REVIEW Microtubule-Based Nanomaterials: Exploiting Nature’s Dynamic Biopolymers George D. Bachand,1 Erik D. Spoerke,2 Mark J. Stevens3 1
Center for Integrated Nanotechnologies, Sandia National Laboratories, Albuquerque 87185-1303, New Mexico; telephone: þ1 505-844-5164; fax: þ1 505-284-7778; e-mail: [email protected] 2 Department of Electronic, Optical, and Nano Materials, Sandia National Laboratories, Albuquerque, New Mexico 3 Department of Computational Materials and Data Science, Sandia National Laboratories, Albuquerque, New Mexico
ABSTRACT: For more than a decade now, biomolecular systems have served as an inspiration for the development of synthetic nanomaterials and systems that are capable of reproducing many of unique and emergent behaviors of living systems. One intriguing element of such systems may be found in a specialized class of proteins known as biomolecular motors that are capable of performing useful work across multiple length scales through the efficient conversion of chemical energy. Microtubule (MT) filaments may be considered within this context as their dynamic assembly and disassembly dissipate energy, and perform work within the cell. MTs are one of three cytoskeletal filaments in eukaryotic cells, and play critical roles in a range of cellular processes including mitosis and vesicular trafficking. Based on their function, physical attributes, and unique dynamics, MTs also serve as a powerful archetype of a supramolecular filament that underlies and drives multiscale emergent behaviors. In this review, we briefly summarize recent efforts to generate hybrid and composite nanomaterials using MTs as biomolecular scaffolds, as well as computational and synthetic approaches to develop synthetic one-dimensional nanostructures that display the enviable attributes of the natural filaments. Biotechnol. Bioeng. 2015;112: 1065–1073. ß 2015 Wiley Periodicals, Inc. KEYWORDS: biomineralization; energy dissipation; molecular dynamics; cytoskeleton; dynamic instability; biomolecular motors
Biopolymers are ubiquitously found throughout living systems, and have received considerable attention due to their intrinsic biological functions, but also in a broader context based on their enviable mechanical properties, unique behaviors, and potential application Correspondence to: G.D.Bachand Received 11 December 2014; Revision received 9 February 2015; Accepted 10 February 2015 Accepted manuscript online 27 February 2015; Article first published online 9 April 2015 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25569/abstract). DOI 10.1002/bit.25569
ß 2015 Wiley Periodicals, Inc.
in engineered nanomaterials. Further, the convergence of biomolecular systems and synthetic nanosystem was apparent even in the early days of nanotechnology, with living systems providing a molecular blueprint for complex nanoengineered systems. Perhaps one of the most intriguing aspects of biomolecular systems is the ability of biopolymers and biomacromolecules to dynamically self-assemble and organize into non-equilibrium nanostructures through the dissipation of chemical energy. Such structures underlie many emergent behaviors, such as self-healing, which remain rather elusive to reproduce in nanosystems composed of only synthetic building blocks. Moreover, the highly parallel nature of such self-assembly is consistent with the ability to mass manufacture complex supramolecular materials that span multiple length scales (Whitesides and Grzybowski 2002). Biomolecular motors are a class of biomacromolecules that are able to efficiently convert chemical energy and/or electrochemical potential energy into useful mechanical work. As such, they have been used as functional components of hybrid nanosystems, and served as inspiration for developing synthetic analogs that can mimic their function and efficiency (Hess et al., 2004). The active transport systems composed of kinesin motors and microtubule (MT) filaments, and myosin motors and actin filaments have received perhaps the greatest attention with respect to developing such bio-enabled nanoengineered materials and systems, and were recently reviewed (Bachand et al., 2014; Månsson 2012). In the present review, we consider the MT filament itself broadly as a biomolecular motor based on its ability to perform work during both polymerization and depolymerization in which chemical energy is actively dissipated. More specifically, we briefly review select literature with respect to MT-based nanomaterials and efforts to mimic MT structure and behavior in synthetic materials.
Microtubules-Structure and Dynamics MTs are one of the three polymeric filaments that, along with actin and intermediate filaments, compose the cytoskeleton of eukaryotic Biotechnology and Bioengineering, Vol. 112, No. 6, June, 2015
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cells. Their functions include (i) providing structural support for the cell, (ii) facilitating cell movement, (iii) segregating chromosomes during mitosis, and (iv) serving as an intracellular “rail” system for motor protein transport (Alberts et al., 2008). The structure and dynamics of MTs have been intensively studied over the past several decades due to their critical function in cellular processes as well as their potential role in neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases (Selkoe 2004). Based on their role in the cell cycle, MTs have also been a rich target for cancer treatment where chemotherapeutic agents interfere with MT dynamics, arrest the cell cycle, and ultimately cause apoptosis (Zhou and Giannakakou 2005). The fundamental building blocks of MTs are a- and b-tubulin monomers, which bind together tightly to form a heterodimer (Fig. 1) with a molecular weight of 110 kDa, diameter of 4 nm, and a length of 8 nm. Each dimer possesses two bound guanosine triphosphate (GTP) molecules, one at the nonexchangeable (N) site on the a subunit and one at the exchangeable (E) site on the b subunit, with the latter being hydrolyzed to GDP during assembly (Nogales et al., 1999). These dimers assemble head-to-tail to form protofilaments, which laterally associate to form a sheet that in turn closes to form a hollow tubule (25 nm diameter, tens of microns in length) with the protofilaments running lengthwise along the tubule
(Fig. 1). The longitudinal bond energy between dimers within a protofilaments is greater than the lateral bond energy between dimers in adjacent protofilaments (Drabik et al., 2007; VanBuren et al., 2002). The hollow, tubular nature of MTs imparts a relatively high flexural rigidity to these filaments, resulting in characteristic persistence lengths ranging from 1 to 10 mm (Hawkins et al., 2010). Because the filaments are formed by regular spacing of asymmetric building blocks (i.e., ab dimer), MTs possess an intrinsic polarity where one end is terminated by an a-tubulin (termed the minusend) and the other end is terminated by a b-tubulin (termed the plus-end). In the cell, MTs are primarily composed of 13 protofilaments with a 9 Å offset between dimers on adjacent protofilaments, which ensures that the protofilaments run parallel to the long axis of the MT (Howard 2001). If a sheet contains fewer than or more than 13 protofilaments, the MT experiences lattice rotation where the protofilaments follow a helical path around the surface of the MTas opposed to running parallel to the MTaxis. The net charge density of MTs has been reported at 260 e- mm1 (Kim et al., 2008; Stracke et al., 2002), while the a- and b-tubulin terminated ends possess positive and negative electrostatic potentials, respectively (Baker et al., 2001). MTs are highly dynamic structures that stochastically switch between states of polymerization and depolymerization, a
Figure 1. Crystal structure of the tubulin dimer (PDB: 1JFF) showing the nonexchangeable (N) and exchangeable (E) GTP-binding sites on the a and b subunits, respectively. Linear chains ab tubulin dimers, called protofilaments (middle, in black box), are formed through the head-to-tail self-assembly of dimers; lateral association of protofilaments forms a hollow tubule (i.e., MT filament; right) with an outer diameter of 25 nm and length in the tens of microns.
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phenomenon known as dynamic instability, which relies on conversion of GTP and is able to produce forces up to 40 pN and 15 pN, respectively (Howard 2001). These forces are used to perform work such as organizing the interior of a cell and pushing/ pulling chromosomes during segregation (Alberts et al., 2008; TolicNorrelykke, 2008). At physiological temperatures, spontaneous nucleation of GTP-tubulin and polymerization of MTs occurs at tubulin concentrations greater than 15 mM (1.7 mg mL1) (Fygenson et al., 1994). GTP occupying the E-site is hydrolyzed during polymerization, which in turn induces structural transitions that affect the longitudinal interfaces and overall lattice stability of the MT (Alushin et al., 2014). When the rate of addition of tubulin exceeds the rate of GTP hydrolysis, the MT will maintain polymerization with a “GTP cap” at the plus-end of the growing filament. If the rate of GTP hydrolysis exceeds the rate of tubulin addition, the MT loses its GTP cap and rapidly depolymerizes (catastrophe) until which time it stochastically switches back to polymerization (rescue). Cells rely on MT-associated proteins (MAPs) such as MAP2 and Tau to modulate dynamic instability particularly with regard to increasing the rate of MT elongation, increasing the frequency of rescue, and suppressing the frequency of catastrophe (Cassimeris 1993). Despite being a stochastic process and relatively energy intensive, dynamic instability has been shown
to be an efficient means of searching the three-dimensional volume of the cell, for example to capture chromosomes during prometaphase (Holy and Leibler 1994).
MT Templates for Nanomaterials Synthesis MTs possess a variety of characteristics that make them attractive as templates for the growth of nanoscale materials. The highly regulated assembly of the tubulin building blocks creates structures with an effectively monodisperse nanoscale diameter (25 nm), but also with tunable length. Moreover, their diverse amino acid composition imparts rich chemical functionality that can be used to coordinate secondary materials interactions. To date, there are a number of examples in which MTs have been explored as templates for the growth of metals, oxides, and even semiconducting chalcogenides (Fig. 2). Metal nanoparticle arrays and nanowires have been templated through the reductive growth of silver, palladium, and gold on preformed MTs (Behrens et al., 2002, 2004; Boal et al., 2004). In these demonstrations, MTs were exposed to solutions containing cationic metal precursors, allowing electrostatic attractions or ionic chelation by amino acid residues in the MT structure to bind and concentrate the cations to the MT. These ion-enriched MTs, then serve as favorable nucleation sites for the
Figure 2. Transmission electron micrographs of various nanowires formed from MT templates. (a) Palladium nanoparticles immobilized on a MT filament display dense packing and helical ordering consistent with the MT structure (arrows); (b) cadmium sulfide (CdS) nanowires showing the hollow core (arrows); and (c) a lepidocrocite (FeOOH) nanowires formed from a MT template. (d and e) Darkfield optical images and schematic depictions illustrating MT-templated CdS nanotubes organized into advanced secondary structures include three-dimensional asters (d) and rings (e). Image adapted with permissions from (a) (Behrens et al., 2002) (Copyright 2002, John Wiley & Sons, Inc.); (b) from (Spoerke et al., 2014) (Copyright 2014, John Wiley & Sons, Inc.); (c) (Boal et al., 2003) (Copyright 2003, John Wiley & Sons, Inc.).
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subsequent templating of the metal nanostructures. Similarly, Boal et al (2003) used cationic binding of iron ions along with oxidative chemistry to produce MTs densely coated with lepidicrocite (iron oxy-hydroxide). Finally, these approaches were further translated for sulfide-based semiconductor templating of ZnS and CdS on MTs (Boal et al., 2004; Spoerke et al., 2014). These sulfide-materials, however, have a propensity to react rapidly, forming poorlyassociated, bulk mineralization rather than well-templated mineral coatings. Therefore, modifications of the relatively simple approaches employed for metal and oxide growth were required to achieve high fidelity materials. For example, MTs decorated with ZnS nanoparticles were produced by alternating exposure of MTs to dilute ZnCl2 and Na2S solutions (Boal et al., 2004). For CdSmineralized MTs, a different approach was applied that relied on cation binding of Cd2þ to the MT templates in the presence of the stable organic sulfur source, thioacetamide. Subsequently raising the pH facilitated the decomposition of the thioacetamide and drove the relatively gradual mineralization of the MTs with CdS (Spoerke et al., 2014). This approach produced MTs densely coated with CdS,
only 1–2 nanocrystals thick that permeated the protein structure but did not invade the naturally-defined MT lumen, forming unique CdS nanotubes. While the majority of these studies have focused largely on simply demonstrating material templating on MTs themselves, the work by Spoerke et al (2014) also explored the organized assembly of the MTs into secondary structures to create unique bio-enabled CdS nanostructures (Fig. 2d and e). In particular, this work demonstrated the use of MAP2, tau, and kinesin motor proteins to direct the assembly of MTs into aligned arrays, three-dimensional asters, and surface-bound rings prior to mineralization (Spoerke et al., 2014). The subsequent templating of CdS resulted in the formation of replicate semiconducting nanostructures that spanned nanometer to multiple micrometer length scales in one, two, and three dimensions, a feat that is difficult or impossible to produce by other methods. In principle, this approach could be readily adapted for the formation of metals, oxides, or other semiconducting materials similar to those described above. Importantly, these demonstrations collectively illustrate how MTs can serve as
Figure 3. (a–c) Fluorescence photomicrograph of individual MTs with bound CdSe quantum dots (upper left) and actively assembled ring nanocomposites composed of MTs and quantum dots. (b and c) Scanning and transmission electron microscopy images of ring composites. (d) Scanning transmission electron microscopy image of MT filaments in a ring nanocomposites showing the organization of quantum dots along the filaments. Image adapted with permissions from (Liu et al., 2008)(Copyright 2008, John Wiley & Sons, Inc.).
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multiscale templates for the synthesis of diverse functional materials.
Dynamic Organization of Nanoparticles In addition to serving as templates for direct material nucleation and growth as described above, MT filaments are also ideal templates for the assembly and organization of nanoparticles based on: (i) the similarity in size of tubulin subunits and nanoparticles, (ii) the large number of approaches for functionalizing MTs (see review by Malcos and Hancock (2011), and (iii) the ability to regulate depolymerization/re-polymerization of filaments through environmental and chemical stimuli. As such, a variety of nanomaterials have been organized using MTs as templates including semiconductor nanocrystals (i.e., quantum dots), and gold, polymer, and magnetic nanoparticles (Bachand et al., 2004a, b, 2005; Platt et al., 2005). It is important to note that these composite structures also retain their ability to depolymerize/repolymerize into filaments (Achermann et al., 2011; Jeong and Hollingsworth 2006; Riegler et al., 2003) and interact with molecular motors (Bachand et al., 2004b, 2005; Hutchins et al., 2006, 2007). Retaining the dynamic behavior of the filaments permits dynamic reorganization of the attached nanoparticles, which in turn can modulate the electronic and/or optical properties of the overall ensemble. With respect to the latter, motor proteinbased transport of quantum-dot functionalized MTs has been used to dynamically assemble nanocomposite rings, in which energy dissipation by kinesin motors enables the formation of highly nonequilibrium structures (Fig. 3) (Bachand et al., 2005; Liu and Bachand 2011; Liu et al., 2008). Similar approaches have been applied to actin filaments for the preparation of metallic nanowires that electrically conductive and are able to maintain their ability to interact with myosin motors (Patolsky et al., 2004). Semiconductor nanowires and fibers have also been fabricated through the conjugation of quantum dots to actin filaments (Henry et al., 2011; Yao et al., 2009). While much of the work in this area has focused on organization of nanoparticles at the individual filament level, MTs in cells are commonly organized into highly complex, two- and three-dimensional structures such as axonal bundles, centrosomes, and mitotic spindles. Thusly mimicking such structures ex vivo provides an intriguing approach for organizing nanoparticles across multiple length scale and in two and/or three dimensions, while exploiting MT dynamics to reconfigure the composite structure. For example, Spoerke et al (2008) demonstrated that through controlled, multistep polymerization of MTs, it was possible to dictate the polar organization of MTs in three-dimensional assemblies (Spoerke et al., 2008). More recently, artificial microtubule asters (AMAs), mimicking the structures of the centrosome, were assembled by functionalized polymer spheres that nucleate MT polymerization. These asters then served as unique templates for the organization of quantum dots on the MT filaments radiating out into three-dimensional space. The temperature responsiveness of the MT templates was subsequently used to cyclically disassemble and re-assemble these composite structures (Fig. 4), demonstrating the ability to dynamically regulate structural morphology (Spoerke et al., 2013). Artificial MT aster structures have also been used to generate self-organized optical
Figure 4.
Fluorescence photomicrographs showing the cyclic (a) assembly, (b) disassembly, and (c) re-assembly of three-dimensional MT-quantum dots asters grown on bio-functionalized polymer microspheres. The asters in these images (a and c) adopt a comet-like morphology due to fluidic shear forces in the assembly chamber. Reprinted with permission from (Spoerke et al., 2013) (Copyright 2013 American Chemical Society).
devices inspired by the melanophore cells that allow certain fish to change color. In this system, motor protein transport enabled the reorganization of pigment granules, changing the distribution of fluorescence in the chambers (Aoyama et al., 2013). A significant challenge moving forward in this work will involve developing novel, increasingly complex approaches to organize MT filaments into deliberate, multi-dimensional assemblies, where the collective behaviors/properties of the nanoparticles may be tuned through dynamic reorganization of the composite.
Toward Artificial Microtubule Filaments Although natural MTs and motor proteins provide a valuable platform for exploring the dynamic materials organization and assembly, there are practical limitations to using such natural materials for scalable technological purposes. For example, MTs and motor proteins have a limited operational range with respect to pH, oxidative conditions, and heavy metals (Bachand and Bachand 2012; Thier et al., 2003). Thus, developing artificial, synthetic MTanalogs may enhance the operation range particularly where robustness is currently problematic (e.g., smart dust sensors; Bachand et al., 2009), as well as potentially impact other technical fields ranging from biomedical research to scalable nanomaterials assembly. Exactly reproducing the complex composition and structure of the natural tubulin proteins in an artificial system, however, is synthetically impractical. More viable approaches aim to identify critical elements of MTstructure, function, and behavior,
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and translate these elements as foundational targets in synthetic systems. Examples of such key MT elements include: (i) selfassembly from nanoscale building blocks, (ii) dynamic, programmable assembly/disassembly, (iii) one-dimensional nanostructure morphology, (iv) controlled secondary organization into multi-scale structures, (v) polarity to direct material function, and (vi) filament motility. While targets such as motility and even integration into secondary functional structures are enticing technical objectives,
achieving these properties is first dependent on the ability to control self-assembly of functional nanoscale building blocks to form onedimensional nanostructures. An understanding of the essential features of monomers that self-assemble into tubular MT-mimics is critical, and theory and simulation have provided a number of valuable insights toward this understanding. For example, molecular dynamics (MD) simulations using a wedge-shaped monomer (Fig. 5a) have recently demonstrated that self-assembly into tubule structures depends on key factors such as molecular
Figure 5. (a) Image of chiral wedge MD model (grey wedge) with ideal pitch 3 tubule. Individual wedge monomers are alternately colored red, blue, and green to aid in visualizing monomers in the tubule assembly. Stage of self-assembly of wedge system into tubules and final tubules of pitch 2 formed from chiral 2 monomer; (b) Geometric model for formation of nanotubes from wedge-shaped lanreotide bilayers, where variations in the sizes of close-contact residues (red and orange dots) influences wedge shape and ultimately nanotube diameter (Tarabout et al., 2011) (Copyright 2011 National Academy of Sciences, USA); (c) Scheme illustrating stacking assembly of cyclized peptides into individual nanotubes and ordered parallel arrays (Hartgerink et al., 1996) (Copyright 1996 American Chemical Society); (d) Illustration of nanotube formation from positively charged amphiphilic peptide bilayers (von Maltzahn et al., 2003) (Copyright 2003 American Chemical Society); and (e) Proposed self-assembly of diphenylalanine into sheets, and the subsequent curling to form nanotubes (Carny et al., 2006) (Copyright 2006 American Chemical Society).
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orientation and the relative influences of both lateral (transverse to the tube) and vertical (along the tube length) molecular interaction strengths (Cheng et al., 2012; Cheng and Stevens 2014). MD has further shown that the pitch of the tubule can be controlled by the chirality of the monomer, but also that a twist deformation tends to yield a range of pitch values. An additional interaction in the vertical direction is required to prevent such twist, a discovery that not only informs synthetic designs, but adds to our understanding of assembly in natural MTs. Meanwhile, self-consistent field theory has provided insight into the potential cooperative roles of intramolecular and intermolecular interactions in determining the self-assembled morphologies (Ting et al., 2014). A number of the key results from these studies were consistent with experimental observations in a recent study of the self-assembly of wedgeshaped, branched peptides into one-dimensional nanofibers (Gough et al., 2014). In general, peptide-based systems are particularly attractive for the self-assembly of MT mimics, although polymers, lipids, DNA, and even nanoparticles have also shown promise for programmable assembly of tubular structures (Bong et al., 2001; Scanlon and Aggeli 2008; Wang et al., 2013; Yin et al., 2008). In peptide-based systems, the rich amino acid chemistry of peptides provides for an extremely versatile synthetic platform, and enables the introduction of biochemical characteristics potentially needed for subsequent biomimetic function. One interesting demonstration used wedge-shaped building blocks formed from b-sheet forming peptides (model shown in Fig. 5b) to successfully generate nanotubes with a tunable diameter (Tarabout et al., 2011), offering enticing parallels to the simulated system described above. There are, however, a remarkable number of additional configurations of peptide structures and compositions that lead to nanotube assembly (Gao and Matsui 2005; Ghadiri et al., 1993; Hamley, 2014; Sakai et al., 2008; Shimizu et al., 2005; Vauthey et al., 2002; Yan et al., 2010). Not only does the composition vary with each of these approaches, but the mechanisms for tube formation vary to include stacking of cyclic D-L peptide rings, rolling of self-assembled sheets or amphiphilic peptide bilayers, twisting of helical ribbons, or even organization of rigid molecular “staves” into tubules (Fig. 5c–e) (Carny et al., 2006; Hartgerink et al., 1996; von Maltzahn et al., 2003). Regardless of the ultimate mechanism involved, factors such as molecular morphology, amphiphilicity (solvation), hydrogen bonding, electrostatics, and the balance of lateral and vertical interactions play strong roles in determining the size, shape, and assembly behavior of these tubular structures, just as they do in the dynamic assembly of natural MTs. Certainly, the diversity of these supramolecular approaches holds promise for the development of biomimetic materials systems aimed at emulating complex materials such as MTs. Learning to control and tune the cooperative and competitive driving forces governing these self-assembling structures, however, will be critical to tailoring the ultimate biomimetic characteristics (Gao and Matsui 2005; Ghadiri et al., 1993; Sakai et al., 2008; Shimizu et al., 2005; Vauthey et al., 2002; Yan et al., 2010). While the experimental studies outlined above do provide compelling examples of tubule formation, for the deliberate design of structural or functional MT mimics, it is clear that the
cooperation of experimental work with theory and modeling stand to significantly streamline molecular design and improve our understanding of these molecular systems.
Conclusion and Future Challenges Biochemical and biophysical characterization of MT structure, dynamics, and function has contributed strongly toward the development of a wide range of multi-dimensional hybrid and/or composite nanomaterials. This review has briefly highlighted a number of efforts to use these remarkable biopolymer filaments as functional templates to direct uniquely bio-enabled nanomaterial growth and/or assembly. To date, the majority of this work, however, has relied on MTs that have been chemically stabilized to prevent depolymerization, which consequently eliminates one of the most unique and enabling properties (i.e., dynamic instability) of these filaments. Whereas cells rely on the highly coordinated action of stabilizers and destabilizers (van der Vaart et al., 2009), a key challenge moving forward will include the realization of ex vivo mechanisms to regulate dynamic instability, enabling novel materials that exploit the intrinsic dynamics of MTs. The use of microfluidics, for example, offers a potential approach to spatially and temporally regulate positive and negative effectors of MT stability, as well as to controllably introduce synthetic materials to form composite structures. Fundamental studies of MTs have also provided a critical knowledge base for designing and generating synthetic one-dimensional nanostructures that mimic various elements of the natural cytoskeletal filaments. The strong focus on recapitulating the tubular structure of MT filaments has established basic platforms and key design rules for generating one-dimensional nanostructures from a diverse array of molecular building blocks. Strategic challenges in the continued development of synthetic MT analogs include developing systems that display selfregulated assembly dynamics (i.e., artificial dynamic instability), as well as the ability to interface with synthetic motors, mimicking biomolecular active transport. Such attributes would significantly expand the functional nature of synthetic one-dimensional nanostructures, and enable the inclusion of revolutionary properties and behaviors. We thank Drs. Nathan Bouxsein and Brad Jones for their useful comments and suggestions in preparing this review. Support for the preparation of the manuscript was provided by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, Project KC0203010. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
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Center for Integrated Nanotechnologies, Sandia National Laboratories, Albuquerque 87185-1303, New Mexico; telephone: þ1 505-844-5164; fax: þ1 505-284-7778; e-mail: [email protected] 2 Department of Electronic, Optical, and Nano Materials, Sandia National Laboratories, Albuquerque, New Mexico 3 Department of Computational Materials and Data Science, Sandia National Laboratories, Albuquerque, New Mexico
ABSTRACT: For more than a decade now, biomolecular systems have served as an inspiration for the development of synthetic nanomaterials and systems that are capable of reproducing many of unique and emergent behaviors of living systems. One intriguing element of such systems may be found in a specialized class of proteins known as biomolecular motors that are capable of performing useful work across multiple length scales through the efficient conversion of chemical energy. Microtubule (MT) filaments may be considered within this context as their dynamic assembly and disassembly dissipate energy, and perform work within the cell. MTs are one of three cytoskeletal filaments in eukaryotic cells, and play critical roles in a range of cellular processes including mitosis and vesicular trafficking. Based on their function, physical attributes, and unique dynamics, MTs also serve as a powerful archetype of a supramolecular filament that underlies and drives multiscale emergent behaviors. In this review, we briefly summarize recent efforts to generate hybrid and composite nanomaterials using MTs as biomolecular scaffolds, as well as computational and synthetic approaches to develop synthetic one-dimensional nanostructures that display the enviable attributes of the natural filaments. Biotechnol. Bioeng. 2015;112: 1065–1073. ß 2015 Wiley Periodicals, Inc. KEYWORDS: biomineralization; energy dissipation; molecular dynamics; cytoskeleton; dynamic instability; biomolecular motors
Biopolymers are ubiquitously found throughout living systems, and have received considerable attention due to their intrinsic biological functions, but also in a broader context based on their enviable mechanical properties, unique behaviors, and potential application Correspondence to: G.D.Bachand Received 11 December 2014; Revision received 9 February 2015; Accepted 10 February 2015 Accepted manuscript online 27 February 2015; Article first published online 9 April 2015 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25569/abstract). DOI 10.1002/bit.25569
ß 2015 Wiley Periodicals, Inc.
in engineered nanomaterials. Further, the convergence of biomolecular systems and synthetic nanosystem was apparent even in the early days of nanotechnology, with living systems providing a molecular blueprint for complex nanoengineered systems. Perhaps one of the most intriguing aspects of biomolecular systems is the ability of biopolymers and biomacromolecules to dynamically self-assemble and organize into non-equilibrium nanostructures through the dissipation of chemical energy. Such structures underlie many emergent behaviors, such as self-healing, which remain rather elusive to reproduce in nanosystems composed of only synthetic building blocks. Moreover, the highly parallel nature of such self-assembly is consistent with the ability to mass manufacture complex supramolecular materials that span multiple length scales (Whitesides and Grzybowski 2002). Biomolecular motors are a class of biomacromolecules that are able to efficiently convert chemical energy and/or electrochemical potential energy into useful mechanical work. As such, they have been used as functional components of hybrid nanosystems, and served as inspiration for developing synthetic analogs that can mimic their function and efficiency (Hess et al., 2004). The active transport systems composed of kinesin motors and microtubule (MT) filaments, and myosin motors and actin filaments have received perhaps the greatest attention with respect to developing such bio-enabled nanoengineered materials and systems, and were recently reviewed (Bachand et al., 2014; Månsson 2012). In the present review, we consider the MT filament itself broadly as a biomolecular motor based on its ability to perform work during both polymerization and depolymerization in which chemical energy is actively dissipated. More specifically, we briefly review select literature with respect to MT-based nanomaterials and efforts to mimic MT structure and behavior in synthetic materials.
Microtubules-Structure and Dynamics MTs are one of the three polymeric filaments that, along with actin and intermediate filaments, compose the cytoskeleton of eukaryotic Biotechnology and Bioengineering, Vol. 112, No. 6, June, 2015
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cells. Their functions include (i) providing structural support for the cell, (ii) facilitating cell movement, (iii) segregating chromosomes during mitosis, and (iv) serving as an intracellular “rail” system for motor protein transport (Alberts et al., 2008). The structure and dynamics of MTs have been intensively studied over the past several decades due to their critical function in cellular processes as well as their potential role in neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases (Selkoe 2004). Based on their role in the cell cycle, MTs have also been a rich target for cancer treatment where chemotherapeutic agents interfere with MT dynamics, arrest the cell cycle, and ultimately cause apoptosis (Zhou and Giannakakou 2005). The fundamental building blocks of MTs are a- and b-tubulin monomers, which bind together tightly to form a heterodimer (Fig. 1) with a molecular weight of 110 kDa, diameter of 4 nm, and a length of 8 nm. Each dimer possesses two bound guanosine triphosphate (GTP) molecules, one at the nonexchangeable (N) site on the a subunit and one at the exchangeable (E) site on the b subunit, with the latter being hydrolyzed to GDP during assembly (Nogales et al., 1999). These dimers assemble head-to-tail to form protofilaments, which laterally associate to form a sheet that in turn closes to form a hollow tubule (25 nm diameter, tens of microns in length) with the protofilaments running lengthwise along the tubule
(Fig. 1). The longitudinal bond energy between dimers within a protofilaments is greater than the lateral bond energy between dimers in adjacent protofilaments (Drabik et al., 2007; VanBuren et al., 2002). The hollow, tubular nature of MTs imparts a relatively high flexural rigidity to these filaments, resulting in characteristic persistence lengths ranging from 1 to 10 mm (Hawkins et al., 2010). Because the filaments are formed by regular spacing of asymmetric building blocks (i.e., ab dimer), MTs possess an intrinsic polarity where one end is terminated by an a-tubulin (termed the minusend) and the other end is terminated by a b-tubulin (termed the plus-end). In the cell, MTs are primarily composed of 13 protofilaments with a 9 Å offset between dimers on adjacent protofilaments, which ensures that the protofilaments run parallel to the long axis of the MT (Howard 2001). If a sheet contains fewer than or more than 13 protofilaments, the MT experiences lattice rotation where the protofilaments follow a helical path around the surface of the MTas opposed to running parallel to the MTaxis. The net charge density of MTs has been reported at 260 e- mm1 (Kim et al., 2008; Stracke et al., 2002), while the a- and b-tubulin terminated ends possess positive and negative electrostatic potentials, respectively (Baker et al., 2001). MTs are highly dynamic structures that stochastically switch between states of polymerization and depolymerization, a
Figure 1. Crystal structure of the tubulin dimer (PDB: 1JFF) showing the nonexchangeable (N) and exchangeable (E) GTP-binding sites on the a and b subunits, respectively. Linear chains ab tubulin dimers, called protofilaments (middle, in black box), are formed through the head-to-tail self-assembly of dimers; lateral association of protofilaments forms a hollow tubule (i.e., MT filament; right) with an outer diameter of 25 nm and length in the tens of microns.
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phenomenon known as dynamic instability, which relies on conversion of GTP and is able to produce forces up to 40 pN and 15 pN, respectively (Howard 2001). These forces are used to perform work such as organizing the interior of a cell and pushing/ pulling chromosomes during segregation (Alberts et al., 2008; TolicNorrelykke, 2008). At physiological temperatures, spontaneous nucleation of GTP-tubulin and polymerization of MTs occurs at tubulin concentrations greater than 15 mM (1.7 mg mL1) (Fygenson et al., 1994). GTP occupying the E-site is hydrolyzed during polymerization, which in turn induces structural transitions that affect the longitudinal interfaces and overall lattice stability of the MT (Alushin et al., 2014). When the rate of addition of tubulin exceeds the rate of GTP hydrolysis, the MT will maintain polymerization with a “GTP cap” at the plus-end of the growing filament. If the rate of GTP hydrolysis exceeds the rate of tubulin addition, the MT loses its GTP cap and rapidly depolymerizes (catastrophe) until which time it stochastically switches back to polymerization (rescue). Cells rely on MT-associated proteins (MAPs) such as MAP2 and Tau to modulate dynamic instability particularly with regard to increasing the rate of MT elongation, increasing the frequency of rescue, and suppressing the frequency of catastrophe (Cassimeris 1993). Despite being a stochastic process and relatively energy intensive, dynamic instability has been shown
to be an efficient means of searching the three-dimensional volume of the cell, for example to capture chromosomes during prometaphase (Holy and Leibler 1994).
MT Templates for Nanomaterials Synthesis MTs possess a variety of characteristics that make them attractive as templates for the growth of nanoscale materials. The highly regulated assembly of the tubulin building blocks creates structures with an effectively monodisperse nanoscale diameter (25 nm), but also with tunable length. Moreover, their diverse amino acid composition imparts rich chemical functionality that can be used to coordinate secondary materials interactions. To date, there are a number of examples in which MTs have been explored as templates for the growth of metals, oxides, and even semiconducting chalcogenides (Fig. 2). Metal nanoparticle arrays and nanowires have been templated through the reductive growth of silver, palladium, and gold on preformed MTs (Behrens et al., 2002, 2004; Boal et al., 2004). In these demonstrations, MTs were exposed to solutions containing cationic metal precursors, allowing electrostatic attractions or ionic chelation by amino acid residues in the MT structure to bind and concentrate the cations to the MT. These ion-enriched MTs, then serve as favorable nucleation sites for the
Figure 2. Transmission electron micrographs of various nanowires formed from MT templates. (a) Palladium nanoparticles immobilized on a MT filament display dense packing and helical ordering consistent with the MT structure (arrows); (b) cadmium sulfide (CdS) nanowires showing the hollow core (arrows); and (c) a lepidocrocite (FeOOH) nanowires formed from a MT template. (d and e) Darkfield optical images and schematic depictions illustrating MT-templated CdS nanotubes organized into advanced secondary structures include three-dimensional asters (d) and rings (e). Image adapted with permissions from (a) (Behrens et al., 2002) (Copyright 2002, John Wiley & Sons, Inc.); (b) from (Spoerke et al., 2014) (Copyright 2014, John Wiley & Sons, Inc.); (c) (Boal et al., 2003) (Copyright 2003, John Wiley & Sons, Inc.).
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subsequent templating of the metal nanostructures. Similarly, Boal et al (2003) used cationic binding of iron ions along with oxidative chemistry to produce MTs densely coated with lepidicrocite (iron oxy-hydroxide). Finally, these approaches were further translated for sulfide-based semiconductor templating of ZnS and CdS on MTs (Boal et al., 2004; Spoerke et al., 2014). These sulfide-materials, however, have a propensity to react rapidly, forming poorlyassociated, bulk mineralization rather than well-templated mineral coatings. Therefore, modifications of the relatively simple approaches employed for metal and oxide growth were required to achieve high fidelity materials. For example, MTs decorated with ZnS nanoparticles were produced by alternating exposure of MTs to dilute ZnCl2 and Na2S solutions (Boal et al., 2004). For CdSmineralized MTs, a different approach was applied that relied on cation binding of Cd2þ to the MT templates in the presence of the stable organic sulfur source, thioacetamide. Subsequently raising the pH facilitated the decomposition of the thioacetamide and drove the relatively gradual mineralization of the MTs with CdS (Spoerke et al., 2014). This approach produced MTs densely coated with CdS,
only 1–2 nanocrystals thick that permeated the protein structure but did not invade the naturally-defined MT lumen, forming unique CdS nanotubes. While the majority of these studies have focused largely on simply demonstrating material templating on MTs themselves, the work by Spoerke et al (2014) also explored the organized assembly of the MTs into secondary structures to create unique bio-enabled CdS nanostructures (Fig. 2d and e). In particular, this work demonstrated the use of MAP2, tau, and kinesin motor proteins to direct the assembly of MTs into aligned arrays, three-dimensional asters, and surface-bound rings prior to mineralization (Spoerke et al., 2014). The subsequent templating of CdS resulted in the formation of replicate semiconducting nanostructures that spanned nanometer to multiple micrometer length scales in one, two, and three dimensions, a feat that is difficult or impossible to produce by other methods. In principle, this approach could be readily adapted for the formation of metals, oxides, or other semiconducting materials similar to those described above. Importantly, these demonstrations collectively illustrate how MTs can serve as
Figure 3. (a–c) Fluorescence photomicrograph of individual MTs with bound CdSe quantum dots (upper left) and actively assembled ring nanocomposites composed of MTs and quantum dots. (b and c) Scanning and transmission electron microscopy images of ring composites. (d) Scanning transmission electron microscopy image of MT filaments in a ring nanocomposites showing the organization of quantum dots along the filaments. Image adapted with permissions from (Liu et al., 2008)(Copyright 2008, John Wiley & Sons, Inc.).
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multiscale templates for the synthesis of diverse functional materials.
Dynamic Organization of Nanoparticles In addition to serving as templates for direct material nucleation and growth as described above, MT filaments are also ideal templates for the assembly and organization of nanoparticles based on: (i) the similarity in size of tubulin subunits and nanoparticles, (ii) the large number of approaches for functionalizing MTs (see review by Malcos and Hancock (2011), and (iii) the ability to regulate depolymerization/re-polymerization of filaments through environmental and chemical stimuli. As such, a variety of nanomaterials have been organized using MTs as templates including semiconductor nanocrystals (i.e., quantum dots), and gold, polymer, and magnetic nanoparticles (Bachand et al., 2004a, b, 2005; Platt et al., 2005). It is important to note that these composite structures also retain their ability to depolymerize/repolymerize into filaments (Achermann et al., 2011; Jeong and Hollingsworth 2006; Riegler et al., 2003) and interact with molecular motors (Bachand et al., 2004b, 2005; Hutchins et al., 2006, 2007). Retaining the dynamic behavior of the filaments permits dynamic reorganization of the attached nanoparticles, which in turn can modulate the electronic and/or optical properties of the overall ensemble. With respect to the latter, motor proteinbased transport of quantum-dot functionalized MTs has been used to dynamically assemble nanocomposite rings, in which energy dissipation by kinesin motors enables the formation of highly nonequilibrium structures (Fig. 3) (Bachand et al., 2005; Liu and Bachand 2011; Liu et al., 2008). Similar approaches have been applied to actin filaments for the preparation of metallic nanowires that electrically conductive and are able to maintain their ability to interact with myosin motors (Patolsky et al., 2004). Semiconductor nanowires and fibers have also been fabricated through the conjugation of quantum dots to actin filaments (Henry et al., 2011; Yao et al., 2009). While much of the work in this area has focused on organization of nanoparticles at the individual filament level, MTs in cells are commonly organized into highly complex, two- and three-dimensional structures such as axonal bundles, centrosomes, and mitotic spindles. Thusly mimicking such structures ex vivo provides an intriguing approach for organizing nanoparticles across multiple length scale and in two and/or three dimensions, while exploiting MT dynamics to reconfigure the composite structure. For example, Spoerke et al (2008) demonstrated that through controlled, multistep polymerization of MTs, it was possible to dictate the polar organization of MTs in three-dimensional assemblies (Spoerke et al., 2008). More recently, artificial microtubule asters (AMAs), mimicking the structures of the centrosome, were assembled by functionalized polymer spheres that nucleate MT polymerization. These asters then served as unique templates for the organization of quantum dots on the MT filaments radiating out into three-dimensional space. The temperature responsiveness of the MT templates was subsequently used to cyclically disassemble and re-assemble these composite structures (Fig. 4), demonstrating the ability to dynamically regulate structural morphology (Spoerke et al., 2013). Artificial MT aster structures have also been used to generate self-organized optical
Figure 4.
Fluorescence photomicrographs showing the cyclic (a) assembly, (b) disassembly, and (c) re-assembly of three-dimensional MT-quantum dots asters grown on bio-functionalized polymer microspheres. The asters in these images (a and c) adopt a comet-like morphology due to fluidic shear forces in the assembly chamber. Reprinted with permission from (Spoerke et al., 2013) (Copyright 2013 American Chemical Society).
devices inspired by the melanophore cells that allow certain fish to change color. In this system, motor protein transport enabled the reorganization of pigment granules, changing the distribution of fluorescence in the chambers (Aoyama et al., 2013). A significant challenge moving forward in this work will involve developing novel, increasingly complex approaches to organize MT filaments into deliberate, multi-dimensional assemblies, where the collective behaviors/properties of the nanoparticles may be tuned through dynamic reorganization of the composite.
Toward Artificial Microtubule Filaments Although natural MTs and motor proteins provide a valuable platform for exploring the dynamic materials organization and assembly, there are practical limitations to using such natural materials for scalable technological purposes. For example, MTs and motor proteins have a limited operational range with respect to pH, oxidative conditions, and heavy metals (Bachand and Bachand 2012; Thier et al., 2003). Thus, developing artificial, synthetic MTanalogs may enhance the operation range particularly where robustness is currently problematic (e.g., smart dust sensors; Bachand et al., 2009), as well as potentially impact other technical fields ranging from biomedical research to scalable nanomaterials assembly. Exactly reproducing the complex composition and structure of the natural tubulin proteins in an artificial system, however, is synthetically impractical. More viable approaches aim to identify critical elements of MTstructure, function, and behavior,
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and translate these elements as foundational targets in synthetic systems. Examples of such key MT elements include: (i) selfassembly from nanoscale building blocks, (ii) dynamic, programmable assembly/disassembly, (iii) one-dimensional nanostructure morphology, (iv) controlled secondary organization into multi-scale structures, (v) polarity to direct material function, and (vi) filament motility. While targets such as motility and even integration into secondary functional structures are enticing technical objectives,
achieving these properties is first dependent on the ability to control self-assembly of functional nanoscale building blocks to form onedimensional nanostructures. An understanding of the essential features of monomers that self-assemble into tubular MT-mimics is critical, and theory and simulation have provided a number of valuable insights toward this understanding. For example, molecular dynamics (MD) simulations using a wedge-shaped monomer (Fig. 5a) have recently demonstrated that self-assembly into tubule structures depends on key factors such as molecular
Figure 5. (a) Image of chiral wedge MD model (grey wedge) with ideal pitch 3 tubule. Individual wedge monomers are alternately colored red, blue, and green to aid in visualizing monomers in the tubule assembly. Stage of self-assembly of wedge system into tubules and final tubules of pitch 2 formed from chiral 2 monomer; (b) Geometric model for formation of nanotubes from wedge-shaped lanreotide bilayers, where variations in the sizes of close-contact residues (red and orange dots) influences wedge shape and ultimately nanotube diameter (Tarabout et al., 2011) (Copyright 2011 National Academy of Sciences, USA); (c) Scheme illustrating stacking assembly of cyclized peptides into individual nanotubes and ordered parallel arrays (Hartgerink et al., 1996) (Copyright 1996 American Chemical Society); (d) Illustration of nanotube formation from positively charged amphiphilic peptide bilayers (von Maltzahn et al., 2003) (Copyright 2003 American Chemical Society); and (e) Proposed self-assembly of diphenylalanine into sheets, and the subsequent curling to form nanotubes (Carny et al., 2006) (Copyright 2006 American Chemical Society).
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orientation and the relative influences of both lateral (transverse to the tube) and vertical (along the tube length) molecular interaction strengths (Cheng et al., 2012; Cheng and Stevens 2014). MD has further shown that the pitch of the tubule can be controlled by the chirality of the monomer, but also that a twist deformation tends to yield a range of pitch values. An additional interaction in the vertical direction is required to prevent such twist, a discovery that not only informs synthetic designs, but adds to our understanding of assembly in natural MTs. Meanwhile, self-consistent field theory has provided insight into the potential cooperative roles of intramolecular and intermolecular interactions in determining the self-assembled morphologies (Ting et al., 2014). A number of the key results from these studies were consistent with experimental observations in a recent study of the self-assembly of wedgeshaped, branched peptides into one-dimensional nanofibers (Gough et al., 2014). In general, peptide-based systems are particularly attractive for the self-assembly of MT mimics, although polymers, lipids, DNA, and even nanoparticles have also shown promise for programmable assembly of tubular structures (Bong et al., 2001; Scanlon and Aggeli 2008; Wang et al., 2013; Yin et al., 2008). In peptide-based systems, the rich amino acid chemistry of peptides provides for an extremely versatile synthetic platform, and enables the introduction of biochemical characteristics potentially needed for subsequent biomimetic function. One interesting demonstration used wedge-shaped building blocks formed from b-sheet forming peptides (model shown in Fig. 5b) to successfully generate nanotubes with a tunable diameter (Tarabout et al., 2011), offering enticing parallels to the simulated system described above. There are, however, a remarkable number of additional configurations of peptide structures and compositions that lead to nanotube assembly (Gao and Matsui 2005; Ghadiri et al., 1993; Hamley, 2014; Sakai et al., 2008; Shimizu et al., 2005; Vauthey et al., 2002; Yan et al., 2010). Not only does the composition vary with each of these approaches, but the mechanisms for tube formation vary to include stacking of cyclic D-L peptide rings, rolling of self-assembled sheets or amphiphilic peptide bilayers, twisting of helical ribbons, or even organization of rigid molecular “staves” into tubules (Fig. 5c–e) (Carny et al., 2006; Hartgerink et al., 1996; von Maltzahn et al., 2003). Regardless of the ultimate mechanism involved, factors such as molecular morphology, amphiphilicity (solvation), hydrogen bonding, electrostatics, and the balance of lateral and vertical interactions play strong roles in determining the size, shape, and assembly behavior of these tubular structures, just as they do in the dynamic assembly of natural MTs. Certainly, the diversity of these supramolecular approaches holds promise for the development of biomimetic materials systems aimed at emulating complex materials such as MTs. Learning to control and tune the cooperative and competitive driving forces governing these self-assembling structures, however, will be critical to tailoring the ultimate biomimetic characteristics (Gao and Matsui 2005; Ghadiri et al., 1993; Sakai et al., 2008; Shimizu et al., 2005; Vauthey et al., 2002; Yan et al., 2010). While the experimental studies outlined above do provide compelling examples of tubule formation, for the deliberate design of structural or functional MT mimics, it is clear that the
cooperation of experimental work with theory and modeling stand to significantly streamline molecular design and improve our understanding of these molecular systems.
Conclusion and Future Challenges Biochemical and biophysical characterization of MT structure, dynamics, and function has contributed strongly toward the development of a wide range of multi-dimensional hybrid and/or composite nanomaterials. This review has briefly highlighted a number of efforts to use these remarkable biopolymer filaments as functional templates to direct uniquely bio-enabled nanomaterial growth and/or assembly. To date, the majority of this work, however, has relied on MTs that have been chemically stabilized to prevent depolymerization, which consequently eliminates one of the most unique and enabling properties (i.e., dynamic instability) of these filaments. Whereas cells rely on the highly coordinated action of stabilizers and destabilizers (van der Vaart et al., 2009), a key challenge moving forward will include the realization of ex vivo mechanisms to regulate dynamic instability, enabling novel materials that exploit the intrinsic dynamics of MTs. The use of microfluidics, for example, offers a potential approach to spatially and temporally regulate positive and negative effectors of MT stability, as well as to controllably introduce synthetic materials to form composite structures. Fundamental studies of MTs have also provided a critical knowledge base for designing and generating synthetic one-dimensional nanostructures that mimic various elements of the natural cytoskeletal filaments. The strong focus on recapitulating the tubular structure of MT filaments has established basic platforms and key design rules for generating one-dimensional nanostructures from a diverse array of molecular building blocks. Strategic challenges in the continued development of synthetic MT analogs include developing systems that display selfregulated assembly dynamics (i.e., artificial dynamic instability), as well as the ability to interface with synthetic motors, mimicking biomolecular active transport. Such attributes would significantly expand the functional nature of synthetic one-dimensional nanostructures, and enable the inclusion of revolutionary properties and behaviors. We thank Drs. Nathan Bouxsein and Brad Jones for their useful comments and suggestions in preparing this review. Support for the preparation of the manuscript was provided by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, Project KC0203010. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
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