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Model substrates for studies of cell mobility
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Model substrates for studies of cell mobility Muhammad N Yousaf Cells do not live in static surroundings, they exist in highly evolving dynamic environments. During cell adhesion and migration, cells adapt and communicate to their environment by numerous methods ranging from differentiation, gene expression, growth, and apoptosis. How and when cells determine to adhere, polarize, and migrate is important to a number of fundamental biological processes such as wound healing, metastasis, inflammation, and development. In order to elucidate the spatial and temporal mechanisms of these important complex processes on a molecular basis, several research groups have generated model substrates that can present nanopatterned ligands, well-defined gradients of ligands and surfaces that can be dynamically modulated where the interaction between cell and material is defined at the molecular level.
Address Department of Chemistry and the Carolina Center for Genome Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, United States Corresponding author: Yousaf, Muhammad N ([email protected])
Current Opinion in Chemical Biology 2009, 13:697–704 This review comes from a themed issue on Model Systems Edited by Marcey Waters and Dan Appella Available online 26th October 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.
cell polarity, migration, and invasion involve a precise but constantly changing subcellular nano-architecture. To fully understand the complex signaling and cytoskeletal aspects of the cellular nano-architecture during migration requires a multidisciplinary coordinated effort. The long-term goal of this research field is to develop and integrate new surface chemistry and cell biological tools to generate a class of tailored dynamic patterned substrates for a variety of cell adhesion, cell polarization, and migration experiments. The integration of dynamic substrates, molecular surface gradients, new migratory cell lines, and in vivo biosensors that are currently being developed will potentially allow for the complete analysis and quantitation of each step of cell migration from initial engagement with extracellular matrix (ECM) ligands, to localized activation of signaling proteins, to organization and activation of the cytoskeleton, to directional polarization to overall movement of the cell. Integrins, signaling, and migration
Both during local invasion and during metastasis to distant sites, tumor cells breach normal tissue boundaries by utilizing a variety of mechanisms [1,2]. The invading tumor cells gain traction by engaging the extracellular matrix via cell surface integrins, often with preferential utilization of certain members of the integrin family [3,4]. Focused degradation of the ECM via matrix metalloproteinases, adamlysins, and tissue serine proteinases is also a key aspect of invasion [5], as are tumor cell–host cell interactions [2].
Introduction
The integrin family of adhesion receptors plays a vital role in cell adhesion and migration. Not only do integrins provide transmembrane linkages from the ECM to the actin cytoskeleton [6,7], but also they make a profound contribution to the signaling events that take place during these processes [8–10].
Active migration, local tissue invasion, and seeding of distant metastases are all characteristic of malignant cells. These complex cellular events require the integration of information derived from soluble growth factors with positional information gained from interactions with the extracellular matrix and with other cells. The network to determine how a cell responds and moves involves complex signaling cascades that guide the directional and contractile functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner that then dynamically shape the cytoskeleton in specific subcellular regions. Therefore
The human integrin family comprises 18 a subunits and 9 b subunits resulting in two dozen integrin heterodimers [11]. This chemical diversity gives rise to biological complexity, and thus integrins have been implicated in numerous functions including cell–ECM and cell–cell adhesion, organization of actin filaments, signal transduction, cell survival, cell growth and differentiation, and unique roles in developmental processes [12–14]. Despite this complexity, most integrins share two key inter-related functions: firstly, promoting the assembly and organization of the actin cytoskeleton and secondly, regulating signal transduction cascades [15–18].
DOI 10.1016/j.cbpa.2009.10.001
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In addition to integrins and cytoskeletal proteins, focal complexes/adhesions are enriched in a variety of signaling molecules including focal adhesion kinase (FAK), Srcfamily kinases, G-proteins, and elements of MAP Kinase cascades [19]. In addition to influencing Erk and other MAP Kinase cascades, there is a complex set of relationships between integrins and Rho-family GTPases and their downstream effectors. In focal adhesions (FAs)/complexes integrins are connected to various forms of filamentous actin arrays ranging from the loose meshwork of filaments at the leading edge to robust actin stress fibers containing thousands of filaments in the interior fibrillar adhesions. With the advent of live-cell imaging, the temporal distribution of integrin complexes could begin to be addressed [20]. It is now clear that the various adhesive structures dynamically mature from nascent structures at the edge of the cell to the larger interior structures such as FAs. In a migrating cell, there is also loss of adhesion at the trailing edge that involves a combination of regulated proteolysis of integrins and associated proteins, as well as physical tearing. While previous studies have provided a great deal of information about how cells dynamically control the cytoskeleton–integrin linkages in space and time, new methodologies will be needed to advance our understanding of this process. Integrin/ECM interactions
A number of experimental approaches have been used to study the details of integrin/ECM interaction. Many in vitro studies have perturbed integrin function by altering the concentration, composition, and presentation of ECM ligands. Addition of anti-integrin antibodies, or of synthetic (Arg–Glyc–Asp) RGD-based peptides, will often block cell spreading and migration on native ECM. However, these synthetic peptides can also be immobilized on the substrate by various chemistries to provide a more defined surface for integrin-based adhesion or migration [21–23]. These relatively crude studies have been extended by the use of patterned surfaces based on pioneering approaches developed by Whitesides and colleagues [24,25]. From a biological view, gene deletion studies in mice show that integrins are required for many, if not all, cell migration events both in vitro and in vivo [26]. Thus a particularly promising approach will be to combine manipulations of the ECM with genetically or epigenetically modified cells to address specific questions about integrin function in cell motility and tumor cell invasion. There is an intricate interplay among integrins, signaling pathways, and the cytoskeleton that influence the dynamic nano-architecture of both normal and tumor cells, with aberrations in the integrin/signaling connections contributing to the abnormal behavior of tumors. However, new tools, such as the dynamic nanopatterned Current Opinion in Chemical Biology 2009, 13:697–704
surfaces described below, will be needed to fully understand these complex events. Haptotaxis and cell polarity
Haptotaxis, or migration on a gradient of ECM protein, is another form of directed migration. Although the mechanisms involved are largely unknown, haptotaxis is dependent on focal contacts (FCs)/FA formation and turnover. Integrin engagement and clustering leads to the formation of FCs, characterized by the recruitment of paxillin and a-actinin and the activation of a number of tyrosine kinases. Tyrosine phosphorylation is a key event in FC/FA formation, as treatment with the tyrosine kinase inhibitor herbimycin A inhibits the formation of both FAs and stress fibers [27]. FAK is one of the first kinases recruited to FCs [28], and the importance of this recruitment is underscored by the fact that in the absence of FAK, FCs fail to mature to FAs [29]. Subsequent activation of FAK creates binding sites for a number of different signaling and scaffolding proteins, including the Src tyrosine kinase and PI3K, which contributes to FA maturation. FAK activation has also been reported to lead to both increased and decreased Rho activity, depending on the cellular context, through the activities of p190RhoGEF and GRAF, respectively [30]. Decreased Rho activity is thought to be necessary for the formation of FCs, while increased Rho activity contributes to FA maturation, probably through increased actomysin contractility. Disassembly of FAs results from repeated microtubule targeting and occurs in a FAKdependent and Dynamin-dependent manner [31]. How these events contribute to haptotactic migration is not known. Cellular polarity is fundamental to a number of cellular processes and it has long been assumed that morphological polarity contributes to directional migration. For example, in vitro wound healing assays demonstrate that both the microtubule-organizing center (MTOC) and the Golgi apparatus become re-oriented to face the direction of migration. The importance of morphological polarity during migration is highlighted by the fact that treatments that inhibit reorientation also inhibit cellular migration. Leading edge dynamics and adhesion to the ECM also play a role in polarity and migration, but whether the leading edge or adhesion serve to establish polarity or are simply required for migration remains unclear. A promising approach is to integrate the use of a polarity sensor cell line that allows for the assessment of morphological polarity in live cells. Using these sensors, it may be possible to quantitatively describe how cellular polarity is established and maintained on molecularly defined surfaces at the single cell level. Integrating surface chemistry and cell biological tools
In order to generate relevant model substrates for detailed mechanistic studies of cell adhesion, polarity, and cell www.sciencedirect.com
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migration, cell culture compatible surfaces that can present multiple ligands or proteins at defined densities in patterns and in gradients are necessary but remain technically challenging. The key criteria to generate model substrates for studies and applications in cell biology are: Firstly, a sophisticated surface chemistry reaction to chemoselectively immobilize multiple ligands with the ability to precisely modulate the density of each immobilized ligand within patterns and gradients. Secondly, compatible with cell culture conditions where the only interaction between attached cell and material is a ligand–receptor interaction that is surfaces that are inert to nonspecific protein adsorption or cell attachment (biospecific surfaces). Thirdly, a robust fabrication technique to generate complex patterns and gradients. Fourthly, dynamic surfaces, where a noninvasive stimuli can modulate cell behavior in real-time. Fifthly, compatible with live-cell high-resolution fluorescence microscopy techniques. Sixthly, amenable to many analytical techniques in order to characterize interfacial associations ranging from nanoparticles to live cells.
Surface chemistry to immobilize ligands Strategies to immobilize biomolecules onto solid supports is important for a wide variety of applications ranging from the development of small molecule and protein microarrays to model substrates for mechanistic studies of cell behavior [29,30]. There have been several immobilization methods developed to tailor surfaces for a variety of diagnostic and high throughput assays [31]. However, there are very few quantitative strategies to determine the yield and therefore density of ligands immobilized to a surface and none to pattern ligands with control of density. In order to extend the utility of surfaces to generate new types of model substrates to study complex biological processes, it is necessary to develop more sophisticated and flexible surface properties. Surfaces for these applications would require the precise control of ligand density, the ability to immobilize multiple ligands and the in situ modulation of ligand activity. We have recently reported a novel chemical approach to couple ligands, proteins and cells onto an electroactive self-assembled monolayer (SAM) of alkanethiolates on gold via chemoselective ligation where we have precise control of the ligand density on the surface [32]. Surface gradients and dynamic substrates
Current strategies seek to modify surfaces with appropriate protein and peptide ligands in order to promote cell adhesion and maintain cell function and viability [32]. These surface engineering methods, together with methods for patterning the attachment of cells, are now important tools for fundamental studies in biology and for the preparation of chip-based systems in biotechnology [33]. www.sciencedirect.com
Although substantial progress has been made in the modification of material structures to control the adhesion of cells, much less effort has been invested in developing ‘dynamic substrates’ — substrates that can actively modulate the behaviors of attached cells. There also has been tremendous investigation in generating surfaces that present gradients of immobilized ligands. However, there have been no methods to combine these methods to generate dynamic gradient substrates for studying cell polarity and directed cell migration. Dynamic substrates that can alter the presentation of ligands to an attached cell will generate immediate opportunities for studies of matrix-dependent cell adhesion, signaling, migration, and differentiation. Dynamic surfaces would allow for the real-time manipulation and study of cells that currently do not exist with other static substrate strategies. These surfaces provide exquisite molecular control of the ligand–receptor interactions between cell and material that can be potentially controlled at the single molecule level. Dynamic substrates may also be important for generating substrates that can control the spatial and temporal interactions between two or more different populations of attached cells (cocultures, multi-cell arrays, tissue engineering). Ultimately, this work might provide new understandings and improved methods to direct the maturation of stem cells or to control the generation of tissue for medicinal applications. Thus, molecularly defined gradient surfaces and dynamically nanopatterned surfaces with ECM type ligands offer exciting new possibilities for cell biological research. Gold as a model substrate platform
Gold has been the metal of choice for generating model substrates for cell biological studies, which utilize SAMs. These types of surfaces are useful due to their resistance to oxidation, biocompatibility, synthetic flexibility to tailor its properties, conductivity, and its ability to be inert to nonspecific protein adsorption and cell attachment. SAMs on gold present a well-defined surface, which is ideally suited to study cell migration. Ligands can be chemoselectively immobilized to the surface in patterns to probe the subtle effects of ligand affinity and density on cell migration. The ability to tailor materials with SAMs to generate diverse chemical and physical properties has proven to be important for a range of research fields, such as biomedical engineering, organic catalysis, molecular electronics, and cell biology [34]. SAMs of alkanethiolates on gold represent one of the most well-studied surface chemistry systems. The major advantages of SAMs on gold include the inherent conductivity of gold that allows substrate compatibility with a variety of surface characterization techniques. Alkanethiols are also synthetically flexible or commercially available, providing opportunities to tailor Current Opinion in Chemical Biology 2009, 13:697–704
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materials for a range of applications [35]. In addition to the direct synthesis of alkanethiols, a number of convergent interfacial chemoselective strategies including Diels-Alder conjugation [36], Click chemistry [37], quinone and aldehyde coupling [38], Staudinger ligation [39], and Michael addition [40] have been developed to immobilize and present a variety of ligands on the surface.
Live-cell high-resolution fluorescence microscopy Fluorescence microscopy is a major research tool in cell biological investigations [41]. It has been used to image organelles, protein dynamics, and study protein–protein interactions. However, integrating SAMs of alkanethiolates on gold with live-cell fluorescence studies has been difficult due to the fact that gold surfaces efficiently quench fluorescence thus limiting access to important tools in cell biology research (such as fluorescently labeled cell lines, and fluorescence resonance energy transfer (FRET) techniques, and fluorescent dyes). In a series of experiments, we recently overcame this limitation for FRET analysis by examining actin assembly at cell protrusions with an inexpensive customized microscopy setup on gold SAM surfaces [42]. For this analysis, the microscopy presented a major technical obstacle, as fluorescent biosensors in living cells must be used at low intracellular concentrations that produce relatively low light levels; gold model substrates are known to be highly absorptive at the wavelengths used for live-cell fluorescence imaging causing efficient quenching of the fluorescent molecules, significantly reducing fluorescence excitation. We combined a surface chemistry approach with a FRET-based biosensor to investigate the effects of the cell microenvironment on RhoA activation in membrane protrusions. The approach is based on the combination of SAMs of alkanethiolates on gold to generate defined hydrophobic and hydrophilic regions on the model surface, and sensitive live-cell fluorescence microscopy that is compatible with gold surfaces. Another major limitation of SAM gold surfaces is the inability to visualize ligand patterns on the substrate during cell adhesion or directed migration experiments. Although patterning gold with different chemistries is straightforward, observing the patterns on a flat two-dimensional surface simultaneously while using compatible microscopies to observe live-cell fluorescent behavior is currently unavailable. One solution to these challenges is to pattern the gold directly to generate gold/ glass hybrid surfaces [43,44]. However, this methodology functions as a binary system and cannot generate topologies such as gradients. For cell motility studies on planar gold surfaces the directional path of migration must be determined ex post Current Opinion in Chemical Biology 2009, 13:697–704
facto because a ligand pattern cannot be independently visualized during cell migration. To study complex cell behavior and especially directed cell polarity and migration, a molecularly well-defined surface that can be patterned with a chemoselective immobilization strategy, compatible with live-cell high-resolution microscopy while simultaneously visualizing the cell path trajectory would be of tremendous utility. A recent report demonstrated the combination of three new straightforward technologies that synergistically provide complete spatial and visual control of directed cell polarity and migration [43]. The strategy uses: Firstly, partially etched gold surfaces to determine the precise track or path of cell migration trajectory. Secondly, microfluidic lithography (mFL) to pattern gold surfaces rapidly with a variety of alkanethiols for biospecific cell interactions. Thirdly, live-cell high-resolution fluorescence microscopy on gold surfaces to visualize internal organelle dynamics during polarity and migration. To provide ligand control over the surface and characterize the patterning, a chemoselective electroactive SAM immobilization methodology was used to pattern ligands on the partially etched gold surfaces. They showed live-cell directed migration of a mammalian cell line where the nucleus and Golgi are fluorescently labeled to determine the role of cell polarity on motility. The strategy is based on using microfluidic cassettes to both partially etch the gold surface and then install a monolayer on the etched regions for subsequent immobilization of biospecific adhesive peptides. By using a slightly modified inverted microscopy set-up [44], access to routine live-cell fluorescence microscopy is now possible on gold surfaces. These results are the first demonstration of live-cell fluorescence microscopy of directed cell migration on tailored gold surfaces. Total internal reflection microscopy
Total internal reflection fluorescence microscopy (TIRFM) is a technique that has been used for the past two decades to study events at the cell plasma membrane [45]. One early application of TIRFM in cell biology was to study the cell–substrate contact regions of adherent cells on glass coverslips [46,47]. TIRFM is based on the principle of total internal reflection (TIR). TIRFM has been used to study a variety of systems, including single molecule fluorescence [48,49] as well as events and structures at the cell surface such as exocytosis [50] and ion channels [51] and has also been integrated with other methods such as interference reflection microscopy [52] and fluorescence correlation spectroscopy [53]. Until recently, it was thought that gold surfaces precluded the use of live-cell high-resolution fluorescence microscopy to study internal cell structure dynamics. There have been conflicting reports on whether the presence of gold impedes the use of TIRFM in studying cell adhesion www.sciencedirect.com
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to gold substrates [54]. Borisy and coworkers have stated that gold surfaces prevent the use of TIRFM [55], while Arima and Iwata have reported studying the adhesion of cells with fluorescently labeled plasma membranes on SAMs of varying properties using TIRFM [56]. However, the latter did not examine the internal structures of the cell, nor were the surfaces tailored with biologically relevant ligands. In a recent report, the combination of microcontact printing or an electroactive chemoselective immobilization strategy, dip-pen nanolithography, and TIRFM was used to visualize cell adhesion on tailored and patterned gold surfaces [57]. This report is the first to show that TIRFM can be used to visualize internal features of a cell on chemoselectively tailored gold SAM surfaces and will open many avenues for integrating TIRFM with material science and cell signaling dynamics to study cell behavior. Dip-pen nanolithography for cell polarity
Dip-pen nanolithography (DPN) has revolutionized nanotechnology and is based on a scanning probe technique in which an atomic force microscopy (AFM) tip is used to pattern molecules on a surface with precise nanometer scale features [58]. A wide variety of materials have been patterned by DPN for applications ranging from the fabrication of nanostructures to ultra high density DNA arrays [59]. More recently, DPN has been extended to the development of protein nanoarrays for high throughput genetic and biomarker analysis [60]. A major area of research in which DPN nanoarray technology will make a significant impact is in cell biology. For example, the spatial presentation of cell adhesive ligands influences the subcellular nano-architecture of adherent cells and affects their behavior. In particular, the number and size of biospecific interactions between extracellular ligands and cell surface receptors is critical for cell adhesion and cell migration and remain poorly understood and elusive due to the lack of molecularly defined nanopatterned model substrates. By immobilizing the adhesive RGD peptide and cyclicRGD peptide on a single cell nanoarray (400 nm spots spaced 600 nm apart) it was shown that cells had larger and more FAs on the higher affinity cyclic-RGD presenting surface [61]. In order to probe how cell polarity is established with the minimum amount of ligand mediated adhesions the dippen nanolithography (DPN) technique was used to generate single cell symmetric and asymmetric nanoarrays. The polarity of a cell can be experimentally measured through the systematic reorientation and alignment of several organelles in the cell including the nucleus, centrosome, and Golgi apparatus, which can be visualized using fluorescent dyes to map the direction of polarity [62,63]. These experiments are important to the cell www.sciencedirect.com
motility research field because previously there was no method to separate the dynamic processes of cell adhesion, cell polarity, and cell migration without the use of genetic or small molecule manipulations. By using the single cell nanoarrays, cells may adhere to the nanoarray pattern, determine if the conditions are met for establishing polarity but are unable to migrate. These surfaces allow for the analysis of cell polarity and determine how the underlying adhesive environment influences cell behavior.
Conclusion Although gold-based SAMs are the most flexible model system for studying biointerfacial science, there remain severe long-term stability and biocompatibility issues. The thiol–gold bond is thermally unstable and upon long durations of air exposure may oxidize, damaging the integrity of the monolayer [64]. Gold also efficiently quenches fluorescence and has limited optical transparency properties that complicate its use for fluorescent based biosensor or cell array technologies. To address these limitations, there has been intense interest in transferring certain surface chemistries developed for gold to other materials to increase their scope of applications. An alternate model system, monolayers of siloxanes on glass have been intensely studied and utilized for cell based assays and biosensor applications [65]. Not only has glass been shown to be robust, its optical transparency allows for the unimpeded observation of fluorescence. However, synthetic strategies to tailor glass surfaces can be difficult, laborious, and expensive, and characterization techniques are limited due to its lack of conductivity. Another material that shows much promise as a biointerfacial platform for cell behavior studies is indium tin oxide (ITO), a common material widely used for applications in optoelectronics and is found as the transparent conductive coatings in plasma, touch, and liquid crystal displays, as well as solar cells and organic light emitting diode (OLED) devices [66]. Its high conductivity permits the characterization of ITO with a variety of analytical techniques. Unlike gold, the optical transparency of ITO presents opportunities for studies involving fluorescence and, in particular, research in cell biology that requires live-cell high-resolution fluorescence microscopy to study cell behavior. However, the major limitation of ITO is the inability to tailor the surface with a variety of surface chemistries. Alkyl-carboxylates and alkyl-phosphonates have been shown to form SAMs on ITO, but the lack of synthetic routes available to append chemical functionalities to these tail groups and to pattern these molecules has limited the overall use of ITO substrates in biosensor and cell biological studies [67]. Currently, methods exist to pattern ITO onto other materials such as glass, aluminum, and silicon for applications in optoelectronics. Microcontact printing has been used to pattern siloxane Current Opinion in Chemical Biology 2009, 13:697–704
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SAMs on ITO, but direct SAM tailoring through chemical activation of a tail group has remained undeveloped [68]. A methodology that could chemically alter a single phosphonate SAM on ITO to multiple functionalities for subsequent ligand immobilization would be cost effective and extremely useful. Not only would this strategy circumvent the difficulties encountered in synthesis, but it would also provide a platform to tailor many other materials for a broad range of interests [69]. The gold SAM surface is one of the most studied and developed model substrate platforms for cell behavior studies, but it is generally planar and does not replicate the complex nanotopology of the extracellular matrix. To study cells sampling a three-dimensional environment, a number of modifications to the SAM substrate have been pursued with varying success [70]. In the future, to understand the complex effects of nanotopology on cell migration and adhesion, a multidisciplinary effort that integrates surface chemistry, microfluidics, and microfabrication may be utilized to simultaneously pattern surfaces while generating different surface topologies. There have been many important contributions to generate model substrates to study cell mobility behavior. Many surface chemistry and analytical tools have been developed and refined to fabricate and characterize materials applied to cell biology. Future work will focus on the continued integration of surface chemistry and cell biological tools with the emphasis on analyzing the biochemistry of signal transduction processes and microscopic observation of cell behavior on bio-responsive or dynamic substrates.
Conflict of interest The authors have no conflict of interest.
Acknowledgements MNY acknowledges support from The University of North Carolina at Chapel Hill, The Burroughs Wellcome Foundation (Interface Career Award), the National Science Foundation (NSF Career Award), and the National Cancer Institute (Carolina Center for Cancer Nanotechnology Excellence).
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61. Hoover DK, Lee EJ, Chan EWL, Yousaf MN: Electroactive nanoarrays for biospecific ligand mediated cell adhesion. ChemBioChem 2007, 8:1920.
42. Hodgson L, Chan EWL, Hahn KM, Yousaf MN: Combining surface chemistry with a FRET-based biosensor to study the dynamics of RhoA GTPase activation in cells on patterned substrates. J Am Chem Soc 2007, 129:9264. The authors showed for the first time that live-cell high-resolution fluorescence microscopy is compatible with tailored and patterned gold surfaces. 43. Lamb BM, Westcott NP, Yousaf MN: Live-cell fluorescence microscopy of directed cell migration on partially etched electroactive SAM gold surfaces. ChemBioChem 2008, 9:2220. 44. Westcott NP, Lamb BM, Yousaf MN: Electrochemical and chemical microfluidic gold etching to generate patterned and gradient substrates for cell adhesion and cell migration. Anal Chem 2009, 81:3297. 45. Steyer JA, Almers W: A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol 2001, 2:268. 46. Axelrod DJ: Cell–substrate contacts illuminated by total internal reflection fluorescence. Cell Biol 1981, 89:141. www.sciencedirect.com
62. Hoover DK, Chan EWL, Yousaf MN: Asymmetric peptide nanoarray surfaces for studies of single cell polarization. J Am Chem Soc 2008, 130:3280. The authors developed a single cell nanoarray composed of cell adhesive RGD peptides to study how the size and distance between cell adhesive nanospots influences cell polarity orientation. 63. Kupfer A, Louvard D, Singer SJ: Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. Proc Natl Acad Sci U S A 1982, 79:2603. 64. Schoenfisch MH, Pemberton JE: Air stability of alkanethiol selfassembled monolayers on silver and gold surfaces. J Am Chem Soc 1998, 120:4502. 65. Ulman A: Formation and structure of self-assembled monolayers. Chem Rev 1996, 96:1533. 66. Paine DC, Whitson T, Janiac D, Beresford R, Yang CO: A study of low temperature crystallization of amorphous thin film indium–tin–oxide. J Appl Phys 1999, 85:8445. 67. Kang M, Ma H, Yip H, Jen AKY: Direct surface functionalization of indium tin oxide via electrochemically induced assembly. J Mater Chem 2007, 17:3489. Current Opinion in Chemical Biology 2009, 13:697–704
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68. Pulsipher A, Westcott NP, Luo W: Rapid microfluidic generation of patterned aldehydes from hydroxy-terminated self-assembled monolayers for ligand and cell immobilization on optically transparent indium tin oxide surfaces. Adv Mater 2009, 21:3082.
The authors combined microfluidics and mild oxidizing conditions to rapidly pattern indium tin oxide surfaces with aldehyde and carboxylic acid groups with controlled functional group density from an inexpensive hydroxy presenting indium tin oxide surface.
69. Pulsipher A, Westcott NP, Luo W: Rapid in situ generation of two patterned chemoselective surface chemistries from a single hydroxy-terminated surface using controlled microfluidic oxidation. J Am Chem Soc 2009, 131:7626.
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Model substrates for studies of cell mobility Muhammad N Yousaf Cells do not live in static surroundings, they exist in highly evolving dynamic environments. During cell adhesion and migration, cells adapt and communicate to their environment by numerous methods ranging from differentiation, gene expression, growth, and apoptosis. How and when cells determine to adhere, polarize, and migrate is important to a number of fundamental biological processes such as wound healing, metastasis, inflammation, and development. In order to elucidate the spatial and temporal mechanisms of these important complex processes on a molecular basis, several research groups have generated model substrates that can present nanopatterned ligands, well-defined gradients of ligands and surfaces that can be dynamically modulated where the interaction between cell and material is defined at the molecular level.
Address Department of Chemistry and the Carolina Center for Genome Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, United States Corresponding author: Yousaf, Muhammad N ([email protected])
Current Opinion in Chemical Biology 2009, 13:697–704 This review comes from a themed issue on Model Systems Edited by Marcey Waters and Dan Appella Available online 26th October 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.
cell polarity, migration, and invasion involve a precise but constantly changing subcellular nano-architecture. To fully understand the complex signaling and cytoskeletal aspects of the cellular nano-architecture during migration requires a multidisciplinary coordinated effort. The long-term goal of this research field is to develop and integrate new surface chemistry and cell biological tools to generate a class of tailored dynamic patterned substrates for a variety of cell adhesion, cell polarization, and migration experiments. The integration of dynamic substrates, molecular surface gradients, new migratory cell lines, and in vivo biosensors that are currently being developed will potentially allow for the complete analysis and quantitation of each step of cell migration from initial engagement with extracellular matrix (ECM) ligands, to localized activation of signaling proteins, to organization and activation of the cytoskeleton, to directional polarization to overall movement of the cell. Integrins, signaling, and migration
Both during local invasion and during metastasis to distant sites, tumor cells breach normal tissue boundaries by utilizing a variety of mechanisms [1,2]. The invading tumor cells gain traction by engaging the extracellular matrix via cell surface integrins, often with preferential utilization of certain members of the integrin family [3,4]. Focused degradation of the ECM via matrix metalloproteinases, adamlysins, and tissue serine proteinases is also a key aspect of invasion [5], as are tumor cell–host cell interactions [2].
Introduction
The integrin family of adhesion receptors plays a vital role in cell adhesion and migration. Not only do integrins provide transmembrane linkages from the ECM to the actin cytoskeleton [6,7], but also they make a profound contribution to the signaling events that take place during these processes [8–10].
Active migration, local tissue invasion, and seeding of distant metastases are all characteristic of malignant cells. These complex cellular events require the integration of information derived from soluble growth factors with positional information gained from interactions with the extracellular matrix and with other cells. The network to determine how a cell responds and moves involves complex signaling cascades that guide the directional and contractile functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner that then dynamically shape the cytoskeleton in specific subcellular regions. Therefore
The human integrin family comprises 18 a subunits and 9 b subunits resulting in two dozen integrin heterodimers [11]. This chemical diversity gives rise to biological complexity, and thus integrins have been implicated in numerous functions including cell–ECM and cell–cell adhesion, organization of actin filaments, signal transduction, cell survival, cell growth and differentiation, and unique roles in developmental processes [12–14]. Despite this complexity, most integrins share two key inter-related functions: firstly, promoting the assembly and organization of the actin cytoskeleton and secondly, regulating signal transduction cascades [15–18].
DOI 10.1016/j.cbpa.2009.10.001
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In addition to integrins and cytoskeletal proteins, focal complexes/adhesions are enriched in a variety of signaling molecules including focal adhesion kinase (FAK), Srcfamily kinases, G-proteins, and elements of MAP Kinase cascades [19]. In addition to influencing Erk and other MAP Kinase cascades, there is a complex set of relationships between integrins and Rho-family GTPases and their downstream effectors. In focal adhesions (FAs)/complexes integrins are connected to various forms of filamentous actin arrays ranging from the loose meshwork of filaments at the leading edge to robust actin stress fibers containing thousands of filaments in the interior fibrillar adhesions. With the advent of live-cell imaging, the temporal distribution of integrin complexes could begin to be addressed [20]. It is now clear that the various adhesive structures dynamically mature from nascent structures at the edge of the cell to the larger interior structures such as FAs. In a migrating cell, there is also loss of adhesion at the trailing edge that involves a combination of regulated proteolysis of integrins and associated proteins, as well as physical tearing. While previous studies have provided a great deal of information about how cells dynamically control the cytoskeleton–integrin linkages in space and time, new methodologies will be needed to advance our understanding of this process. Integrin/ECM interactions
A number of experimental approaches have been used to study the details of integrin/ECM interaction. Many in vitro studies have perturbed integrin function by altering the concentration, composition, and presentation of ECM ligands. Addition of anti-integrin antibodies, or of synthetic (Arg–Glyc–Asp) RGD-based peptides, will often block cell spreading and migration on native ECM. However, these synthetic peptides can also be immobilized on the substrate by various chemistries to provide a more defined surface for integrin-based adhesion or migration [21–23]. These relatively crude studies have been extended by the use of patterned surfaces based on pioneering approaches developed by Whitesides and colleagues [24,25]. From a biological view, gene deletion studies in mice show that integrins are required for many, if not all, cell migration events both in vitro and in vivo [26]. Thus a particularly promising approach will be to combine manipulations of the ECM with genetically or epigenetically modified cells to address specific questions about integrin function in cell motility and tumor cell invasion. There is an intricate interplay among integrins, signaling pathways, and the cytoskeleton that influence the dynamic nano-architecture of both normal and tumor cells, with aberrations in the integrin/signaling connections contributing to the abnormal behavior of tumors. However, new tools, such as the dynamic nanopatterned Current Opinion in Chemical Biology 2009, 13:697–704
surfaces described below, will be needed to fully understand these complex events. Haptotaxis and cell polarity
Haptotaxis, or migration on a gradient of ECM protein, is another form of directed migration. Although the mechanisms involved are largely unknown, haptotaxis is dependent on focal contacts (FCs)/FA formation and turnover. Integrin engagement and clustering leads to the formation of FCs, characterized by the recruitment of paxillin and a-actinin and the activation of a number of tyrosine kinases. Tyrosine phosphorylation is a key event in FC/FA formation, as treatment with the tyrosine kinase inhibitor herbimycin A inhibits the formation of both FAs and stress fibers [27]. FAK is one of the first kinases recruited to FCs [28], and the importance of this recruitment is underscored by the fact that in the absence of FAK, FCs fail to mature to FAs [29]. Subsequent activation of FAK creates binding sites for a number of different signaling and scaffolding proteins, including the Src tyrosine kinase and PI3K, which contributes to FA maturation. FAK activation has also been reported to lead to both increased and decreased Rho activity, depending on the cellular context, through the activities of p190RhoGEF and GRAF, respectively [30]. Decreased Rho activity is thought to be necessary for the formation of FCs, while increased Rho activity contributes to FA maturation, probably through increased actomysin contractility. Disassembly of FAs results from repeated microtubule targeting and occurs in a FAKdependent and Dynamin-dependent manner [31]. How these events contribute to haptotactic migration is not known. Cellular polarity is fundamental to a number of cellular processes and it has long been assumed that morphological polarity contributes to directional migration. For example, in vitro wound healing assays demonstrate that both the microtubule-organizing center (MTOC) and the Golgi apparatus become re-oriented to face the direction of migration. The importance of morphological polarity during migration is highlighted by the fact that treatments that inhibit reorientation also inhibit cellular migration. Leading edge dynamics and adhesion to the ECM also play a role in polarity and migration, but whether the leading edge or adhesion serve to establish polarity or are simply required for migration remains unclear. A promising approach is to integrate the use of a polarity sensor cell line that allows for the assessment of morphological polarity in live cells. Using these sensors, it may be possible to quantitatively describe how cellular polarity is established and maintained on molecularly defined surfaces at the single cell level. Integrating surface chemistry and cell biological tools
In order to generate relevant model substrates for detailed mechanistic studies of cell adhesion, polarity, and cell www.sciencedirect.com
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migration, cell culture compatible surfaces that can present multiple ligands or proteins at defined densities in patterns and in gradients are necessary but remain technically challenging. The key criteria to generate model substrates for studies and applications in cell biology are: Firstly, a sophisticated surface chemistry reaction to chemoselectively immobilize multiple ligands with the ability to precisely modulate the density of each immobilized ligand within patterns and gradients. Secondly, compatible with cell culture conditions where the only interaction between attached cell and material is a ligand–receptor interaction that is surfaces that are inert to nonspecific protein adsorption or cell attachment (biospecific surfaces). Thirdly, a robust fabrication technique to generate complex patterns and gradients. Fourthly, dynamic surfaces, where a noninvasive stimuli can modulate cell behavior in real-time. Fifthly, compatible with live-cell high-resolution fluorescence microscopy techniques. Sixthly, amenable to many analytical techniques in order to characterize interfacial associations ranging from nanoparticles to live cells.
Surface chemistry to immobilize ligands Strategies to immobilize biomolecules onto solid supports is important for a wide variety of applications ranging from the development of small molecule and protein microarrays to model substrates for mechanistic studies of cell behavior [29,30]. There have been several immobilization methods developed to tailor surfaces for a variety of diagnostic and high throughput assays [31]. However, there are very few quantitative strategies to determine the yield and therefore density of ligands immobilized to a surface and none to pattern ligands with control of density. In order to extend the utility of surfaces to generate new types of model substrates to study complex biological processes, it is necessary to develop more sophisticated and flexible surface properties. Surfaces for these applications would require the precise control of ligand density, the ability to immobilize multiple ligands and the in situ modulation of ligand activity. We have recently reported a novel chemical approach to couple ligands, proteins and cells onto an electroactive self-assembled monolayer (SAM) of alkanethiolates on gold via chemoselective ligation where we have precise control of the ligand density on the surface [32]. Surface gradients and dynamic substrates
Current strategies seek to modify surfaces with appropriate protein and peptide ligands in order to promote cell adhesion and maintain cell function and viability [32]. These surface engineering methods, together with methods for patterning the attachment of cells, are now important tools for fundamental studies in biology and for the preparation of chip-based systems in biotechnology [33]. www.sciencedirect.com
Although substantial progress has been made in the modification of material structures to control the adhesion of cells, much less effort has been invested in developing ‘dynamic substrates’ — substrates that can actively modulate the behaviors of attached cells. There also has been tremendous investigation in generating surfaces that present gradients of immobilized ligands. However, there have been no methods to combine these methods to generate dynamic gradient substrates for studying cell polarity and directed cell migration. Dynamic substrates that can alter the presentation of ligands to an attached cell will generate immediate opportunities for studies of matrix-dependent cell adhesion, signaling, migration, and differentiation. Dynamic surfaces would allow for the real-time manipulation and study of cells that currently do not exist with other static substrate strategies. These surfaces provide exquisite molecular control of the ligand–receptor interactions between cell and material that can be potentially controlled at the single molecule level. Dynamic substrates may also be important for generating substrates that can control the spatial and temporal interactions between two or more different populations of attached cells (cocultures, multi-cell arrays, tissue engineering). Ultimately, this work might provide new understandings and improved methods to direct the maturation of stem cells or to control the generation of tissue for medicinal applications. Thus, molecularly defined gradient surfaces and dynamically nanopatterned surfaces with ECM type ligands offer exciting new possibilities for cell biological research. Gold as a model substrate platform
Gold has been the metal of choice for generating model substrates for cell biological studies, which utilize SAMs. These types of surfaces are useful due to their resistance to oxidation, biocompatibility, synthetic flexibility to tailor its properties, conductivity, and its ability to be inert to nonspecific protein adsorption and cell attachment. SAMs on gold present a well-defined surface, which is ideally suited to study cell migration. Ligands can be chemoselectively immobilized to the surface in patterns to probe the subtle effects of ligand affinity and density on cell migration. The ability to tailor materials with SAMs to generate diverse chemical and physical properties has proven to be important for a range of research fields, such as biomedical engineering, organic catalysis, molecular electronics, and cell biology [34]. SAMs of alkanethiolates on gold represent one of the most well-studied surface chemistry systems. The major advantages of SAMs on gold include the inherent conductivity of gold that allows substrate compatibility with a variety of surface characterization techniques. Alkanethiols are also synthetically flexible or commercially available, providing opportunities to tailor Current Opinion in Chemical Biology 2009, 13:697–704
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materials for a range of applications [35]. In addition to the direct synthesis of alkanethiols, a number of convergent interfacial chemoselective strategies including Diels-Alder conjugation [36], Click chemistry [37], quinone and aldehyde coupling [38], Staudinger ligation [39], and Michael addition [40] have been developed to immobilize and present a variety of ligands on the surface.
Live-cell high-resolution fluorescence microscopy Fluorescence microscopy is a major research tool in cell biological investigations [41]. It has been used to image organelles, protein dynamics, and study protein–protein interactions. However, integrating SAMs of alkanethiolates on gold with live-cell fluorescence studies has been difficult due to the fact that gold surfaces efficiently quench fluorescence thus limiting access to important tools in cell biology research (such as fluorescently labeled cell lines, and fluorescence resonance energy transfer (FRET) techniques, and fluorescent dyes). In a series of experiments, we recently overcame this limitation for FRET analysis by examining actin assembly at cell protrusions with an inexpensive customized microscopy setup on gold SAM surfaces [42]. For this analysis, the microscopy presented a major technical obstacle, as fluorescent biosensors in living cells must be used at low intracellular concentrations that produce relatively low light levels; gold model substrates are known to be highly absorptive at the wavelengths used for live-cell fluorescence imaging causing efficient quenching of the fluorescent molecules, significantly reducing fluorescence excitation. We combined a surface chemistry approach with a FRET-based biosensor to investigate the effects of the cell microenvironment on RhoA activation in membrane protrusions. The approach is based on the combination of SAMs of alkanethiolates on gold to generate defined hydrophobic and hydrophilic regions on the model surface, and sensitive live-cell fluorescence microscopy that is compatible with gold surfaces. Another major limitation of SAM gold surfaces is the inability to visualize ligand patterns on the substrate during cell adhesion or directed migration experiments. Although patterning gold with different chemistries is straightforward, observing the patterns on a flat two-dimensional surface simultaneously while using compatible microscopies to observe live-cell fluorescent behavior is currently unavailable. One solution to these challenges is to pattern the gold directly to generate gold/ glass hybrid surfaces [43,44]. However, this methodology functions as a binary system and cannot generate topologies such as gradients. For cell motility studies on planar gold surfaces the directional path of migration must be determined ex post Current Opinion in Chemical Biology 2009, 13:697–704
facto because a ligand pattern cannot be independently visualized during cell migration. To study complex cell behavior and especially directed cell polarity and migration, a molecularly well-defined surface that can be patterned with a chemoselective immobilization strategy, compatible with live-cell high-resolution microscopy while simultaneously visualizing the cell path trajectory would be of tremendous utility. A recent report demonstrated the combination of three new straightforward technologies that synergistically provide complete spatial and visual control of directed cell polarity and migration [43]. The strategy uses: Firstly, partially etched gold surfaces to determine the precise track or path of cell migration trajectory. Secondly, microfluidic lithography (mFL) to pattern gold surfaces rapidly with a variety of alkanethiols for biospecific cell interactions. Thirdly, live-cell high-resolution fluorescence microscopy on gold surfaces to visualize internal organelle dynamics during polarity and migration. To provide ligand control over the surface and characterize the patterning, a chemoselective electroactive SAM immobilization methodology was used to pattern ligands on the partially etched gold surfaces. They showed live-cell directed migration of a mammalian cell line where the nucleus and Golgi are fluorescently labeled to determine the role of cell polarity on motility. The strategy is based on using microfluidic cassettes to both partially etch the gold surface and then install a monolayer on the etched regions for subsequent immobilization of biospecific adhesive peptides. By using a slightly modified inverted microscopy set-up [44], access to routine live-cell fluorescence microscopy is now possible on gold surfaces. These results are the first demonstration of live-cell fluorescence microscopy of directed cell migration on tailored gold surfaces. Total internal reflection microscopy
Total internal reflection fluorescence microscopy (TIRFM) is a technique that has been used for the past two decades to study events at the cell plasma membrane [45]. One early application of TIRFM in cell biology was to study the cell–substrate contact regions of adherent cells on glass coverslips [46,47]. TIRFM is based on the principle of total internal reflection (TIR). TIRFM has been used to study a variety of systems, including single molecule fluorescence [48,49] as well as events and structures at the cell surface such as exocytosis [50] and ion channels [51] and has also been integrated with other methods such as interference reflection microscopy [52] and fluorescence correlation spectroscopy [53]. Until recently, it was thought that gold surfaces precluded the use of live-cell high-resolution fluorescence microscopy to study internal cell structure dynamics. There have been conflicting reports on whether the presence of gold impedes the use of TIRFM in studying cell adhesion www.sciencedirect.com
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to gold substrates [54]. Borisy and coworkers have stated that gold surfaces prevent the use of TIRFM [55], while Arima and Iwata have reported studying the adhesion of cells with fluorescently labeled plasma membranes on SAMs of varying properties using TIRFM [56]. However, the latter did not examine the internal structures of the cell, nor were the surfaces tailored with biologically relevant ligands. In a recent report, the combination of microcontact printing or an electroactive chemoselective immobilization strategy, dip-pen nanolithography, and TIRFM was used to visualize cell adhesion on tailored and patterned gold surfaces [57]. This report is the first to show that TIRFM can be used to visualize internal features of a cell on chemoselectively tailored gold SAM surfaces and will open many avenues for integrating TIRFM with material science and cell signaling dynamics to study cell behavior. Dip-pen nanolithography for cell polarity
Dip-pen nanolithography (DPN) has revolutionized nanotechnology and is based on a scanning probe technique in which an atomic force microscopy (AFM) tip is used to pattern molecules on a surface with precise nanometer scale features [58]. A wide variety of materials have been patterned by DPN for applications ranging from the fabrication of nanostructures to ultra high density DNA arrays [59]. More recently, DPN has been extended to the development of protein nanoarrays for high throughput genetic and biomarker analysis [60]. A major area of research in which DPN nanoarray technology will make a significant impact is in cell biology. For example, the spatial presentation of cell adhesive ligands influences the subcellular nano-architecture of adherent cells and affects their behavior. In particular, the number and size of biospecific interactions between extracellular ligands and cell surface receptors is critical for cell adhesion and cell migration and remain poorly understood and elusive due to the lack of molecularly defined nanopatterned model substrates. By immobilizing the adhesive RGD peptide and cyclicRGD peptide on a single cell nanoarray (400 nm spots spaced 600 nm apart) it was shown that cells had larger and more FAs on the higher affinity cyclic-RGD presenting surface [61]. In order to probe how cell polarity is established with the minimum amount of ligand mediated adhesions the dippen nanolithography (DPN) technique was used to generate single cell symmetric and asymmetric nanoarrays. The polarity of a cell can be experimentally measured through the systematic reorientation and alignment of several organelles in the cell including the nucleus, centrosome, and Golgi apparatus, which can be visualized using fluorescent dyes to map the direction of polarity [62,63]. These experiments are important to the cell www.sciencedirect.com
motility research field because previously there was no method to separate the dynamic processes of cell adhesion, cell polarity, and cell migration without the use of genetic or small molecule manipulations. By using the single cell nanoarrays, cells may adhere to the nanoarray pattern, determine if the conditions are met for establishing polarity but are unable to migrate. These surfaces allow for the analysis of cell polarity and determine how the underlying adhesive environment influences cell behavior.
Conclusion Although gold-based SAMs are the most flexible model system for studying biointerfacial science, there remain severe long-term stability and biocompatibility issues. The thiol–gold bond is thermally unstable and upon long durations of air exposure may oxidize, damaging the integrity of the monolayer [64]. Gold also efficiently quenches fluorescence and has limited optical transparency properties that complicate its use for fluorescent based biosensor or cell array technologies. To address these limitations, there has been intense interest in transferring certain surface chemistries developed for gold to other materials to increase their scope of applications. An alternate model system, monolayers of siloxanes on glass have been intensely studied and utilized for cell based assays and biosensor applications [65]. Not only has glass been shown to be robust, its optical transparency allows for the unimpeded observation of fluorescence. However, synthetic strategies to tailor glass surfaces can be difficult, laborious, and expensive, and characterization techniques are limited due to its lack of conductivity. Another material that shows much promise as a biointerfacial platform for cell behavior studies is indium tin oxide (ITO), a common material widely used for applications in optoelectronics and is found as the transparent conductive coatings in plasma, touch, and liquid crystal displays, as well as solar cells and organic light emitting diode (OLED) devices [66]. Its high conductivity permits the characterization of ITO with a variety of analytical techniques. Unlike gold, the optical transparency of ITO presents opportunities for studies involving fluorescence and, in particular, research in cell biology that requires live-cell high-resolution fluorescence microscopy to study cell behavior. However, the major limitation of ITO is the inability to tailor the surface with a variety of surface chemistries. Alkyl-carboxylates and alkyl-phosphonates have been shown to form SAMs on ITO, but the lack of synthetic routes available to append chemical functionalities to these tail groups and to pattern these molecules has limited the overall use of ITO substrates in biosensor and cell biological studies [67]. Currently, methods exist to pattern ITO onto other materials such as glass, aluminum, and silicon for applications in optoelectronics. Microcontact printing has been used to pattern siloxane Current Opinion in Chemical Biology 2009, 13:697–704
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SAMs on ITO, but direct SAM tailoring through chemical activation of a tail group has remained undeveloped [68]. A methodology that could chemically alter a single phosphonate SAM on ITO to multiple functionalities for subsequent ligand immobilization would be cost effective and extremely useful. Not only would this strategy circumvent the difficulties encountered in synthesis, but it would also provide a platform to tailor many other materials for a broad range of interests [69]. The gold SAM surface is one of the most studied and developed model substrate platforms for cell behavior studies, but it is generally planar and does not replicate the complex nanotopology of the extracellular matrix. To study cells sampling a three-dimensional environment, a number of modifications to the SAM substrate have been pursued with varying success [70]. In the future, to understand the complex effects of nanotopology on cell migration and adhesion, a multidisciplinary effort that integrates surface chemistry, microfluidics, and microfabrication may be utilized to simultaneously pattern surfaces while generating different surface topologies. There have been many important contributions to generate model substrates to study cell mobility behavior. Many surface chemistry and analytical tools have been developed and refined to fabricate and characterize materials applied to cell biology. Future work will focus on the continued integration of surface chemistry and cell biological tools with the emphasis on analyzing the biochemistry of signal transduction processes and microscopic observation of cell behavior on bio-responsive or dynamic substrates.
Conflict of interest The authors have no conflict of interest.
Acknowledgements MNY acknowledges support from The University of North Carolina at Chapel Hill, The Burroughs Wellcome Foundation (Interface Career Award), the National Science Foundation (NSF Career Award), and the National Cancer Institute (Carolina Center for Cancer Nanotechnology Excellence).
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