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ISSN 1672-9145 Acta Biochimica et Biophysica Sinica 2004, 36(9): 637–643 CN 31-1940/Q

Preliminarily Investigating the Polymorphism of Self-organized Actin Filamentin Vitro by Atomic Force Microscope

Jun ZHANG1,2, Yuan-Liang WANG1*, Xin-Yong Chen3, Chuang-Long HE1, Chao CHENG1, and Yang CAO1

1Key Lab of Biomechanics & Tissue Engineering, Chongqing University, Chongqing 400044, China;2Biology Department, Chongqing University of Medicine Sciences, Congqing 400016, China;

3Laboratory of Biophysics and Surface Analysis, University of Nottingham, Nottingham NG7 2RD, United Kingdom

Abstract With the atomic force microscope (AFM), we preliminarily investigated the large-scalestructure of actin filaments formed in low concentration protein solution (5 µg/ml) via self-organizationwithout the presence of any F-actin dynamic interfering factors (such as phalloidin) in vitro. It was foundthat the G-actin could be polymerized into ordered filamentous structures with different diameter fromthe slimmest filament of single F-actin to giant filament in tree-like branched aggregates. The observedpolymerized actin filaments, to which our most intense attention was attracted, was discretely distributed andshowed obvious polymorphism distinctly different from those in the presence of phalloidin or actin bindingproteins (fimbrin, gelsolin, etc.) in previous experiments. Latter structures were mainly composed of singleF-actin and/or multifilaments clearly consisting of several single F-actin. The experimental results clearlydemonstrated that non-interference with the F-actin intrinsic dynamics in self-organizing could lead to thepolymorphism of actin filamentous structures, and further analysis implied that the disturbance of normalF-actin dynamics by many factors could prevent the emergence of structural polymorphism, more often thannot, give rise to formation of specific structures instead and different interference would bring about variousparticular structures under certain conditions.

Key words actin; self-organization; filamentous structure; polymorphism; atomic force microscope

Actin is a major structural component of eukaryoticcytoskeleton and exists in monomer G-actin and filamen-tous F-actin. G-actin consists of 375 amino acid residueswith molecular weight 43 kD and is a highly conservedprotein expressed in most living organisms [1,2]. Actinmonomer can be polymerized into long right-handeddouble helical F-actin induced by Mg2+, K+, Na+, and ATP[3,4]. F-actin can interact with an array of cytoplasmicproteins to meet different biological functions undervarious conditions in vivo [5]. Actin not only can take partin myosin-based motility as an essential component [6,7],but also can alone mediate motility through controlledpolymerization [8] or gel-sol transitions [9].

In recent years, more and more researches revealedthat actin was involved in more extensive and complicatedbiological activities than we thought before, such as celladhesion, signal transduction, modulation of ion channel,and so on [10–15]. So the molecular structure and physio-logical functions of actin have been being importantsubjects of research in cell and structural biology. Notonly has the atomic structure of G-actin been solved inmore than one crystal form [16–20], but also atomicmodels of the F-actin have been proposed based on fiberX-ray diffraction [21,22] and electron microscopy [23–25]. These studies help us understand the actin functionsunder various conditions, the essential correlation betweenits functions and structures, and the role of actin structuraldiversity in their functional versatility in vivo [26–29].In the recent decades, the polymorphism in actin bundles[5,30,31], paracrystals [32,33], crystalline sheet [34,35],cross-linked networks [36,37], and F-actin assembly[38,39] have been reported. However, little attention has

Received: April 6, 2004 Accepted: August 6, 2004This work was supported by the grants from the National Natural Sci-

ence Foundation of China [No. 19732030 (Key Program), No. 19732003,No. 19872080], and the Visiting Scholar Foundation of Key Lab inChongqing University

*Corresponding author: Tel, 86-23-65102508; Fax, 96-23-65316247; E-mail, zhangjun1017@sohu.com

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been paid to the structural versatility in vivo or the struc-tural polymorphism of polymerized actin filaments byself-organization without any interference with F-actindynamics by any factors, such as chemical reagents andmicrofilament stabilizing reagents (phalloidin, etc.) thatwere widely employed in actin researches or any otheractin-bind proteins involved in controlling F-actindynamics. In fact, investigation on the structural polymor-phism of actin filaments and polymerization dynamics inself-organizing could play a fundamental role in profoundlyexpounding the formation of actin structural diversity viaself-assembly dynamic process modulated by manyfactors in living cells and in turn, revealing its functionalversatility, and also have great theoretical implication inexploring the molecular dynamics of proteins and theirpolymerized structures in self-organizing.

In this research, we preliminarily investigated the large-scale structure of actin aggregates by self-organization insimple thermodynamic environment (with only necessaryreagents) near to physiological conditions in vitro withatomic force microscope (AFM) which had been widelyapplied in actin researches [40], focusing on the highorder structure of actin filaments and the filamentousstructure polymorphism. It is observed that actin could formdifferent types of filamentous structure in absence of anyF-actin dynamics interfering factors in F-buffer. Under theexperimental conditions, actin could form large-scaleordered structure of long filaments with different dia-meters in tree-like branched aggregates. The polymerizedfilaments are distinctly different from those formed inthe presence of F-actin dynamic interfering factors suchas phalloidin [41], gelsolin [38,39], or fimbrin [31] inprevious experiments, in which the observed actinfilaments are mainly composed of the single F-actin ormultifilament aggregates (actin bundles, raft, etc.) clearlyconstituted of several single actin filaments, and the highorder filamentous structures are rarely seen.

Materials and Methods

Materials

G-actin was purified from bovine skeletal musclefollowing the method of Spudich and Watt [42]. A singleband was observed on SDS-PAGE gel with no evidenceof contamination by other proteins in the final extract.G-actin was stored in G-buffer at 4 °C for following use.Tris-base and Tris-HCl were products of Promega; Na2ATPand DTT were purchased from Sigma-Aldrich Co.. Other

chemical reagents of analytic grade were made in China.The Si3N4 tip of AFM was model NP, NANOPROBETM.

Solution preparation

G-buffer and F-buffer were prepared according totraditional method with a little modification for AFMassay. G-buffer contained 2 mM Tris-HCl (pH 7.5), 0.2mM CaCl2, 0.5 mM DTT, 0.2 mM ATP; F-buffer contained5 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 100 mM KCl,1 mM DTT, and 1 mM ATP. All freshly prepared solventsand solutions were passed through 0.22 µm filter in orderto remove particles.

Preparation of protein specimen

1 ml purified G-actin extract solution was accuratelydiluted to 100 ml with F-buffer, the final concentration ofG-actin was 5 µg/ml. The prepared sample was incubatedfor polymerization at 37 °C for 30 min. Transfer 5 µlG-actin solution onto the surface of freshly cleaved micathat was already soaked by F-buffer with micropipette.After the polymer sunk for 15 min, the excess droplet wascarefully sucked, and the specimen was naturallydehydrated.

AFM imaging

The prepared sample, without any physical and che-mical treatment in order to keep the sample in a poly-merizing state near to physiological conditions, wasimmediately observed under AFM (CSPM-2000we, BenYuan Nano Instrument Ltd.) in contact mode at roomtemperature (the humidity was 30%; the main operationparameters I.G., P.G., HVS, and Ref were “125”, “125”,“open” and “–0.25”, respectively). The atomic forcemicroscopic images were captured and saved in *.bmp for-mat for future analyses, and the ruler unit was nanometer(nm) in all AFM images. All experimental operations abovewere carried out under super-clean conditions to avoidcontamination.

Results

In this research, it was found that G-actin could bepolymerized into long filaments with different morpho-logies in various scales, from simple single F-actin tocomplicated filaments with multiple order branches, whichshowed distinct structural polymorphism and were signi-ficantly different from those formed in the presence ofphalloidin [41], gelsolin [38,39] or fimbrin [31] under therelative more complicated thermodynamic conditions.

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Sep., 2004 Jun ZHANG et al.: Polymorphism of Self-organized Actin Filament in Vitro 639

Branch structure of actin filaments

The tree-like filament seemed to be composed of mul-tiple-order helix filament with branches, which actuallyindicated the possible constructed manner of high orderactin filaments by self-organization (Fig. 1), and the branchorder could determine the filament diameter. Thickerfilament could also be made in this way. It was preli-minarily inferred that the giant filament should be madefrom single F-actin via non-covalent interaction amongmicrofilaments such as van der Waals force, static elec-tronic force, hydrogen bond, and so on.

form a complicated radial branched structure like afilamentous joint that could come from the cooperativeaction of polymer sinking and interaction among branchedfilaments [Fig. 2(C)].

Random coil structure of actin filaments

Many filaments existed in clustered distribution states(Fig. 3). It was thought that the high density of condensedfilaments throngs have had homology with the branch fila-ment structure in origin, and they were probably the re-sultants in different phase of polymerization, and so haddifferent topological conformations.

In much larger scales, the branch filaments could windone another and form new filament, and some could branchonce again [Fig. 2(A)]. It was also observed that manystacked branch structures clustered together, which mightbe produced in the process of specimen preparation [Fig.2(B)]. In another image, filaments winding one another

Fig. 2 Complicated branch structures(A) Branch filaments could wind one another and form new filament, and somecould branch once again. (B) Stacked branch structures clustered together. (C)Radial branched structure.

Various long actin filaments with different diameters

Long filament with different diameter was formed fromF-actin in different way, and could be easily observed.Possibly the side interaction among the microfilamentscould played a key role in the forming of this high orderstructure, which remained unknown and needed furtherresearches.Structural analysis of single actin filament The pitch ofthe helix of single filament was variable, with publishedvalue around 36 nm, and the width was 5–7 nm or so [23,27,43–46]. In our experiment, single F-actin was judgedaccording to the structural parameters. The slimmestfilament was similar to the single F-actin in size, with7.31 ± 1.21 nm in width and 35.64±3.43 nm in pitch (mean± SD, n=8), which indicated that such filament was a singleF-actin (Fig. 4). The result also demonstrated that the AFMtip was so sharp that it brought no obvious widen effect,which was generally produced when the tip curvatureradius was near to the size of the examined sample.High order structure of actin filaments with much thickerdiameter Some polymerized filaments had much largerdiameter than that of the single F-actin (Fig. 5), which

Fig. 1 Tree-like branch structure with fractal feature Fig. 3 Random coil filaments cluster

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640 Acta Biochimica et Biophysica Sinica Vol. 36, No. 9

suggested that the structure of actin filaments formed inprocess of self-organizing was far more different andcomplicated than those in the presence of phalloidin oractin binding proteins, in which the polymerized filamentswere mainly made of single F-actin or/and simplemultifilament. Although it is clearly that the high orderfilaments are identically made from F-actin, the structuralconstitution manner remains unclear and waits to be solved.

Statistical analysis of branch angle and filamentdiameter

The frequency distribution of branch angle and filamentdiameter has been analyzed with SPSS 10.0 for windows(SPSS Inc.). It was found that the branch angle was mainlyin the scope of 40–65 degree with an average value of52.08±9.42 degree [Fig. 6(A)], and diameter of filamentsapproximately varied from 7 nm to 90 nm, and mainlydistributed in the scope of 20–50 nm with an average valueof 36.61±18.07 nm [Fig. 6(B)]. The frequency distri-bution demonstrated that the aggregated filaments had

Fig. 4 Structural analysis of single F-actin(A) A negatively stained STEM image of single F-actin using annular dark-fielddetector, the scale bar was 20 nm [27]. (B) AFM image of the single F-actin. (C)A simplified model of single F-actin was used to illustrate the AFM image.

Fig. 5 Thicker single filament with different diameter(A) The branch filament could be constituted of two enlaced slimmer filaments,forming the branch in the joining point of another filament. (B) Two filamentswere aligned side by side, and there might exist certain interaction between them,which could be the early step for further assembly of filaments. (C) Filamentwith short fork structure at the end was also likely composed of much thinnerfilaments winding with each other. (D–F) Filaments had much larger diametersthan the single F-actin.

Fig. 6 Frequency distribution of branch angle and filamentdiameterBoth the frequencies showed distinct characteristics of normal distribution, whichsuggested that they could be the indirect reflection of “the rule of minimal en-ergy” in actin self-organization.

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dominant distributed scopes in branch angle and filamentdiameter, which implied that the filament structure in thesescopes was easier to form than that out of these scopes. Infact, the polymerized filament structure should alsoconform to “the rule of minimal energy” when formed inself-organization, so it is very possible that the filamentsin the dominant scopes stand at much lower energy level,thus more stable. Whereas those out of the dominant scopescould have higher energy level, thus less stable and harderto form, so as to result in less magnitude.

Discussion

In previous researches, the tree-like actin filamentousstructure has never been observed in vivo and thestructural polymorphism of actin filaments in vitro has alsonot been paid much attention. Considering the hugedifference in thermodynamic environment between in vivoand in vitro, the phenomena should not be difficult to beunderstood. Further careful analyses revealed that, in manyexperiments on F-actin structure exploration in vitro before,the F-actin stabilizing reagents, generally phalloidin, wereusually employed to stabilize the structure of polymerizedfilamentous actin for the benefit of analysis on its sta-tionary structure. Although the stabilizing reagent couldprovide convenience in these studies and help to clarifythe structure of single actin filament, it can also interferewith the normal dynamics of actin polymerization throughinhibiting the depolymerization process. Furthermore,it could prevent actin from bringing about high orderstructure and structural polymorphism, which accidentallycause us to ignore the polymorphism of actin filament byself-organization in vitro for a long period that, in fact,should be paid enough attention as well.

The present experimental results demonstrate thatthe high order structural polymorphism of actin filamentcan be easily produced in process of undisturbed self-organization in vitro, which is propelled by its inherentthermodynamic properties (which refer to the integratedphysical and chemical characters including the nativeconformation, molecular weight, owned net chargesand their distribution, biomechanics features, residuedistribution on molecular surface, and so on) essentiallydetermined by its amino acid sequence. Together withprevious research results [31,38,39,41,47], further ana-lysis can find that the intact non-linear dynamics of F-actin is the necessary condition in formation of the struc-tural polymorphism, from which it is inferred that thedestruction of the normal F-actin dynamics could lead to

emergence of particular structure and different inter-ference with the dynamics could give rise to differentspecific structures. Obviously the insight can perfectlyexplain the structural diversity of polymerized actinproducing in the living cells.

At present, the nucleation-condensation mechanism ofF-actin polymerization dynamics based on “upper dimer”(UD) pathway has been commonly accepted, which isgenerally believed to involve three distinct steps: (1) fastG-actin activation; (2) rate-limiting nucleation; (3) mode-rately fast elongation conforming to a pseudo-first orderkinetics [3,7,48]. Although there is a general agreementthat dimerization of a significant fraction of G-actin wasinvolved in F-actin dynamics, “lower dimmer” (LD) wasunable to polymerize into F-actin filament by itself [49].Hence it is thought that LD formation may representan unproductive side reaction [27]. But Steinmetz et al.[27,50] recently demonstrates that LD, while beingunproductive by itself, can add to growing F-actinfilament via one of its subunit. It is revealed that LD candirectly incorporate into the growing filaments in thepresence of K+ or divalent cation (Mg2+, Ca2+), therebyproducing ragged filaments that frequently branch inthe early stages of polymerization and the shift from LDto UD occurs approximately after several to 15 min[27,28,50]. Interestingly in the presence of phalloidin,no significant amount of LD can be detected duringpolymerization, and the phalloidin-induced polymerizationpathway appears to be dominated by a simple nucleation-condensation mechanism [27,50]. According to theexperiments [28,50], it is definitely confirmed that theUD- and LD-based pathway co-exist under the conditionof undisturbed self-organization. On the basis of the noveldiscovery, a possible mechanism for the structuralpolymorphism of actin filaments via self-organization isput forward and schematically described in Fig. 7(A).

It is well known that the F-actin dynamic process canbe modulated by many factors, such as icon concentration,ATP, and actin-binding proteins in vivo [1,3,4,47]. So it isnaturally induced that actin can be kinetically assembledinto various particular structures with the modulation ofbiomechanics properties, space conformations and topo-logical features via interaction with related cytologicalproteins, and most important, with the regulation of theF-actin dynamics by icon concentration, ATP, and chieflyby actin-binding proteins [Fig. 7(B)]. Furthermore, it isself-evident that the actin-binding proteins could playa key role in controlling actin to form different structure(after all, reagents such as phalloidin etc. don’t exist invivo) and providing actin with functional versatility, and

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are thought to exert their functions mainly throughaffecting the polymerization dynamic mechanism orpathway and altering biomechanics properties, spaceconformations and topological features via direct orindirect interaction with actin in order to meet the demandof different cytological functions in vivo.

Although we qualitatively analyzed the possible mecha-nism for the structural diversity of actin in vivo based onprevious and our current experiments, we didn’t know themolecular dynamic process and regulatory details yet,and we would continue the exploration in the future. Theresolution of the high order filament structure andmeasurement of their molecular mechanic features will bealso an important and interesting subject in the followingresearches.

Fig. 7 Schematically representation of actin polymerization(A) Self-organization without interference. (B) Self-assembly in complicated thermodynamic system. ABPs, the actin binding proteins; OE, other effectors; the symbolof “?” denoted the probability.

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Edited byHong-Yu HU