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From isolated structures to continuous networks: A categorization of cytoskeleton‐based motile engineered biological microstructures

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As technology at the small scale is advancing, motile engineered microstructures are becoming useful in drug delivery, biomedicine, and lab‐on‐a‐chip devices. However, traditional engineering methods and materials can be inefficient or functionally inadequate for small‐scale applications. Increasingly, researchers are turning to the biology of the cytoskeleton, including microtubules, actin filaments, kinesins, dyneins, myosins, and associated proteins, for both inspiration and solutions. They are engineering structures with components that range from being entirely biological to being entirely synthetic mimics of biology and on scales that range from isotropic continuous networks to single isolated structures. Motile biological microstructures trace their origins from the development of assays used to study the cytoskeleton to the array of structures currently available today. We define 12 types of motile biological microstructures, based on four categories: entirely biological, modular, hybrid, and synthetic, and three scales: networks, clusters, and isolated structures. We highlight some key examples, the unique functionalities, and the potential applications of each microstructure type, and we summarize the quantitative models that enable engineering them. By categorizing the diversity of motile biological microstructures in this way, we aim to establish a framework to classify these structures, define the gaps in current research, and spur ideas to fill those gaps. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Nanotechnology Approaches to Biology > Cells at the Nanoscale Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies
The motile cytoskeleton. Schematics of the filaments and motors that make up the motile cytoskeleton. Microtubules are comprised of α‐ and β‐tubulin dimers (red and orange), and actin filaments are comprised of g‐actin subunits (orange). Representatives of each motor protein superfamily are shown: Kinesin‐1 (dark blue) is a homodimer, outer arm axonemal dynein (green) is a heterotrimer, and a myosin V (light blue) is a homodimer that is shown decorated with associated light chains. Below are images of example cytoskeletal microstructures found in cells. Shown are the two flagella of a Chlamydomonas reinhardtii (green algae) cell (Geyer, Sartori, Friedrich, Jülicher, & Howard, ) (Reprinted with permission from Geyer et al. (). Copyright 2016 Elsevier); lamellipodia (LP) and filopodia (FP) in migrating goldfish fibroblast cells (Reprinted with permission from Nemethova, Auinger, and Small (). Copyright 2008 Royal Society of Chemistry); the mitotic spindle of a rat‐kangaroo epithelial kidney cell at metaphase with microtubules (red), kinetochores (green) and DNA (blue) all stained for structured illumination microscopy (Stout & Walczak, ); and the cytokinetic ring (red) of a Drosophila sperm cell undergoing meiosis (Giansanti, Sechi, Frappaolo, Belloni, & Piergentili, ) used by license under a Creative Commons Attribution‐NonCommercial 3.0 Unported license
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Examples of synthetic isolated structures. (a) Artificial cilia. The cilia in this scanning electron micrograph are composed of maghemite nanoparticles embedded in PDMS pillars. While engineered to function like biological cilia, this construct contains no biological building blocks (Reprinted with permission from Shields et al. (). Copyright 2010 Proceedings of the National Academy of Sciences of the United States of America; PNAS). (b) a nanoparticle (yellow) functionalized with a DNA‐based molecular motor comprised of a catalytic core (green) and two recognition arms (red) moves along a carbon nanotube track (black), shown as a molecular model (left) and series of schematics (right). The motor converts the chemical energy of RNA into mechanical motion through a series of DNA conformation changes, walking along the nanotube track processively and autonomously, similar to intracellular protein motors. (Reprinted with permission from Cha et al. (). Springer Nature Customer Service Centre GmbH: Springer Nature Nanotechnology)
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Examples of hybrid isolated structures. (a) Structures formed by conjugation of quantum dots onto a microtubule‐motor protein structure, resulting in a self‐assembling ring. In this case, an otherwise entirely biological structure is being used as a carrier for a synthetic particle, making the structure a true hybrid (Reprinted with permissions from Liu et al. (). Copyright 2008, John Wiley & Sons., Inc.) (b) WASP‐coated beads were incubated with rhodamine‐labeled actin and Arp2/3 complexes to allow for actin assembly. These isolated aster‐like structures formed upon the addition of fascin (left, scale bar is 5 μm). Electron microscopy of the star bundles revealed a filopodia‐like isolated microstructure (right, scale bar is 100 nm). (Panels were reprinted with permission from Vignjevic et al. (). Copyright 2003. Journal of Cell Biology and Rockefeller University Press)
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Examples of hybrid clusters. (a) Tri‐block copolymer‐stabilized droplets are comprised of non‐ionic PFPE–PEG–PEPE surfactant and contain microtubules (red) and kinesin‐14 motor proteins functionalized with non‐motor microtubule binding domains (green). Under the correct conditions, hybrid clusters that resemble asters can be formed. Scale bar is 100 μm. (Reprinted with permission from Juniper, Weiss, Platzman, Spatz, and Surrey (). Copyright 2018 The Royal Society of Chemistry and Creative Commons Attribution 3.0 Unported License). (b) Interconnected actin‐based clusters formed from Arp2/3 coated beads in brain extract. Scale bar is 50 μm. (Reprinted with permission from Vignjevic et al. (). Copyright 2003 Journal of Cell Biology and Rockefeller University Press)
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Examples of modular clusters. (a) Multimeric kinesin‐1 and minus‐end directed Ncd motors crosslink microtubules and organize them into aster‐like clusters in the presence of ATP. By modulating native kinesin‐1 motors, which normally bind to only one microtubule, into multimeric complexes and changing the concentration of these and other elements of the system, microscale modular clusters resembling vortices, asters, and interconnected networks can be generated (Reprinted with permission from Surrey et al. (). Copyright 2001 AAAS). (b) Time series evolution of modular cluster formation using taxol‐stabilized ATTO647N‐labeled microtubules (magenta), ATTO565‐labeled microtubules (yellow), and KIF5Bhead‐Eg5tail chimera motor proteins (cyan). (This panel was reprinted with permission from Torisawa et al. (). Copyright 2016 Attribution‐NonCommercial‐NoDerivatives 4.0 International Creative Commons License (CC BY‐NC‐ND 4.0)
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Examples of entirely biological isolated structures. Askew (left) and parallel (right) structures made from axonemal dynein motor proteins and rhodamine‐labeled microtubules. These structures form when motors and filaments are combined in solution. Imaged using fluorescence microscopy and the scale bar is 10 μm in both images
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Example entirely biological networks. (a) Nearly undiluted meiotic Xenopus egg extract plus taxol rapidly forms an essentially continuous, stabilized microtubule network. Dynein‐driven motility leads to a contraction of the microtubule network confined in a flow channel over time. W(t) is 1.51 mm, 1.22 mm, and 0.78 mm at t = 0, 2, and 4 minutes, respectively, and the scale bar is 500 μm. (Reprinted with permission from Figure 2(a) of Foster et al. () and reproduced under the CC by 4.0 international license ‐ https://creativecommons.org/licenses/by/4.0/). (b) Reconstituted contractile actomyosin cortex. Alexa‐568–labeled F‐actin associates with a supported lipid bilayer coated surface. (c) F‐actin (red) and myosin II (green) are shown in the reconstituted cortex immediately after myosin thick filament formation. Panels (b) and (c) are reprinted with permission from Figure 1 of Murrell and Gardel (), and the scale bars on both panels are 10 μm
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Schematics of engineered biological microstructure types. (a) Entirely biological network: Dynein motor proteins accumulate at the minus end of microtubules (inset), which causes a microtubule network to contract as these dyneins walk toward the minus end of neighboring microtubules upon the addition of ATP (Foster, Fürthauer, Shelley, & Needleman, ). (b) Entirely biological cluster: Kinesin‐5 motor proteins crosslink and slide neighboring microtubules (inset), which causes otherwise dissociated microtubules to coalesce into aster‐like structures (Torisawa, Taniguchi, Ishihara, & Oiwa, ). (c) Entirely biological isolated structure: Axonemal dynein motor proteins crosslink microtubules (inset) into parallel or askew single structures. (d) Modular network: Truncated kinesin‐1 motor proteins are complexed using biotin and streptavidin (inset) to form a tunable motile gel that exists in a dynamic, turbulent state limited only by the availability of ATP (Henkin, DeCamp, Chen, Sanchez, & Dogic, ). (e) Modular cluster: Truncated kinesin‐1 motor proteins are complexed using biotin and streptavidin and used in conjunction with multimeric minus‐end‐directed kinesin‐14 (ncd) motor proteins complexed using GST domains and anti‐GST antibodies (inset) to cause the formation of interconnected asters (Surrey, Nédélec, Leibler, & Karsenti, ). (f) Modular isolated structure: A liposome attached to a microtubule with single‐stranded DNA in a kinesin‐driven gliding assay‐like system (inset) transports cargo (Hiyama et al., ). (g) Hybrid network: A tunable gel‐like network, similar to panel (d), functionalized with synthetic nanoparticles (inset) could be used to deliver drugs conjugated to the nanoparticles throughout the network. (h) Hybrid cluster: MAPs (shown in teal) attach microtubules to synthetic microspheres that are functionalized with nanocrystals to form a non‐motile hybrid cluster (Spoerke, Boal, Bachand, & Bunker, ). (i) Hybrid isolated structure: Artificial cilia are created by fixing microtubules with kinesin‐1 motors conjugated to a polystyrene bead (purple). Neighboring microtubules are crosslinked, and motility is driven by the kinesins. Methylcellulose (teal) is used as a crowding agent to promote crosslinking of neighboring microtubules (Sasaki et al., ). (j) Synthetic network: Polymer gels with spirobenzopyran chromophores and ruthenium catalysts (blue and green, inset) drive the Belousov‐Zhabotinsky reaction leading to oscillatory motility in the active material (Kuksenok & Balazs, ). (k) Synthetic cluster: Pluroinic F127‐DA micelles, which form in the presence of the activated photo‐initiator Irgacure 2,959 (purple), crosslink to form potentially functional clusters (X. Liu et al., ). (l) Synthetic isolated structure: Biomimetic cilia made from a magnetic nanoparticle‐polydimethysiloxane (PDMS) composite material (inset) can be actuated with a magnetic field (Shields et al., ). In all panels, dyneins (shades of green dots), kinesins (dark blue), synthetic particles (shades of purple dots), DNA (red lines), synthetic filaments (dark blue and dark green lines), and microtubules (orange lines) are as shown. Arrows in all panels indicate the direction of motility, and schematics are not to scale
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Gliding assays are motile engineered biological microstructures. (a) Schematic of an example gliding assay in which kinesin‐1 motor proteins (dark blue) are attached to a microscope coverslip (black). Upon the addition of ATP, the kinesins walk along a microtubule (red and orange) toward its plus end (indicated), and the microtubule moves with its minus end (indicated) leading. The arrow indicates the direction of microtubule motility. (b) Schematic of a self‐powered microtubule‐creatine phosphate kinase (CPK) engineered cargo delivery system gliding on the kinesin‐modified surface (Reprinted with permission from Jia, Dong, Feng, Li, and Li (). Copyright 2014 Royal Society of Chemistry). (c) Rhodamine‐labeled (red) stable microtubule seeds were extended with fluorescein‐labeled (green) tubulin to demonstrate that microtubules can self‐assemble into a completely overlapping network in confining channels. Scale bars are 15 μm. (Reprinted with permission from Doot, Hess, and Vogel (). Copyright 2007 Royal Society of Chemistry)
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