Self-Organized Optical Device Driven by Motor Proteins

Self-organized optical device driven by motor proteins

Susumu Aoyama, Masahiko Shimoike, and Yuichi Hiratsuka1

School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved August 29, 2013 (received for review April 3, 2013) Protein molecules produce diverse functions according to their the methods required to create such systems, we decided to combination and arrangement as is evident in a living cell. create unique optical devices, inspired by fish melanophores. Therefore, they have a great potential for application in future Some species of fish, such as killifish and zebrafish, change devices. However, it is currently very difficult to construct systems their skin color depending on their surroundings (Fig. 1 A and in which a large number of different protein molecules work B). This camouflage phenomenon is related to the pigment cells, cooperatively. As an approach to this challenge, we arranged “melanophores,” that exist on the fish skin. The cell has a radial protein molecules in artificial microstructures and assembled an array of microtubules that elongate from the center; the minus optical device inspired by a molecular system of a fish melano- ends of the microtubules are located at the center and the plus phore. We prepared arrays of cell-like microchambers, each of ends are located along the periphery (18). Along these micro- which contained a scaffold of microtubule seeds at the center. By tubule networks, specific motor proteins deliver black pigment polymerizing tubulin from the fixed microtubule seeds, we granules, “melanosomes,” and alter their distribution. When obtained radially arranged microtubules in the chambers. We cytoplasmic dynein (a minus end-directed motor) is activated, subsequently prepared pigment granules associated with dynein the pigment granules are transported to and concentrated at the motors and attached them to the radial microtubule arrays, which center of the cell, and the melanophore becomes transparent. In made a melanophore-like system. When ATP was added to the contrast, activation of kinesin II (a plus end-directed motor) system, the color patterns of the chamber successfully changed, induces dispersion of the pigments, which darkens the melano- due to active transportation of pigments. Furthermore, as an phore (19–21) (Fig. 1 C–F and Movie S1). application of the system, image formation on the array of the In this study, using a microstructure that supports the arrange- optical units was performed. This study demonstrates that a prop- ment and self-organization of protein components, we created a erly designed microstructure facilitates arrangement and self- melanophore-like optical device, “artificial melanophore” (Fig. 1G). organization of molecules and enables assembly of functional The combination of microelectromechanical systems (MEMS) molecular systems. technology and self-organization of the proteins enables the easy fabrication of thousands of identical artificial melanophores. bioengineering | microdevice | molecular robotics We tried to create images on the array of artificial melanophores by controlling the pigment distribution of each unit (Fig. 1H). ithin a cell, motor proteins work as mechanical compo- Wnents that efficiently convert chemical energy to me- Results and Discussion chanical energy. Major motor proteins, such as myosin, kinesin, Design and Fabrication of Supporting Microstructures. A key struc- and dynein, travel unidirectionally along specific filamentous ture of a melanophore is the radial array of microtubules sur- protein polymers, actin filaments, or microtubules, using the rounded by the cell membrane. To construct this microtubule chemical energy derived from ATP. Although the action of structure, we referred to a previous microtubule-arranging motor proteins itself is rather simple, they are involved in nu- method, in which microtubules were elongated from short merous functions in living cells such as cell division, muscle microtubules (microtubule seeds) fixed on a glass surface (22). contractions, ciliary beating, and melanophore color changes (1). When the microtubule seeds are fixed within a small spot area, These diverse and elaborate functions are realized through the spot should function as an artificial microtubule-organizing highly ordered molecular systems that consist of not only the motor proteins but also various types of protein molecules. For Significance example, myosin and actin form alternatively arranged bundles with tens of other proteins to construct aligned sarcomeres, the Certain nanomaterials, such as protein molecules, produce basic units of the muscle, which produce efficient contractions various advanced functions when incorporated into an ordered + under strict Ca2 regulation (1). Likewise, in the cilium or flagel- system and, therefore, have large potential for use in engi- lum, dynein molecules are integrated into the “9 + 2” arrangement neering devices. To explore how to assemble a functional device of microtubules and generate oscillatory bending (1). Thus, di- from protein components, we have tried to create a molecular versity of in vivo functions of motor proteins is achieved by the device inspired by a fish pigment cell, “melanophore.” We in- variety of manners in which motor proteins are organized into duced ordered assembly of protein molecules through self- fi fi specific higher order systems. organization of the proteins in a speci c arti cial microstructure In the last decade, remarkable progress has been made in the and thereby succeeded in producing a melanophore-like optical applications of motor proteins in microscale and nanoscale en- device. We believe that self-organization of molecules in micro- gineering, which has enabled the control of motor protein structures can be a powerful method for assembling functional molecular systems in future nanotechnology. movements and the transport of artificial objects by motor pro- – tein (2 14). These microtransportation systems are expected to Author contributions: Y.H. designed research; S.A. and M.S. performed research; and S.A. be a shuttle for micrototal analysis systems and other simple and Y.H. wrote the paper. tools (15–17). To fully use the potential of motor proteins in The authors declare no conflict of interest. artificial systems, it is necessary to develop higher-level func- This article is a PNAS Direct Submission. tional systems. However, simply mixing protein components Freely available online through the PNAS open access option. rarely forms an ordered system, and, therefore, organizing motor 1To whom correspondence should be addressed. E-mail: [email protected]. protein molecules with other associated proteins into highly or- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dered structures is a key bioengineering challenge. To explore 1073/pnas.1306281110/-/DCSupplemental.

16408–16413 | PNAS | October 8, 2013 | vol. 110 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1306281110 Downloaded by guest on October 2, 2021 to the glass surface of circular hydrophilic patterns due to their negative charge, we used a nonmotile mutant kinesin T93N, which binds to both microtubules and hydrophilic surfaces without motility (26). By including a surfactant (0.1% Brij35), which selectively blocks protein attachment to hydrophobic surfaces (4), we were able to attach the T93N kinesin molecules selectively to the circular hydrophilic patterns (Fig. 2 A2 and F, Left, and Fig. S1F). We subsequently introduced microtubule seeds and anchored hun- dreds of them on the circular hydrophilic patterns via the mutant kinesin molecules (Fig. 2 A3 and F, Center Left, and Fig. S1G). We will refer to the circular hydrophilic patterns as seeding zone. Next, we polymerized tubulin from the fixed seeds to assemble the radial microtubule arrays. In the ordinary tubulin polymerization method, microtubules are grown toward both plus and minus ends (27), which causes a mixed polarity in the resultant radial microtubule arrays. To avoid this problem, we polymerized tubulin in the presence of N-ethylmaleimide–modified tubulin, which inhibits microtubule growth from the minus ends (28, 29) so that the microtubules elongated only toward their plus ends (Fig. 2C). Because length of microtubules was almost proportional to polymerization time (Fig. 2 D and E and Fig. S2A), we optimized the polymerization time so that the plus ends of the majority of microtubules just reach the edge of the chambers. As a result, radial arrays of polarity-arranged microtubules were successfully assembled in the chambers (Fig. 2 A4 and F, Center Right and Right, Figs. S1H and S2B, and Movie S2 and Movie S3). In a typical radial microtubule array, the number of microtubules was 200–500, as estimated from the fluorescence Fig. 1. Molecular system of fish camouflage and design of an optical sys- intensity of the microtubule seeds. The length of the micro- tem. (A and B) Zebrafish in a dark and a bright field. (Scale bar: 1 cm.) (C and tubules was about 15–20 μm. Most of the microtubules (about D) Microscopic images of the skin of a zebrafish. Pigment cells (melano- 70%) were elongated approximately horizontally (<30°) and phores) change the color of the skin by dispersion (C) or aggregation (D)of were contained in the chamber. pigment granules (melanosomes). (Scale bar: 50 μm.) (E and F) The mecha- nisms of color change of a melanophore. In a melanophore, motor proteins Preparation of Motor-Associated Pigment Granules. Previously, many transport pigment granules along radially arranged microtubules, which researchers have succeeded in transporting artificial microobjects, SCIENCES induces dispersion (E) or aggregation (F) of pigments. (G) Design of an op- fi such as polystyrene beads, Au- or Q-dots, or photolithographically tical system mimicking a sh melanophore. With the support of a micro- fabricated microstructure, with motor protein physically absorbed APPLIED BIOLOGICAL structure, the protein components form a melanophore-like ordered system in a self-organizing manner. (H) The concept of a protein-based display. In or chemically linked to the surface of them (3, 6, 7, 10, 13). an array of melanophore-like optical units, activation of a specific group of However, our system required all granules to be transported along units produces a picture. a microtubule toward its minus end without detachment, which necessitated developments of unique, robustly motile granules. This prompted us to focus on the complex of the axonemal center (MTOC). By growing microtubules from those MTOCs, dynein and the microtubules. As reported previously, flagellar radially arranged microtubules can be obtained (23, 24). We outer-arm dynein molecules are aligned on a microtubule in a self- have adopted this strategy and fabricated microchambers that organizing manner with the interval of 24 nm (30, 31). Further- included a scaffold to fix microtubule seeds at the center (details more, the dense cluster of dynein molecules on a microtubule of fabrication methods are available in SI Materials and Methods). enables the complex to travel a long distance along another mi- By reference to the size of fish melanophores, we designed a crotubule (>10 μm) toward the minus end in the presence of ATP microchamber hexagonally surrounded by partition walls whose (32, 33). For example, ∼40 dynein molecules can be mounted radius and height were 26 and 7 μm, respectively, and photo- linearly even on 1-μm microtubule fragments and interact with lithographically fabricated the chambers of thick photoresist SU8 a track microtubule cooperatively. We thought such dynein– on a glass surface (Fig. S1A). To create scaffolds for seeds, we microtubule complexes could be used as robustly motile granules overlaid the removable photoresist on the chamber structures suitable in our system (Fig. 3A). and executed oxygen plasma etching through circular patterns of We prepared fluorescently stained microtubule fragments from overlaid photoresist (Fig. S1 B–D). Removing the overlaid pho- fluorescent tubulin as “pigments.” We mixed the microtubule toresist, we eventually obtained hydrophobic microchambers, each fragments with crude axonemal dynein extracted from Chlamy- containing a hydrophilic circular scaffold area (5.5-μmradius)at domonas. At this point, outer-arm dynein molecule should be the center (Fig. 2 A1 and B and Fig. S1E; the hydrophilic scaffold associated with a microtubule at its ATP-dependent motor stalk area cannot be discerned in Fig. 2B). The difference between head and/or at its ATP-independent stem (31). To dissociate the hydrophilic and hydrophobic surfaces was used to control the ATP-dependent binding, we added ATP to the mixture of dynein selectivity of protein attachment to the surfaces in the later and microtubules. The resulting complexes should interact with processes. a track microtubule at the motor domain of dynein molecules. Immediately after the removal of the ATP by ATPase ac- Assembly of a Radial Array of Microtubules. We prepared micro- tivity of apyrase, we introduced the motile pigment granules to tubule fragments (seeds) by polymerizing tubulin with guanosine- the chambers containing a radial microtubule array. The pig- 5′-(α,β-methyleno)triphosphate (GMPCPP), which promotes ments were randomly attached to the arranged microtubules, nucleation of tubulin but inhibits the elongation of microtubules mimicking the basic structure of a melanophore consisting of (25). Because the microtubule fragments could hardly get attached a radial microtubule array, motor proteins, and pigment granules

Aoyama et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16409 Downloaded by guest on October 2, 2021 Fig. 2. Fabrication of a radial microtubule array in a microchamber. (A) Fabrication process of a radial microtubule array (side view of a wafer). (A1) Partition walls of the microchambers were photolithographically fabricated on a glass surface. The fabricated walls formed hexagonal chambers and each chamber had a hydrophilic pattern (seeding zone) at the center. (A2) Nonmotile mutant kinesin (blue dots), which serves as an anchor between the surface and the seeds, was selectively attached to the seeding zone in the presence of a surfactant. (A3) Microtubule fragments (red dots) were fixed via the anchor molecules as seeds of microtubule assembly. (A4) Microtubules (green lines) were grown from seeds, which made a radial microtubule array in the chamber. (B) An SEM image of the fabricated microchambers. (Scale bar: 10 μm.) (C) Microtubules (green lines) elongated from the microtubule seeds (yellow dots). Note that the microtubules were grown only from one end (the plus end) of short microtubules. (D) A histogram of the length of the microtubules that were polymerized for different times. (E) The time course of the length (the average and SD) of microtubules in tubulin polymerization. (F) Microscopic images of a radial microtubule array fabricated in a chamber. T93N kinesin-CFP (blue; Left), microtubule seeds (red; Center Left), a microtubule array (green; Center Right), and a merged image (Right) are shown. Hexagonal fluorescent pattern is the autofluorescence of photoresist. (Scale bar: 10 μm.)

(Fig. 3B and Fig. S1I). In our optimized condition, about 0 to estimated to be about 500–1,000. Despite the high density of three pigments were attached to each elongated microtubule, arranged microtubules at the center, the distribution of pigments and the total number of pigments in a single chamber was was not much biased probably because the high density of

Fig. 3. Transportation of the pigment granules along the radial microtubule array. (A) Illustration of the pigment transportation on an elongated microtubule. Dynein molecules transport a pigment (ﬂuorescent microtubule fragment) toward the minus end of the microtubule (toward the center of the chamber). (B) Fluorescence images of pigment attachment to a radial microtubule array. A radial microtubule array (Left) and pigments attached to the array (Center) and a merged image (Right) are shown. (C) Time-lapse microscopic image sequence of transportation of pigments in a radial microtubule array. ATP was generated by photolysis of caged ATP, which was included in the chamber, to activate the motor protein dynein. The number in each picture represents the time (in seconds) after the UV ﬂash. (Scale bar: 10 μm.)

16410 | www.pnas.org/cgi/doi/10.1073/pnas.1306281110 Aoyama et al. Downloaded by guest on October 2, 2021 microtubules prevented the pigments from diffusing into the device. We fabricated a honeycomb array of about 7,500 artificial dense array. melanophores on a 4 mm × 4 mm area of a glass surface. In this array, each melanophore chamber, activatable with a UV flash, Color Pattern Change in a Melanophore-Like System. To activate the acts as one pixel of a display. dynein-associated pigments without exchanging the solutions, we We aimed to create an image on the melanophore array by added photoreleasable ATP (0.5 mM caged ATP) to the buffer. irradiating selected artificial melanophores with UV light (Fig. When ATP was released by 350-nm UV light irradiation, almost 4A). For that purpose, we first prepared several photomasks all of the pigment granules were transported toward the seeding through which UV was flashed (Fig. S4A). When an artificial zone at the center of the chambers along the radially arranged melanophore array was exposed to patterned UV through microtubules, although some pigments were motionless or dis- a mask, the mask pattern was copied on the array transiently, but sociated from the microtubules (Fig. 3C and Movie S4). Velocity the picture was rapidly blurred presumably due to diffusion of of pigment transportation was about 13 μm/s, and almost all released ATP (Fig. S4 B, D, and F). Therefore, we covered the motile pigments aggregated in the seeding zone within 10 s. The top of chambers with pentadecane oil and obtained an array of aggregated pigment granules continued to move in random artificial melanophores that were separated from each other directions within the seeding zone as long as ATP existed, which (Fig. S1J). Using this array, we succeeded in changing the color presumably resulted from repetition of transportation, dissociation, patterns only in the chambers that were exposed to UV (Fig. 4B, and reassociation of pigment granules in the dense microtubule Figs. S1 K and L and S4 C, E, and G, and Movie S5). Thus, we network. When ATP was exhausted by dynein and apyrase, pig- were able to accurately copy the mask pattern, such as simple ments were immobilized in the seeding zone with the rigor cross- graphics or English letters, on the screen of the artificial mela- bridges between dynein molecules and microtubules. As a result nophores (Fig. 4C and Fig. S5 A and B). of pigment aggregation, distribution of fluorescence was changed in the chambers, and the brightness was greatly increased only Contrast of Images Displayed on the Artificial Melanophore Array. at the center of the chambers (Fig. S3). We call this system The contrast between the bright and dark pixels of displayed “artificial melanophore.” images depended on several factors, related to microtubule arrays and pigment granules that composed each artificial me- Portrayal of Pictures on an Array of Optical Units. As an application lanophore (Fig. S6). Regarding a microtubule array, the number of the artificial melanophore, we tried to create an image display and length of aligned microtubules were important. A low SCIENCES APPLIED BIOLOGICAL

Fig. 4. Image portrayal on an array of artificial melanophores. (A) Schematic demonstrating the technique used to display a picture on an array of artificial melanophore chambers. When the UV light was flashed through a patterned photomask on a melanophore layer, which contained caged ATP, the color patterns in the chambers exposed to UV were changed, thereby making a copy of the mask pattern on the melanophore screen. (B) The boundary between the UV-exposed area and the UV-unexposed area of an artificial melanophore array. The pigment granules aggregated only in the UV-exposed area (Right) because the chambers were sealed with oil to prevent the ATP from diffusing. This figure is a magnified image of a part of C.(C) Biodisplay composed of thousands of artificial melanophore chambers. Each 50-μm chamber functioned as a pixel, and the two colors in the chambers created the pictures on the screen (Fig. S5 A and B). The picture was captured as ∼300 contiguous images and was subsequently reconstituted (Materials and Methods or SI Materials and Methods), and the figure image has “seams” as the boundaries between capture images. (Scale bar: 1 mm.)

Aoyama et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16411 Downloaded by guest on October 2, 2021 number (<100 per chamber) of microtubules resulted in paucity aligned microtubules had the same polarity as that of living cells of attachment of the pigment granules, which attenuated the and functioned well as a fixed track for transportation. In addi- color change of a chamber. To avoid this problem, we had to fix tion, the number, the length, or the position of microtubules is a sufficient number of microtubule seeds on the seeding zone by controllable to some extent. However, our method consists of adjusting the concentration and the size of the seeds. In addition, several distinctive steps and is not very simple to perform. A the appropriate length of microtubules was required so that the possible alternative to assemble microtubule arrays is to induce array just fits in the chamber (25-μm radius). On one hand, too self-organization of microtubules with oligomers of motor pro- short microtubules generated areas devoid of pigments along the tein. Nédélec et al. (37) reported that a mixture of microtubules periphery of the chamber. On the other hand, when micro- and kinesin oligomers tethered with tetrameric streptavidin tubules were too long, some pigments lost motility, presumably could be dynamically arranged into a radial microtubule array in because long microtubules protruded into the oil area. Although a microchamber. Furthermore, in this system, motor oligomers we were able to control the average length of microtubules by aggregate at the center of the microtubule array during the as- changing the time of polymerization, the size of the seeding zone sembly processes. Therefore, simply associating pigment gran- (5.5-μm radius) created an additional problem. The distance ules with the motor oligomers may enable the motile system to from the periphery of a chamber to the near edge of the seeding accomplish color change. This is certainly a simple and attractive zone was 19.5 μm, whereas that to the far edge was 30.5 μm, so alternative approach that is worth pursuing in the future. that the optimum length of a microtubule varies depending on As to the system design, reversibility is the most important where and to which direction it elongates within the seeding subject to be challenged. Our artificial melanophore system can zone. Smaller seeding zones have less of this problem but result display any monochrome image but cannot show a second image in fewer attached seeds and, consequently, fewer microtubules. after erasing the first image. To make this display reversible, we The size of the current seeding zone (5.5 μm) was chosen as need to add a system to redisperse pigment granules. In a fish a compromise between those two factors that influence the melanophore, a pigment granule is associated with not only dy- contrast of the images. The number of pigment granules also nein, but also kinesin and myosin, which enables bidirectional significantly affected the clarity of images: not only the paucity of transport of pigments in the network of cytoskeleton and thereby pigments but also their excess degraded the contrast of the pic- accomplishes reversible color change of the cell (19, 34, 35). ture image. This is because too many pigment granules were not Although it is currently difficult to disperse pigments actively contained within the seeding zone after aggregation, which using motor protein, which requires independent regulation of resulted in poor convergence of brightness. The current number multiple motor proteins within the chamber, Brownian diffusion (0 to three pigments per microtubule), achieved by adjusting the can also be used to disperse pigments. For example, our pigment concentration of pigments and incubation time for attachment, granules should be detached from track microtubules by addition was optimized for the 5.5-μm seeding zone. of ATP analogs, such as ADP-vanadate or adenosine-5′-(β,γ-imido) Our present optical system has three main areas for im- triphosphate (AMPPNP), because binding affinity between an ax- provement of image quality, which, in fish melanophores, are onemal dynein molecule and a microtubule significantly decreases properly solved. One is that the total amount of brightness in in the presence of those compounds (38). If the concentration of each chamber does not change much before and after the ag- those chemicals can be changed by external stimuli such as light, gregation of pigments, attenuating the contrast of images. In fish temperature, or magnetic field, pigment granules will be dispersed melanophores, the total amount of brightness changed signifi- by diffusion throughout the chamber within 10 min. Thus, the cantly by the aggregation of pigment granules because the color system will be reset to the original state and become ready to change is generated by movements of granules that absorb light, show a second image. rather than those that emit light. Hence, the development of nonluminous, light-absorbing “pigment” in its original sense should Potentials of Molecular Assembly Supported by Artificial Microstructures. greatly improve the image quality in our system. Second, a lower In living organisms, functional molecular systems are assembled density of arrayed microtubules in the peripheral region of in a self-organizing manner involving many regulatory proteins. a chamber is reducing the magnitude of color changes in that area. However, it is extremely difficult to artificially build protein-based In fish melanophores, network of actin filaments is used to fill the functional systems through similar processes because the bio- gaps between the microtubules, along which pigments associated logical assembly process is complicated and is not yet adequately with myosin V are transported (34, 35). Although it is not easy to understood. Hence, we have induced arrangement and self- incorporate actin–myosin motile system in our current system, it organization of protein molecules with the support of an artificial may be feasible to form similar microtubule networks that fill the microstructure. In this method, once the supporting micro- gaps between arrayed microtubules. For example, γ-tubulin adds structures are prepared, functional molecular systems can be branched microtubules on existing microtubules (36), which will created by simple addition of components. We believe that capture pigments in the peripheral region and support their molecular assembly supported by artificial microstructures will transport to the seeding zone when ATP is supplied. Third, the enable us to fabricate a variety of micromachines in broad en- ratio between the area of seeding zone and the total chamber area gineering fields. should affect the extent of color change. The ratio in our system (10–15%) was similar to or smaller than that in fish melanophores Materials and Methods (10–25%). Therefore, the superior performance of the fish mel- Details on experimental materials and methods are presented in SI anophores indicates that factors mentioned above have greater Materials and Methods. A summary is given below. impact on the performance than the area ratio. Partition wall of the hexagonal chambers was fabricated by photoli- thography of negative photoresist SU8-5 (MicroChem). Hydrophilic circular Further Sophistication of Our Optical Device. In this study, we suc- patterns located at the center of the chamber were created by O2 plasma cessfully assembled an optical device from protein components etching through a temporal patterned layer of S1818 photoresist (Rohm and combined with MEMS technologies. However, there are a num- Haas Electronic Materials). The gene of T93N mutant human conventional kinesin was obtained using QuikChange mutagenesis kit (Stratagene), and ber of ways for further sophistication in terms of assembly was expressed in Escherichia coli Rosetta (DE3) pLysS (Novagene). Dynein methods and the design. was extracted from the flagellar axoneme of Chlamydomonas reinhardtii We assembled the radial microtubule arrays by polymerizing 137c in 0.6 M KCl buffer. Tubulin was extracted from porcine brains by cycles tubulin from artificial MTOCs. We think that this is one of the of polymerization and depolymerization, and was purified using phospho- most useful ways to assemble microtubule arrays. In fact, the cellulose P11 (Whatman). Purified tubulin was stained with 10 mM reactive

16412 | www.pnas.org/cgi/doi/10.1073/pnas.1306281110 Aoyama et al. Downloaded by guest on October 2, 2021 fluorescent dye carboxytetramethylrhodamine (Invitrogen; C1171) or Alexa fluorescence of the artificial melanophores was observed using an inverted Fluor 488 (Invitrogen; A20000). The tubulin was polymerized in buffer microscope (IX71; Olympus) equipped with a highly sensitive color CCD including 1 mM GMPCPP and was stabilized with 10 μM Taxol to obtain camera (MLX, nac). The pictures displayed on the 4 × 4 mm array of the microtubule fragments (short microtubules). Artificial melanophores were artificial melanophores were captured as 300 contiguous images and were fabricated in the microchambers by sequential introduction of protein subsequently reconstituted. components (see Assembly of a Radial Array of Microtubules and Prepara- tion of Motor-Associated Pigment Granules). UV light, which was used to ACKNOWLEDGMENTS. We thank C. Tatsumi for technical assistance. This study fi fl photolyze caged ATP and activate the arti cial melanophores, was ashed was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI through an objective lens (UPlanFLN60X or UPlanSApo4X; Olympus) from Grant 20651038 and partially supported by Japan Science and Technology a mercury arc lamp equipped with an electronic shutter. The change in Agency (JST) Precursory Research for Embryonic Science and Technology.

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