechanical support for the cell and nucleus [5,6]. 2.1.5. Tensegrity It is becoming widely accepted that the cytoskeleton is critical in the cell’s action and reaction to external mechanical stimuli. Researchers have tried to develop models of how

Fig. 1. Fluorescent cytoskeletal labelling. (A) Actin stress fibres, contractile stress fibres are seen throughout the cells. (B) Tubulin microtubules—note the microtubules radiating from the microtubule organising centre just below the nucleus. (C) Vimentin intermediate filaments—note they completely surround the nucleus. (D) Vinculin labelling of focal adhesions.

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the cytoskeleton may act to transduce the mechanical stimuli. Perhaps the most convincing thus far is the tensegrity theory [7]. Ingber proposes a model where microfilaments (actin) and microtubules (tubulin) form a tensegrity structure in the cell, with microtubules acting as load-bearers and microfilaments under tension. In this model, intermediate filaments also act in a tensile manner and are associated with microtubules [8]. Tensegrity (tensional integrity) structures are stabilised through continuous tension, rather than compression (such as an arch) [9]. All agree that actin/myosin motors are used to apply prestress to the tensegrity structure and that microtubules form the compressive element, but as will be discussed later, proof has been forwarded that intermediate filaments may, in fact, form the tensile element of the tensegrity structure [10,11]. In support of the tensegrity theory, biophysical studies with isolated microfilaments and microtubules showed the microfilaments are better at resisting tension, whereas microtubules are better at withstanding compression [12]. When in solution, microtubules are straight (bent in living cells), microfilaments are bent (straight in living cells) and intermediate filaments highly entangled (bent in living cells) [13–15]. These observations are consistent with engineering rules stating that tension straightens and compression bends—again implying a compressive role for microtubules and tensional roles for microfilaments and intermediate filaments. Ingber describes the general role of intermediate filaments as providing stiffness to the cell. They interconnect frequently with along microtubules, microfilaments and the nuclear surface, hence acting as suspensory elements—they have certainly been linked to shielding the nucleus from sudden shock (see Section 3.3) [11] (Fig. 1). In his description of tensegrity, Fuller stated that there are two broad classes of tensegrity—pre-stressed and geodesic [9]. In cell biology, we mainly discuss pre-stressed (Fig. 2A), with actin–myosin interactions providing the pre-stress. However, examples of geodesic tensegrity may often be seen in cell biology (Fig. 2B). Here, structural members are orientated along minimal paths. Additional contributions to prestress may come from distension through focal adhesions

(possibly acting on cortical actin (see Section 3.3)), osmotic and hydrostatic forces (pressure exerted by the cytoplasm on the cell membrane). Later, using a rather clever method of microindentation with an atomic force microscope and cytoskeletal poisoning, Charras and Horton proposed that whilst microtubules and intermediate filaments were required for cell response to mechanical strain, microfilaments were not. This study agrees that microfilaments act as cellular ‘guy-wires’, anchoring the internal tensegrity unit and applying pre-stress to it—as has been described above, but implies that it intermediate filaments form the tensile part of the tensegrity structure [10] (Fig. 3). As well as the many biochemical studies on cellular mechanotransduction through the cytoskeleton, there have been a number of theoretical manuscripts looking at mechanotransduction. Perhaps the most recent is by McGarry and Prendergast [16]. They describe the development of a finite element model of a eukaryotic cell adherent to a substrate incorporating cellular components likely to be structurally significant (nucleus, cytoplasm, membrane components, cytoskeleton). Their results concurred that the cytoskeleton plays a critical role in determining the structural properties of the cell. They further showed that the cell cytoplasm was the next most important component of the cell, offering – after the cytoskeleton – the largest resistance to deformation. Their results – and those of other theorists – tie in well with the tensegrity model [16,17]. Ingber has recently reviewed the topic of tensegrity in two commentaries, and these form two excellent reference texts [11,18]. 2.1.6. Percolation Percolation describes a network system based on interconnected (or percolation) networks for transducing mechanical signals (Fig. 4). Forgacs writes in his 1995 [19] review of percolation that it can be likened to a spiders web. He describes how a spider receives information on victims through the web network—even if there is slight damage to the web. However, if there is too much damage, the spider cannot ac-

Fig. 2. Tensegrity would require either pre-stress or geodesic actin arrangement. (A) A cell with contractile stress fibres which could apply the pre-stress to the cells. (B) Geodesic actin arrangement.

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Fig. 3. One possible tensegrity arrangement. Here, microtubules are under compression, microfilaments under tension in the tensegrity structure. Intermediate filaments interact with the tensegrity structure and lamins in the nucleus.

cumulate the information it needs—i.e. if the network ceases to be interconnected (or just before). This demonstrates redundancy (i.e. that a message may travel through the web by more than one route) and speed delivered through an interconnected network. Percolation can be applied to many situations (see [19]) such as polymer gel formation, forest fire propagation, spread of infectious diseases. Basically, for percolation you need enough components (e.g. cytoskeletal fibres) to form a critical concentration (percolation threshold). If the concentration is high enough, mechanical signals will be sent through the cytoskeleton to the nucleus, a fire would be able to spread from tree to tree and a disease spread from person to person with redundancy. Forgacs writes that biological networks are dynamic in nature, changing their configurations by removing links and establishing others, but at any instant, there is a well-defined distribution of cluster sizes [20]. Notably, the cytoskeleton resembles a percolation network spanning from membrane to nucleus. Forgacs continues by suggesting that whole cellular environment may function through percolation, with the interconnecting extracellular matrix (e.g. collagen [21], fibronectin, etc.) connected to the cytoskeletal network through focal adhesions and the cytoskeleton connected to the nucleus through the cytoskeletal intermediate filament network directly to the nuclear intermediate filament network (lamins) (see Section 3). As well as redundancy in mechanical signal propagation, there would be redundancy in chemical mechanotransduction, with changes in cytoskeletal configuration resulting in changes in cytoskeleton-linked g-protein and kinase signalling (Juliano and Haskill [22] and then Burridge and Chrzanowska-Wodnicka [23] review signalling mediated by focal contacts).

Forgacs continues to say that there are several problems with the tensegrity theory. Firstly that tensegrity structures contain only the absolute minimum in terms of structural elements that are needed to achieve the specified stability or load bearing capacity—i.e. they need strict rules for stability. They also have less redundancy than percolation structures – redundancy is a must for reliable signalling [24]. The next problem is effective signal transduction through pre-stressed structures. Since pre-stress will constrain the deflection of each fibre, it will dampen the propagation of a mechanical signal. However, Ingber does add some flexibility to the tensegrity model by allowing the fibres to be of differing lengths—Forgacs writes that this makes the structures more percolative. However, percolation models do have structures similar to tensegrity, structures that retain their rest, or pretension even when the network is uncoupled from the cell wall [19,25]. It should be noted, however, that whilst it is almost certain that the cytoskeleton forms an interconnected network through which mechanical signals can be passed from focal adhesions to the nucleus, tensegrity and percolation may only be a part of the story and the whole picture could be far more complicated—comprising pre-stressed structures, geodesic structures and more random percolation structures. These will all in turn be influenced by substratum stiffness, hydrostatic cytosol pressure and a myriad of other influences.

3. The cytoskeleton and direct mechanotransduction 3.1. Direct transduction of mechanical signals Maniotis et al. [26] reported that in reaction to tension, the intermediate filament cytoskeleton re-orients, the nucleus distorts and that the nucleoli rearrange along the applied axis.

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Thus, it was concluded that the nucleus is mechanically integrated within the physical entity of the cell via intermediate filaments and that active or passive cell extension can lead to passive nuclear deformation. Another study has shown that microtubules specifically invade regions of applied mechanical stress [27], tying in with previous work that demonstrated that the number of kinetochore microtubules increased with applied tension to the cell [28]. It is also thought that endothelial cells lining the walls of arteries respond to changes in hemodynamic shear stress through cytoskeletal adaptation [29], and that smooth muscle cell microtubule assembly is also linked to external forces [30]. Microtubules have also been associated with the proteins (G-proteins) of the Rho family, with increased contractil-

ity (characterised by formation of actin stress fibres and focal adhesions) being shown to occur after depolymerisation of microtubules [31]. Rho has also been shown to be required for the selective stabilisation of microtubules. Recent evidence also indicates that effects of externally applied strain on the distribution and activation of the Rho family require changes in the state of microtubule polymerisation [32]. Endothelial intermediate filaments have been shown to become significantly displaced at the onset of shear stress, with average filament displacement magnitude becoming greater with increased height in the cell and becoming magnified in down-stream regions of the cell [29]. Mapping of intermediate filament strain by Helmke et al. [33], showed strain

Fig. 4. Representation of percolation through an interconnected cytoskeletal network. Note the inbuilt signal amplification, or redundancy. Also note that the network can be rearranged during signal transduction. Adapted from [19].

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focusing close to the basal cell membrane, with localized strain magnitudes in areas where the intermediate filaments were dense. This indicates that intermediate filament local mechanics are determined principally by mechanical interactions with specialised features such as focal adhesions. Furthermore, local alterations in mechanical properties at sites of intermediate filament strain focusing also result from interactions with cross-linking proteins and other cytoskeletal elements [34]. In addition, Helmke et al. [33] showed peaks in strain magnitude in the intermediate filaments in response to shear stress were often located at the nuclear boundary, or in perinuclear regions, again suggesting that extracellular forces can act directly on the nucleus and again suggests a role for intermediate filaments in direct mechanotransduction. Thus, it is seen that microtubules and intermediate filaments are considered to play the main roles in direct mechanotransduction from the plasma membrane to the nucleus, efficiently sending tensile signals to the nucleus [10,26–30,35,36]. Compressive forces, however, seem rather more difficult for transmittal by cytoskeletal transduction due to the value of Youngs’ moduli of the cytoskeletal elements. Perhaps, however, compressive forces may be transmitted via tensegrity structures. Ingber writes that the feature of the tensegrity model that most troubles investigators is the presence of compression struts within the cell, with some researchers arguing that the cytoskeleton is like a network of ligaments, tendons and muscles without any bones [18,37]. He then writes that it seems likely that compression may be transmitted by a partnership of focal adhesions and microtubules. It may be that intermediate filaments act as lateral supports to prevent compressive microtubule buckling [11,38]. Recently, in an excellent article in the Journal of Cell Science, Dahl et al. [39] showed the nuclear lamina network to be highly elastic, but to have a compression limit, suggesting that the lamins act as a molecular shock absorber. Using dextrans to swell the nucleus and micropipettes to try and compress the nucleus they showed that the nuclear envelope is stiffer and more resilient than the cell plasma membrane. Their results indicate that the lamina forms a shell of interconnected rods that is extensible but limited in compressibility (Fig. 5). They conclude that the nuclear envelope offers a degree of protection for the DNA inside, while being flexible enough to transmit some forces when the cell’s shape changes. This is clear evidence for the nucleus being able to detect tensile forces through nuclear deformation rather than contraction. Studies with chondrocytes, however, have shown changes in nucleus morphology and cytoskeleton in response to compression of the cell body, and state that mechanotransduction via nuclear deformation may be important in mechanical conditioning of tissue engineering constructs [40,41]. These changes in shape could perhaps be by tension at 90◦ from the direction of compression, thus resulting in deformation rather than compression.

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Fig. 5. Lamin arrangement during nucleus swelling. The native lamina is naturally in a compressed state. This can be expanded to an orthogonal arrangement. Adapted from [39].

3.2. The cytoskeletal—nucleus connection Intermediate filaments seem to be particularly important in mechanotransduction as they form part of a continuous network of cytoskeletal and nucleoskeletal filaments linking the nuclear membrane directly with the plasma membrane [42]. This supports the reported evidence for collaborative cytoskeletal transmission of signals. Getzenberg [43] writes that tissue specific gene expression is intriguing as regulation by single transcription factors cannot be explained simply by DNA sequence, i.e. the same transcription factor interacting with DNA of different cell types results in different gene expressions despite the similar genome in all cells. Getzenberg then suggests the three-dimensional organisation of the genome, structural components of the nucleus and nuclear matrix in different tissues may alter specific gene regulation. In 1996, Bloom et al. [44] provided microscopical evidence for intermediate filaments (desmin) transmitting stress messages to chromatin in mechanically stressed myocytes. Their data allowed them to hypothesize that stretch associated increases in sarcomere length change the special arrangement of the desmin–lamin filament network linking Z-discs to chromatin. This then alters the configuration of the laminbound chromatin, hence positioning it to permit transcription of previously inactive genes. Whilst these findings fit well with our hypothesis, it must be remembered that these results are in muscle cells, a distinct cell type with sarcomeres, which are contractive units rich in actin and myosin. A sarcomere lies between Z-disks. See Fig. 6 which represents their theory.

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Fig. 6. How sarcomere length may alter chromatin positioning in the nucleus. Adapted from [44].

lamin A/C gene (LMNA) causes increased nuclear deformation, defective mechanotransduction and impaired viability under mechanical strain [49]. Aberrations in nuclear morphology, more so than in cell morphology, can identify cancerous tissue [50]. A number of diseases have been linked to mutations in A-type lamins of the nuclear inner membrane—such as Emery–Dreyfuss muscular dystrophy [51]. Heterozygous mutations in lamin B receptor cause the dominant Helger–Puet anomaly of white blood cell nuclear shape, homozygous mutations are linked to bone and cartilage disorders [52]. For such diseases, mechanisms such as defects in tissue specific gene expression coupled to mechanical weakness of the nuclear envelope have been proposed [53–55]. Thus, it is seen that nuclear shielding and lamin function are critical for cell function and it is also shown that the nucleus is highly mechanosensitive. 3.4. Cortical cytoskeleton

The other cytoskeletal elements are important to nuclear positioning, but do not appear to have the same direct – and continuous – connection. At the start of their 1998 commentary, Reinsch and G¨onczy [45] state that whilst the eukaryotic cell is represented with a centrally located nucleus. It is not always appreciated that this position depends on active mechanisms which move the nuclei and maintain the correct position. This is considered to be a microtubule dependant process, with the nucleus closely associated with the microtubule organising centre (Fig. 1B, the organising centre can be seen with emanating microtubules just below where the nucleus would be), with the nucleus following the organising centre. Very little is known about actin involvement in nuclear positioning. There is evidence, however, that it is important, but again indirect [46]. 3.3. Stress shielding A problem associated with observations of nuclear deformation due to mechanotransduction is stress shielding, whereby the nucleus is protected from sudden shock by the intermediate filament cytoskeleton; thus the cytoskeleton can act as a damper as well as a transmitter, further complicating the situation! As with mechanical conditioning, grooved topography can be used to align cells [47] and their nuclei [48]. It is noted that for cells experiencing contact guidance on the grooves, the nuclei are less deformed even though the overall morphology of the cells showed that they stretched themselves considerably. This difference in nuclear and cellular extension shows that the nucleus, although integrated in the cytoskeleton remains to some extent shielded from the overall stresses, which probably develop within the cell. This could be in part due to lamins, for example, which support the nuclear membrane and maintain the shape and integrity of the nucleus. It has been shown that removal of the

The cortical cytoskeleton is important for maintaining the cells relationship with its mechanical surroundings. The cortical cytoskeleton provides strength to the cell membrane, thus removing the need for a cell wall and hence loss of mechanosensitivity. The cortical cytoskeleton also facilitates rapid changes in cell shape, allowing, in fact, cells to deviate from spherical [56]. Cells do, in fact, have a huge excess of lipid-bilayer membrane, which is folded into microvilli, filopodia, membrane folds and caveolae. This, and the expandability of the cytoskeleton allow cytoskeletal expansion in response to mechanical stimuli without the need for membrane insertion. Hamhill and Martinac write an extensive review on how the cell membrane is involved in mechanotransduction [56]. 3.5. Focal adhesions The focal adhesion, a way of describing the cell ‘feet’ (Fig. 1D), is the likely starting place for mechanotransductive events, and thus needs special consideration. Transmembrane integrin proteins connect the cytoskeleton to the extracellular matrix (ECM). They transmit mechanical stresses across the plasma membrane and represent two way signal mediators; transmitting tractional forces developed in the cytoskeleton to the ECM and passing stresses applied in the ECM to the cell [2]. It must also be considered that as part of a focal adhesion complex linked to G-proteins and tyrosine kinases, integrins are also able to facilitate the transduction of physical signals into chemical signals [57]. Initially, integrin interaction with ECM ligands is independent of force, but this quickly changes. Swift attachment to actin cytoskeleton includes a 2 pN talin-dependant increase in adhesion strength [58]. Initial adhesion initiates cdc42 and Rac G-protein pathways resulting in the formation of filopodia and lamellipodia. This is the start point of cells exertion of tractional forces, with lamellipodia producing a 0.8–0.9 nN/␮m2 force on the ECM [59].

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In fact, the whole transition from nascent close contact to focal contact to focal adhesion appears to be force dependant, with integrins taking a leading role. A recent review by Katsumi et al. [1] gives excellent in-depth instruction on this subject. Briefly, the transition from close contact to focal contact requires recruitment of paxillin, phosphoproteins and vinculin. Vinculin levels at adhesion correlate in a linear manner with tractional force; it has further been shown that in order for vinculin recruitment, tension needs to be developed between the ECM (in the experiment, a fibronectin coated bead was used) and the adhesion [59]. Furthermore, loss of cytoskeletal tension leads to vinculin dissociation from the adhesion. This shows that focal contacts require and exert tension for formation and stabilisation [1]. Through Rho activation and increases in cellular contractility (through myosin light chain activation), mature focal adhesions are developed. This occurs largely through integrin gathering via increased force generated by the cytoskeleton at adhesion sites [60]. In order to support the larger focal adhesions, the substrate surface must be suitable stiff; this again, shows that focal adhesions need to both transmit and require tension to stabilise. Considering substrate stiffness, cells experience durotaxis, where cells on flexible substrates has increased migration compared to those on stiff substrates. Focal adhesion kinase (FAK) is thought to be crucial to this process. Phosphorylation of FAKs’ tyrosine 397 is sensitive to adhesion to rigid substrates [61]. FAK is also involved in regulation of Rho and Rac, suggesting FAK may be implicated in durotactic cell movement [62,63]. (Note: In this issue, Schwarz and Bischofs write a fascinating and in-depth review of focal adhesions and discuss substrate stiffness in depth.) Thus, the ability of a cell to form mature focal adhesions will affect the cells ability to form cytoskeletal tensegrity structures, and thus transmit forces to the nucleus. As well as altering the cells ability to deliver direct, mechanical forces to the nucleus via the cytoskeleton, the modulation of focal adhesion shape will change indirect signalling by integrins within the adhesions. These signals are transmitted to the nucleus via G-protein and tyrosine kinase cascades (see [23] for a review on integrin mediated signalling).

4. Centromere positioning Thus, a review of the literature shows that the cell cytoskeleton is vital in mechanotransduction leading to nuclear deformation and changes in cell metabolism, proliferation and differentiation. This has been best described in situations of applied forces (see [64,65] for reports on biochemical changes to mechanical stimulation). Further to this, it was shown by Curtis [66] that mechanical tensions causing the alignment of cells can rearrange centromeres through nucleus deformation. While these experiments describe applied mechanical forces and topography presents encountered mechanical

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Fig. 7. The cell cycle. The dots within the cell nucleus represent the number of centromeres per chromosome at each phase of the cell cycle.

forces (i.e. stationary objects), it is possible that alignment by topography may also lead to changes in centromere arrangement and subsequent down-stream effects. During interphase (Fig. 7), genes in the cells DNA are transcribed and translated into proteins essential for cell function. It has traditionally been thought that during interphase the chromosomes are randomly arranged. Highly convincing evidence, however, is being put forwards by the HeslopHarrison group in Leicester showing that chromosomes, in fact, have a relative consistency of position [67–71]. In fact, it appears that nuclear architecture may be organised and that fine changes in chromatin position may be important in genome regulation. Movements due to, for example, the coming together of enzyme complexes and promoter regions, chromosome pairing and chromatin becoming accessible to polymerases are fundamental to correct cell function [72]. A number of early investigators have observed filaments (possibly of DNA) connecting interphase chromosomes [73–75]. Later, Fey et al. [76] showed that in interphase cells, the nuclear matrix appears to interconnect different nuclear components, such as nucleoli, to each other and the surrounding cytoskeleton. More recently, it has been shown that the human endothelial cell genomes behave as a continuous, elastic structure and it has been suggested that DNA contributes to the cells mechanical continuity in interphase and mitosis [26,35]. Cellular cytosketetal arrangement and nuclear morphology may be important in understanding cellular response to surfaces (Fig. 8).

5. Topography 5.1. Introduction to cell response to nanotopography It has been know for almost one hundred years that cells will react to the topographic structure of their environment, and in 1952 Weiss and Garber first used the term contact

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Fig. 8. Nanotopographies. (A) Polymer demixed topography with 13 nm high nanoislands (image courtesy of Dr. S. Affrossman, University of Strathclyde, UK); (B) 160 nm high nanocolumns produced by colloidal lithography (image courtesy of Dr. D. Sutherland, Chalmers University, Sweden); (C) 120 nm diameter nanopits produced by electron beam lithography (image courtesy of Dr. N. Gadegaard, University of Glasgow, UK).

guidance [77–79]. Further from this, the phenomenon of cell guidance has been well documented, especially in reaction to grooved topography (in many combinations of widths and step heights) [47,80–83]. Cell guidance occurs when cells are inhibited from crossing a step, and rather follow the step edge becoming elongated and thus, highly polarised [47]. Many different cell types have now been shown to respond to topographies of varying shapes and sizes [48,81,84–97]. It is thought that topography can influence cellular responses from initial attachment and migration through to differentiation and production of new tissue [98–100]. It has indeed been shown that explanted and ruptured tendon can be repaired in an ordered manner using grooved topography as a cell guide [84]. It is noted, however, that there are many intermediate inter- and intra-cellular processes occurring during tissue repair, and it is important to understand these processes. Researchers have first looked at cell cytoskeletal response to topography, with observations of actin microfilament alignment to grooved substrates and differential organisation to other surface features [80,101]. As has been discussed, the formation of adhesion plaques and development of cell cytoskeleton is of great importance for subsequent cell activity. Integrin proteins located within the adhesion plaques, and actin cytoskeleton, which is linked to focal contacts, are involved in signal transduction. The expression of focal contacts and the organisation of stress fibres in reaction to a material has long-term effects on cell differentiation [22,23,102–105]. With advances in technologies, such as electron beam lithography [100] (please see our recent preview in European Journal of Cell Biology [106] for a basic explanation (written for biologists by biologists!) of electron beam lithography and other miniaturisation techniques such as colloidal lithography and polymer demixing), biologists are now able to delve into cell response to the nanoworld. It is rapidly becoming apparent that nanoscale topography may be of great importance when considering how cells respond to their environment, and certainly appears to alter significantly the morphology and cytoskeleton of endothelial cells, epithelial cells, epitenon cells, macrophages, osteoblasts and fibroblasts [89–92,101,107–113].

It should be noted that every cell type that has been tested has responded to both nano- and microscale topographies. 5.2. Topographical regulation of gene expression A 1718 gene microarray was used to look at gene expression profiles for fibroblasts cultured on nanoislands (produced by polymer demixing), nanocolumns (produced by colloidal lithography) microgrooves (produced by photolithography). The results demonstrate that contact reactions of cells can results in large gene regulatory changes. The first topography looked at was 13 nm high nanoislands (Fig. 8A). Up-regulation of genes involved in cytoskeleton, proliferation, transcription, translation, extracellular matrix production and inter- and intra-cellular signalling were noted. In total, 584 up-regulations were reported [48]. Whilst there are a growing number of reports focusing on adhesion, morphology and cytoskeleton on nanotopography [85,90,91,93,94,101,109–111,114–116], the 13 nm islands are the only shape that are nano in all dimensions that have been tested by microarray in the literature [97]. However, for recently published work looking at microarray data from fibroblasts cultured on the nanocolumns shown in Fig. 8B see [117]. Whilst on the 13 nm islands, which increased cell spreading and cytoskeletal formation, the consensus of array data was up-regulation, on the nanocolumns, which decrease cell spreading and cytoskeletal organisation, the average shift showed down-regulation. As with the 13 nm high island nanotopography, reproducible changes were noted in the areas of cytoskeleton, proliferation, transcription, translation, extracellular matrix production and inter- and intra-cellular signalling. Changes in cell response in these precise areas have almost certainly arisen from changes in cell spreading and morphology imposed by substrate shape. More convincing evidence comes if microarray results are compared to results obtained on microgrooves with 10 ␮m width and 200 nm depth, where fibroblasts experience contact guidance and are forced to take on an elongated morphology, following the grooves. The gene response to this large change in shape was again, in the same areas of cytoskeleton, pro-

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liferation, transcription, translation, extracellular matrix production and inter- and intra-cellular signalling. The changes were, however, large when compared to the cells on the 13 nm high islands which were more spread, but maintained a normal fibroblast morphology and on the nanocolumns which were less spread, but maintained a normal fibroblast morphology [48,97]. This suggests that the more a cell is deformed by topographic guidance, the larger the genome response.

6. How does topography exerts mechanotransductive effects on cells Tying in all the above observations on nucleus/ cytoskeleton/focal adhesion/topographic interactions, the author and colleagues put forward a hypothesis of selfinduced mechanotransduction, where surface topography alters nucleus morphology and the position of the chromosomes, leading to changes in the probability of gene transcription. This is really an extension of the HeslopHarrison hypothesis. A rational approach to resting this hypothesis would be to observe changes in chromosome positioning in the interphase nucleus and work has started by looking at chromosome 3 (C3) positioning in fibroblasts on nanocolumns (Fig. 8B). The initial results are very promising and are best explained in a preview [106]. Chromosome paints designed to hybridise to C3 centromeres (Fig. 9) were used and the authors have calculated nuclear shape. In G2 of the cell cycle, the cells have synthesised new DNA, thus the cell has two sets of DNA ready for mitosis. If cells are imaged in G2 interphase with painted centromeres, two centromeres are visible and the relative distances can be calculated. By averaging a large number of chromosome pairs, to account for different times in G2, details of chromosome position can be derived. Fig. 9 shows chromosome

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3 painted centromeres for fibroblasts on nanocolumns. The centromeres spacing is significantly closer together for cells on nanocolumns compared to those on planar controls. Also, by comparing the measurements for nuclear depth and area, it was seen that the nuclei of fibroblasts on the nanocolumns resembled football-like spheres, whereas on flat controls, the nuclei resembled rugbyball-like ovoids. If it is assumed that spherical is natures ‘relaxed’ morphology, this suggests that the oval nuclei of the well spread cells on the flat substrate are encountering applied forces. If then, the chromosomes in the interphase nucleus are mechanically linked, these forces (although shielded) will be transmitted to the chromosomes and thus change their relative positions. The use of nanotopography to study cell function is at a very early stage, however, the preliminary results appear to show the possibility of direct mechanotransduction (i.e. transmission of forces encountered by cells to the nucleus via the cytoskeleton, for topography, these forces may be generated by changes in spreading). In the case of fibroblasts on the nanocolumns, it appears that the cells cultured on the nano-columns are in a relaxed conformation. Under similar conditions, poor formation of focal contacts on top of the nano-columns was observed and, the cells studied were less spread and had a more diffuse cytoskeleton observed. Thus, tension applied to the nucleus would be reduced, resulting in a rounder nucleus and a reduction of distance between the chromosome centromeres. It would be hard to imagine, in this case, the cells to be experiencing compression due to the problems associated with transmission of forces through a badly organised cytoskeleton. If it is considered that most of the effects observed in cells on topography stem from changes in ability to spread, these results help to support, although not decisively prove, the theory that changing chromosome position during interphase will alter the probability of gene transcription. In order to establish this hypothesis more chromosome centromeres need to be viewed on a larger range of topographies, and for instance chromosomes 13 and 16 during interphase on electron beam fabricated nanopits (Fig. 8C) are presently under investigation.

7. Conclusion

Fig. 9. Changes in centromeres distance (X). Here a fibroblast cell in G2 interphase with chromosome 3 centromere labelled is shown.

The eukaryotic cell is unimaginably complex and remarkable. This review attempts to show a pathway by which topography regulates cell behaviour. However, it is acknowledged that a lot of the theory presented here is just that—theory, some developed by Adam Curtis and then latterly myself after becoming interested in work by Heslop-Harrison, the majority developed by scientists such as Donald Ingber who was first introduced to tensegrity through studying three-dimensional design in a sculpture course [8]. It is also acknowledged that there will be many other mechanotransductive routes i.e. indirect pathways.

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Acknowledgements Matthew Dalby is a BBSRC David Phillips Fellow and is supported though that route. He would like to thank Prof. Adam Curtis and Prof. J.S. ‘Pat’ Heslop-Harrison for help with developing ideas. He would also like to thank Prof. Chris Wilkinson and Dr. Mathis Riehle for their support and discussion.

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