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ISSN 0582-9879                             ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(6): 489-494                             CN 31-1300/Q

 

Mini Review

 

Atomic Force Microscopy of Actin

ZHANG Jun1,2, WANG Yuan-Liang1*, GU Li1, PAN Jun1

( 1 Key Lab for Biomechanics & Tissue Engineering under the State Ministry of Education, Chongqing University, Chongqing 400044, China; 2 Biology Department, Chongqing University of Medicine Sciences, Congqing 400016, China )

 

Abstract        Atomic force microscope (AFM) is a powerful and novel tool to investigate the surface and submembranous structures of living cells under physiological conditions at high resolution, and has force measure at nano-Newton level. AFM can image and manipulate samples (single macromolecule, cells, and so on) at very high, sometimes atomic resolution by scanning a fine tip over the surface of interest and detecting physical interactions between the tip and sample. Actin is one of the most important proteins, involved extensively in various cell physiological activities and functions in eukaryotes. The present paper clarified the crucial significance and the enormous applicable prospects of AFM in modern biological sciences by reviewing the usage of AFM in actin study, which can be taken as a good example for expounding almost all functions that AFM owns, and can be utilized in life sciences exploration.

 

Key words     atomic force microscopy (AFM); actin; application

 

       Atomic force microscope (AFM) was invented by Binnig et al.[1] in 1986 who was also one of the inventors of the scanning tunnel microscope (STM). AFM is a powerful and novel tool to investigate the surface and submembranous structures of living cells under physiological conditions at high resolution[2]. AFM can image and manipulate samples at very high, sometimes atomic resolution by scanning a fine tip over the surface of interest and detecting physical interactions between the tip and sample. Since the AFM was put into use in life sciences, it has been widely applied in structural analyses and function determination of biological macromolecules, in observation of dynamics of intra- and inter-cellular events in vitro and in vivo in real time and space, and in assay of force spectroscopy produced in processes of biological macromolecule unfolding or stretching, which can provide the fundamental data for further calculation and theoretical analysis of molecular structure, conformation, function and process of self-organization. At present, AFM has become a very important research instrument in exploration of molecular structure and function together with X-ray diffraction, multiple dimensions NMR and other sophisticated technology, and it has special superiority in single molecular investigation, force spectroscopy determination, and study of supramolecular complex[3－5].

       Actin is a highly conserved ubiquitous protein expressed in most living organisms, and is a major structural component of the cytoskeleton[6,7], and is the most abundant protein with very important cytologic physiological functions in eukaryotes. It consists of 375 amino acid residues with a molecular weight of 43 kD or so, and exists in monomer and polymer. Its monomer (globular actin, G-actin) could be polymerized into long right-handed helical polymer (filamentous actin, F-actin) induced by Mg2+, K+, Na+, and ATP[8,9], and its filaments could interact with many cytoplasmic proteins[10]. Actin not only took part in myosin-based motility as an essential component[11,12], but also mediated motility through controlled polymerization[13] or gel-sol transitions[14]. In addition, more and more researches revealed that actin was involved in more extensive and complicated biological activities than we thought before, such as adhesion of cell, signal transduction, modulation of ion channel and so on[15－20]. Due to its diverse and critical functions in eukaryotes, the structure and physiological function of actin had been important subjects of research in cell and structural biology since its discovery. The atomic structure of the monomeric G-actin had been solved in more than one crystal form[21－23], and its filamentous structure had also been proposed based on fiber X-ray diffraction[24,25] and electron microscopy[26－28]. Such structural elucidation has played a pivotal role in our understanding of the function of actin under various conditions.

       In the following, the application of atomic force microscope in actin study would be introduced, which could be taken as a good example in expounding the usage of atomic force microscopy in biological macromolecule study.

 

1    Structure Study of Actin by AFM

1.1   Molecular structure of actin investigated by AFM in vitro

       The high-resolution crystal structure of biological macromolecule could be generally obtained by X-ray diffraction, by which the structure of F-actin and G-actin in complex and uncomplex states have been resolved together with transmission electron microscope[24,29]. And the multiple dimensions NMR, which was usually used in advanced chemical and physical researches, has been employed to determine the structure and conformation of biological macromolecules in solution. The appearance of AFM provided another powerful tool in the exploration of structural biology, and it could be controlled more easily than X-ray or NMR methods. AFM had been successfully applied to directly observe the 3D structure of actin filaments and G-actin within F-actin.

1.1.1       Structure of actin filament     In a recent study[30], the cryoatomic force microscopy (cryo-AFM), which usually runs near to liquid nitrogen temperature so as to enhance the stability of images, was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filament was clearly shown to be right-handed helical structure with a periodicity of approximately 38 nm. The narrow, branched rafts of actin filaments and larger aggregates had also been observed at a moderate concentration (approximately 10 μg/L). The resolution achieved was sufficient to resolve actin monomers within the filaments. A closer examination of the images showed that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of approximately 13 nm. The implications of these higher-order structures had been discussed in terms of X-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicated that the recently proposed model of left-handed F-actin[31] was likely to be an artifact of preparation and/or low-resolution AFM imaging. In another study[32], the surface structure of F-actin in large paracrystals prepared on positively charged lipid monolayers was visualized at high resolution by atomic force microscopy in aqueous solution. The increased stability of these closely packed specimens allowed the investigators to show that both the long pitch (38 nm) and the monomer (5.8 nm) could be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high-resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was (10±1) nm, whereas the smaller inter-filament distance, (8±1) nm, was consistent with interdigitation of the filaments. The (10±1) nm F-actin diameter was in good agreement with the results of fibre X-ray diffraction and three dimensional reconstruction of scanning transmission electron microscopic images[24,33].

       specialized equipment compared with X-ray or electron microscope, the method of AFM provided more applications in the study of the thin filaments containing F-actin-associated proteins.

1.1.2       Imaging of actin monomer in disassociated state  It is rather difficult to directly observe G-actin in disassociated state with AFM because of the limitations of the size of actin monomer, sample preparation on the surface of the substrate, as well as the resolution that the current AFM can actually obtain. Almost no reports on the 3D structural analysis of disassociated G-actin explored by AFM at the single molecule level were published so far. But it could be a very important application field for an improved AFM to determine the delicate surface 3D structure of single molecules in nanometer size with higher resolution, combining with computerized 3D reconstitution technique in the near future, so that the functional sites on the surface of biological macro- or supra-molecule could be easily discerned at atomic resolution, which could either be interaction sites between macromolecules or other important activity domains. Furthermore, even the kinetic process of conformation thermal fluctuation in biological molecules could also be tracked in real space and time with the improved high-end AFM facilities in the near future.

1.2   Actin structure in cell by AFM

       AFM had also been used to directly observe the actin in vivo. Swihart et al.[34] had studied the erythrocyte membrane skeleton with AFM, and successfully resolved the spectrin-actin network texture of membrane skeleton. Their study also demonstrated the applicability of AFM in imaging the erythrocyte membrane skeleton at a resolution that appears to be adequate to identify major components of the membrane skeleton under near-physiological conditions. In an earlier report on imaging subcellular structures of rat mammary carcinoma cells by AFM[35], the cytoskeletal network was resolved as a complex mesh of actin-containing fiber bundles interwoven with a filigree arrangement of thinner filaments. Actually there were many researches of direct observations on cells, subcellular structures and single molecules in vivo with AFM, and it had become another effective technique to observe the living cells at high resolution besides high voltage environmental electron microscope (HVEEM) in order to gain more useful information about morphology, ultrastructure, cytological physiological activities, and other key biology phenomena in vivo.

 

2    Dynamics Detection of F-actin by AFM

       Dynamics of F-actin is a continuous focusing point in cytological research field, because it is critical for explaining the motility function and other equal important ones exerted by actin filaments. But observation of F-actin in living cells was currently limited to the resolution of light microscope. Higher resolution procedures required sample fixation and precluded dynamic studies, which might produce a lot of defects and difficulties, sometime even cause artificial images. However, AFM can image and manipulate samples at very high resolution over the surface of interest and physical interactions, and so can directly trace the dynamical processes in vitro or in biological system. Together with AFM, many modern techniques are already employed in this explioting area. In order to study the dynamics of F-actin in vitro, samples of supported planar lipid-protein membranes and F-actin on mica were imaged by AFM. The samples were fully submerged in buffer at room temperature during imaging. Weisenhorn et al.[36] found that individual protein bound to the reconstituted membrane was distinguishable, some structural details could be resolved. What’s more, surface-induced, self-assembling of F-actin on mica could also be observed. Monomeric subunits on individual actin filaments were imaged. The filaments could even be manipulated on or removed from the surface by the AFM tip. In this experiment, the process of the decoupling of the filamentous network from the surface upon changing the ionic conditions was also imaged in real time. Although the quality of images were not completely satisfying, the kinetic change of F-actin were directly observed in vitro by continuous scanning and data recording. Another study carried out by Henderson et al.[37] also demonstrated that F-actin could be readily resolved in living cells with the AFM and that the dynamic properties of F-actin could be easily observed. Their work testified that the AFM was capable of imaging subsurface features under the plasma membrane within living cells, so AFM provided direct research equipment for observation on events going on under membrane.

 

3    Study of Actin Function

       AFM has also been successfully applied in actin function determination. Previous studies had demonstrated that actin filament organization controls the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel function. The precise molecular nature of the interaction between actin and CFTR, however, remains largely unknown. By ATM, Chasan et al.[38] directly assessed the interactions between actin and purified human epithelial CFTR by reconstitution of the channel protein in a lipid bilayer system. In this experiment, CFTR-containing liposomes in solution were deposited on freshly cleaved mica and imaging was performed by tapping-mode AFM. CFTR function was also determined through this identical preparation. Images of single CFTR molecules were obtained. It was found that when G-actin was below its critical concentration, the formation of F-actin associated with CFTR was detected. Furthermore, the data indicated that there existed a direct interaction between actin and CFTR, which may explain the regulatory role of the cytoskeleton in ion channel function. These results were confirmed by functional studies of CFTR single-channel currents which were regulated by adding actin of various conformations. These studies indicated that CFTR may directly bind actin and this interaction helps affect the functional properties of this channel protein.

       In study of Ishijima et al.[39], a Schizosaccharomyces pombe cps8 mutant, which encoded a mutant actin with an amino acid substitution of Asp for Gly273, was used to determine the role of the actin cytoskeleton in cell wall formation in this cps8 mutant cells, atomic force microscopic and scanning electron microscopic images showed abnormal depolarized and branched morphology. Fibrous material covered partial surface of growing cps8 cells. Transmission electron microscopic images showed variable thickness of the cell wall due to multilayering of cell wall materials, and aberrant multisepta due to diagonal growth of the primary septum, whereas the normal primary septum grew at a right angle from the cortex. This abnormal septum formation may induce abnormality of the cell with multinuclei and/or multisepta, caused by non-separation of daughter cells. These results collected from AFM and electron microscope indicated that actin played an important role in cell wall and septum formation.

       Bhadriraju et al.[40] examined the relationships among cell function, spreading, and stiffness by AFM. Cell stiffness increased when spreading on a high density of fibronectin (10 mg/m2), but remained low if staying rounded on a low fibronectin density (0.01 mg/m2). Disrupting actin or myosin had the same effect of inhibiting spreading, but had different effects on stiffness. Disrupting F-actin assembly lowered both stiffness and spreading, while inhibiting myosin light chain kinase inhibited spreading but increased cell stiffness. However, disrupting either actin or myosin inhibited DNA synthesis. These results demonstrated the relationship existed between cell stiffness and spreading in hepatocytes, and normal actin and myosin function were required for hepatocyte spreading and DNA synthesis, and disruption of actin and myosin had opposite effects on cell stiffness from spreading. All these demonstrated that AFM could be used not only in morphology researches, but also in physiological function identification of cells, supramolecule complex and macromolecule with the help of rational, ingeniously designed experiment scheme.

 

4    Interactions between Actin and Cytoplasmic Proteins

4.1   Interaction of actin-myosin

       How close should two interacting protein molecules be to recognize each other before association? How strongly should a force field be exerted between two proteins at the recognition distance? How extensive could the association interfaces be? How strong should necessary force be to pull the associated proteins apart? These fundamental and intriguing questions were answered with the muscle proteins actin and myosin, by means of atomic force microscopy at a truly single molecule level. Nakajima et al.[41] studied the actin-heavy meromyosin (HMM) interaction using a homemade AFM integrated with an epifluorescence microscope. Utilizing the two microscopes’ integration, they first developed a method to capture a truly single molecule of HMM at an AFM probe tip and observed the interaction force between the captured HMM and actin fixed on the surface. They had observed not only the unbinding event but also the binding event, from which the knowledge of the inter-proteins force field was obtained for the first time at a single molecular level. They successfully assayed the mechanics magnitude, interaction distance and force curves between actin and myosin, and analyzed the molecular mechanics specificity of the force interaction while pulling the myosin associated with actin apart. Further analysis of the unbinding event yielded the unitary unbinding force and the effective rupture length (the maximum distance at which the binding is kept) on the basis of the force spectroscopy determined by AFM. Their experiments fully illustrated that any other extant laboratory equipments used in molecular mechanics could not replace AFM, which can directly assay the physical forces between targeted molecules.

4.2   Interactions between actin and actin-binding proteins

       AFM is also a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Therefore direct correlations can be made between structural and functional states of individual biomolecules in an aqueous environment. In recent years, AFM had been successfully used to collect more information about normal or disease-associated biological processes, especially about the interaction between the interesting macromolecules in these processes[42]. It is well known that, in the cell, G-actin polymerized into filaments, F-actin depolymerized, and both underwent interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli[10,14]. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction, the dynamic organization of the cytoskeleton, and many other important biological activities in which actin plays a key role. With the support of AFM, researchers would readily study the interactions between actin and actin-binding proteins, including the kinetics and molecular mechanics of the interactions, and clarify the possible biological implication. In the coming future, the applications of AFM will be anticipated to inevitably enter into the exploration of interaction between actin and actin-binding protein owing to its excellent technical specificity in the field.

 

5    Discussion

       As a powerful tool, the atomic force microscope had been widely employed to undertake various experiments in biological sciences. The applications of AFM in actin study were not thorough, but only a part scope where the AFM were used in biological macromolecule study on conformation, function and their relationship. In fact, its applications could be extended to more research fields, for example, structural and functional study of nucleic acid, polynucleotide, polysaccharide and other supramolecule complex, and so forth. However, the application of AFM in actin study could essentially stand for almost all usages of AFM in biology study. In brief, the functions AFM could provide the biological researchers were classified to three types. First, the high resolution of AFM in real space and time was its general advantage significantly different from the other bulk measure technology, which made it possible for imaging the single molecule at atomic size level in vivo or in vitro. Second, as an extended function, AFM could manipulate and control bio-molecules or cells. Last, AFM could assay the force spectroscopy through recording the force-distance curves when pulling away the fine sharp AFM tip, to which the targeted molecule’s one end or certain segment was sticked while its other end being firmly attached to the substrate. These three functions outlined all that the AFM were capable of being used in present actin studies. By reviewing the application of AFM used in actin researches, we try to provide some new ideas and methods of atomic force microscopy technology in modern cytology exploitation, which might give some very important enlightening, or practical solutions. For the wide and great applications of AFM in modern life sciences, there were enough reasons to conclude that the AFM would play a more important role with its popularity in worldwide.

 

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Received: February 21, 2003Accepted: April 7, 2003

This work was supported by a grant from the National Natural Science Foundation of China (Key Program) (No. 19732030)

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