NANO LETTERS
Photopatterning of Actin Filament Structures
2005 Vol. 5, No. 4 625-628
Allen P. Liu† and Daniel A. Fletcher*,†,‡ Biophysics Program & Bioengineering Department, UniVersity of California at Berkeley, and Physical Biosciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received December 20, 2004
ABSTRACT We report a general strategy for spatiotemporal control of actin polymerization in vitro using photoactivatable actin. Caged actin was synthesized by chemically modified lysine residues on monomeric actin and released with focused ultraviolet (UV) illumination. Epifluorescence microscopy revealed nucleation and elongation of individual actin filaments (8 nm in diameter) after localized release of caged actin. We also used this strategy to generate branched filament structures by releasing caged actin in the presence of actin binding proteins. Controlled self-assembly of actin filaments represents a versatile “bottom-up” technique for constructing structural building blocks and functional templates for nanoscience applications.
Nanoscale devices have the potential to improve technology in fields ranging from information technology to health care, but synthesis, organization, and integration of nanoscale building blocks to create working devices remain a challenge.1 The size limitations of traditional “top-down” fabrication methods based on lithography and etching are being addressed by the emerging “bottom-up” fabrication paradigm. Functional nanoscale devices based on components such as nanotubes (NTs) and nanowires (NWs) have been created, including field effect transistors, sensors, light emitting diodes, and address decoder.2-5 More recently, biological macromolecules such as DNA, microtubules, and actin, which offer the advantage of specific attachment sites for functionalization, have been used as templates for computing, barcodes, transport, and nanowires.6-11 However, the complexity and density of integrated nanoscale devices based on either semiconductor materials or biological macromolecules is limited by the ability to spatially control assembly of individual components. Here we describe a method for generating spatially localized actin-based structures by photolysis of caged actin. In the presence of specific actin binding proteins, local release of actin by ultraviolet (UV) light illumination is shown to create stabilized single filaments as well as branched structures. While uniform growth of actin filaments in vitro is well understood,12,13 optical control of filament growth provides a new method for spatially and temporally organizing filaments for nanoscale fabrication applications. * Corresponding author. E-mail:
[email protected]. † University of California at Berkeley. ‡ Lawrence Berkeley National Laboratory. 10.1021/nl0478878 CCC: $30.25 Published on Web 03/03/2005
© 2005 American Chemical Society
Actin participates in numerous protein-protein interactions,14 making it an attractive template for constructing complex structures at the nanoscale. A ubiquitous cytoskeletal protein in eukaryotes, actin monomers (G-actin) selfassemble into double helical filaments (F-actin) that are 8 nm in diameter with lengths up to several microns. F-actin is a major constituent of the cell’s cytoskeleton that plays an essential role in motility and is functionally and structurally different on its two ends. In cells, actin organizes into dendritic networks and stabilized bundles through interaction with various actin binding proteins. Assembly of F-actin in vitro can be qualitatively described as having three phases: (i) nucleation, (ii) elongation, and (iii) steady state, similar to the preparation scheme of semiconducting nanoscale wires in which a nanometer-scale catalyst (nucleator) can form a liquid solution with the nanowire material whose axial growth (elongation) requires the addition of material.15,16 Prior to elongation of actin filaments, a nucleus consisting of three actin monomers must form. Formation of the nucleus is thermodynamically unfavorable, causing the lag phase observed during actin polymerization.17 The favorable elongation phase follows nucleation and continues until the local concentration of actin decreases to the critical concentration, which is 0.1 µM for the fast-growing end of the filament.12 Caged actin is polymerization incompetent, meaning it is unable to form nuclei or elongate, until illumination with UV light removes the modified group (Figure 1A). By spatially controlling UV illumination, the conditions for F-actin nucleation and elongation can be spatially controlled. As released actin monomers assemble into filaments, auxiliary proteins in solution that bind to the growing filament
Figure 1. Photo patterning of actin filaments. (A) Control of actin polymerization with UV illumination. Caged actin (purple around blue circle) is polymerization incompetent until the caging group is removed by UV illumination. The polymerized filament (blue circles) is subsequently stabilized and visualized by the fluorescent phalloidin (green double circles). (B) Schematic diagram of the photolysis experiment. UV light is delivered through a 100× objective over a region controlled by the field stop aperture in the illumination path. Elongation of filaments (blue lines) is observed in the field of view where UV illuminates, whereas regions away from UV illumination do not contain actin filaments. (C) Calculated concentration profile of diffusing actin vs position at the onset of photolysis and at later times, assuming initial release of 0.5 µM. Black, time 0; blue, 1 min; red, 10 min. The dotted green line depicts the critical concentration below which actin filaments do not elongate.
can be used to control geometry. In our experiments, phalloidin, a heptadicyclopeptide from the mushroom Amanita phalloides that binds specifically and tightly to F-actin but not G-actin,18 was present in solution for two reasons: (i) when phalloidin is bound to F-actin, it stabilizes the actin filament by reducing the dissociation rate constants for monomers from the ends of filaments19 and (ii) fluorescently labeled phalloidin can be used as tracer molecules to monitor filament growth. Additional actin binding proteins can be used to organize actin filaments into higher order structures, similar to the architecture of cellular actin networks. For example, branched structures were generated in our experiments by binding of Arp2/3 complex to growing filaments. Arp2/3 complex is a branching protein that binds to F-actin and creates a 70° branch between the mother and the 626
daughter filaments. It is believed that the Arp2/3 complex mediates the dendritic nucleation of the actin network in a cell.20 As an initial demonstration of the technique, photo patterning of caged actin was accomplished on a conventional epifluorescence microscope with a Xenon arc lamp. Bandpassed UV light (1.6 × 10-2 mW/µm2 between 320 and 400 nm) was focused through a 100× objective onto a region in the sample plane controlled by the field stop aperture (Figure 1B). Upon photoactivation of caged actin, the uncaged actin diffused away from the illumination region, and the local actin concentration was reduced over time. In Figure 1C, we plot the calculated diffusion profiles from a 200-µmdiameter region with an initial step increase in actin concentration of 0.5 µM. In the calculated example, actin concentration is maintained above the critical concentration for over 10 min after initial release. Full kinetic modeling of the diffusion-reaction process, in which actin monomers are captured by the growing filaments, would lead to a smaller effective radius than that due only to diffusion. The probability of nuclei formation reduces cubically with decreasing actin concentration in the first order approximation of a trimolecular reaction to form trimer nuclei, while elongation rates reduce linearly until the critical concentration is reached (denoted by the dashed line). This concentration dependence confines formation and growth of actin structures to a region controlled by both illumination area and initial actin concentration. We purified G-actin from rabbit muscle acetone powder according to the method of Spudich and Watt21 and prepared caged actin by conjugating a photodeprotection group [(nitroveratryl)oxy]chlorocarbamate (NVOC-Cl) to lysine residues of G-actin according to Marriott.22 Successful caged actin synthesis was confirmed by the presence of a new absorbance peak at 350 nm, and the number of caged groups per monomer was estimated to be three by using electrospray mass spectrometry (data not shown). When caged actin is photolyzed by UV illumination, the caged group undergoes a photoisomerization reaction that results in the breakage of the carbamate bonds, thereby freeing the lysine residues and enabling polymerization. To immobilize growing actin filaments for visualization, coverslips were coated with 100 µg/mL of N-ethylmaleimide modified myosin for 1 min and followed by 1% bovine serum albumin (BSA) for another. Actin filaments were also observed to stick to glass coverslips by nonspecific interaction when uncoated coverslips were used. We observed dynamic polymerization of single filaments within the UV illumination region using time lapse epifluorescence microscopy (Figure 2A). Under ionic conditions favorable for polymerization, a caged actin solution with a low concentration of fluorescent phalloidin was sandwiched between a coverslip and a glass slide (see Supporting Information). Upon UV illumination, the filament elongated for approximately 10 min, consistent with an actin concentration that remained above the critical concentration for that time. The observed elongation is believed to be growth of individual filaments, as opposed to a bundle of filaments, Nano Lett., Vol. 5, No. 4, 2005
Figure 3. Actin-based nanostructures created by photolysis of caged actin. (A) Diagram and fluorescence image of an actin heterostructure. Green is Alexa Fluor 488 phalloidin, and red is rhodamine phalloidin. Alexa Fluor 488 phalloidin stabilized filaments were added as seeds upon which caged actin assembled after photolysis. The newly polymerized actin was visualized by binding of rhodamine phalloidin. Scale bar is 1 µm. (B) Diagram and fluorescence image of Arp2/3 complex (red) mediated branching of actin filaments after photolysis. White arrows indicate branch points. Scale bar is 1 µm.
Figure 2. (A) Elongation of a single actin filament by release of caged actin. White arrows mark beginning and ending filament lengths, and time after UV illumination is indicated below the pictures. Scale bar is 1 µm. (B) Temporal control of actin polymerization. Change in length of a single filament remains constant until UV illumination, indicated by the curvy arrow, initiates rapid elongation of the filament. (C) Spatial control of actin polymerization. Solid line denotes the average elongation of five single filaments that were within the UV illumination region (gray squares). Dotted line denotes the average elongation of five single filaments that were outside of the illumination region (gray triangles).
since negatively charged actin filaments should be repelled from each other at the experimental actin and ionic concentrations in the absence of bundling proteins. Furthermore, filaments do not show bifurcation along their lengths and exhibit similar dynamics. We also note that this provides a method for observing the growth of actin filaments in real time that does not require total internal reflection fluorescence microscopy.23 To illustrate temporal control, we showed elongation of filaments before and after UV illumination (Figure 2B). One Nano Lett., Vol. 5, No. 4, 2005
hundred minutes after the sample was prepared, 15 s of UV illumination caused the filament to elongate 2 µm in approximately 10 min. In a normal actin polymerization time course, steady-state length is reached within 45 min at the experimental actin concentration, indicating that the uncaged actin is responsible for the observed growth. Since one micron of an actin filament contains 370 actin monomers, the elongation rate is ∼1.2 monomers per second over 10 min. The average elongation rate within the first minute after UV illumination was measured to be 3.0 s-1 (n ) 22, S.D. ) 1.8 s-1) compared to 0.1 s-1 for filaments not exposed to UV (n ) 11, S.D. ) 0.2 s-1) (Figure 2C). Miki has previously shown that addition of phalloidin recovers the polymerizability of Lys-61 labeled actin at a ratio of phalloidin/actin of 2:1.24 However, we found that at a low phalloidin/actin concentration (1:100), the effect of phalloidin-induced polymerization is insignificant compared to the UV-induced polymerization. Heterostructures fabricated out of actin filaments, analogous to the metal/semiconductor nanowire heterostructures,25 could be useful in creating sequences of specific binding regions. Using two different fluorescent phalloidin species, we showed that localized growth of actin will occur on existing filaments (Figure 3A). This was accomplished by seeding the solution with small amounts of Alexa Fluor 488 phalloidin stabilized filaments and then photoactivating the caged actin in the presence of dilute rhodamine phalloidin. This observation also supports our conclusion that uncaged actin behaves the same as unmodified actin. To further illustrate the richness of spatially controlled structures that can be generated with caged actin, we included Arp2/3 627
complex in solution to construct branched assemblies of actin filaments (Figure 3B). An advantage of using biological macromolecules as building blocks is that they can be decorated with a wide range of other molecules, increasing the potential complexity of self-assembly to include controlled construction of 3-D nanostructures from 1-D building blocks. We have demonstrated the ability to spatially localized actin polymerization and create heterostructures and branched structures in the presence of actin-binding proteins. Nucleation and elongation of uncaged actin can be optically controlled by tuning the initial actin concentration and illumination area. The synthesis, characterization, and control of photoactivatable actin can be performed with conventional laboratory equipment, and this strategy can be used for spatially controlling actin concentration in biological experiments as well as for fabrication of nanostructures. Furthermore, ongoing development of new photoremovable groups with different activation wavelengths has the potential to enable control over where and when biological building blocks are assembled. The versatility of actin to bind to other proteins and functionalized entities makes it an ideal template for complex nanostructure fabrication. Acknowledgment. We gratefully acknowledge Christophe Le Clainche for help with the purification of actin, David King for help of mass spectrometry, and Carl Co for providing Arp2/3 complex. This work was supported in part by a fellowship from the Natural Sciences and Engineering Research Council of Canada (A.L.), a National Science Foundation CAREER Award (D.A.F.), and a Lawrence Berkeley National Laboratory LDRD grant (D.A.F.). Supporting Information Available: Materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org.
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NL0478878
Nano Lett., Vol. 5, No. 4, 2005
