Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Changes in the stiffness of human mesenchymal stem cells with the progress of cell death as measured by atomic force microscopy

This note reports observations of the change of stiffness of human mesenchymal stem cells (hMSCs) with the progress of cell death as measured by AFM. hMSC with impaired membrane, dead and viable cells were labelled with Annexin V and Propidium Iodide after 24 h cold storage, followed by AFM measurement and Young's modulus of cells was derived. Viable hMSCs have a Young's modulus (E) in the range of 0.81–1.13 kPa and consistent measurement was observed when different measurement locations were chosen. E of cells with partially impaired membrane was 0.69±0.17 kPa or in the range of 2.04–4.74 kPa, depending upon the measurement locations. With the loss of membrane integrity, though there was no variation on measured E between different locations, a mixed picture of cell stiffness was observed as indicated by cells with E as low as 0.09±0.03 kPa, in a mid-range of 4.62±0.67 kPa, and the highest of up to 48.98±19.80 kPa. With the progress of cell death, the highest stiffness was noticed for cells showing a more granular appearance; also the lowest stiffness for cells with vacuole appearance. Findings from this study indicate that cell stiffness is significantly altered with the progress of cell death.     

Force and compliance measurements on living cells using atomic force microscopy (AFM)

We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study. Published: January 15, 2004     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Imaging microtubules and kinesin decorated microtubules using tapping mode atomic force microscopy in fluids

The atomic force microscope has been used to investigate microtubules and kinesin decorated microtubules in aqueous solution adsorbed onto a solid substrate. The netto negatively charged microtubules did not adsorb to negatively charged solid surfaces but to glass covalently coated with the highly positively charged silane trimethoxysilylpropyldiethylenetriamine (DETA) or a lipid bilayer of 1,2-dipalmitoyl-3-dimethylammoniumpropane. Using electron beam deposited tips for microtubules adsorbed on DETA, single protofilaments could be observed showing that the resolution is up to 5 nm. Under conditions where the silane coated surfaces are hydrophobic, microtubules opened, presumably at the seam, whose stability is lower than that of the bonds between the other protofilaments. This led to a “sheet” with a width of about 100 nm firmly attached to the surface. Microtubules decorated with a stoichiometric low amount of kinesin molecules in the presence of the non-hydrolyzable ATP-analog 5′-adenylylimidodiphosphate could also be adsorbed onto silane-coated glass. Imaging was very stable and the molecules did not show any scan-induced deformation even after hundreds of scans with a scan frequency of 100 Hz. Received: 23 February 1999 / Revised version: 19 July 1999 / Accepted: 17 August 1999     

Actin microridges characterized by laser scanning confocal and atomic force microscopy

We have characterized the cell surface of zebrafish stratified epithelium using a combined approach of light and atomic force microscopy under conditions which simulate wound healing. Microridges rise on average 100 nm above the surface of living epithelial cells, which correlate to bundles of cytochalasin B-insensitive actin filaments. Time-lapse microscopy revealed the bundles to form a highly dynamic network on the cell surface, in which bundles and junctions were severed and annealed on a time scale of minutes. Atomic force microscopy topographs further indicated that actin bundle junctions identified were of two types: overlaps and integrated end to side T- and Y-junctions. The surface bundle network is found only on the topmost cell layer of the explant, and never on individual locomoting cells. Possible functions of these actin bundles include cell compartmentalization of the cell surface, resistance to mechanical stress, and F-actin storage.     

Tracking peptide-membrane interactions: insights from in situ coupled confocal-atomic force microscopy imaging of NAP-22 peptide insertion and assembly

Elucidating the role that charged membrane proteins play in determining cell membrane structure and dynamics is an area of active study. We have applied in situ correlated atomic force and confocal microscopies to characterize the interaction of the NAP-22 peptide with model membranes prepared as supported planar bilayers containing both liquid-ordered and liquid-disordered domains. Our results demonstrated that the NAP-22 peptide interacts with membranes in a concentration-dependent manner, preferentially inserting into DOPC (ld) domains. While at low peptide concentrations, the NAP-22 peptide formed aggregate-like structures within the ld domains, at high peptide concentrations, it appeared to sequester cholesterol into the ld domains and recruited phosphatidyl-myo-inositol 4,5-bisphosphate by inducing a blending effect that homogenizes the phase-segregated domains into one liquid-ordered domain. This study describes a possible mechanism by which the NAP-22 peptide can affect neuronal morphology.     

Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized. Published: June 29, 2004.     

Double minute chromosomes in mouse methotrexate-resistant cells studied by atomic force microscopy

Double minute chromosomes (DMs) are acentric, autonomously replicating extra-chromosomes and frequently mediate gene amplification in tumor and drug resistant cells. Atomic force microscopy (AFM) is a powerful tool in microbiology. We used AFM to explore the ultrastructure of DMs in mouse fibroblasts 3T3R500. DMs in various phases of cell cycle were also studied in order to elucidate the mechanisms of their duplication and separation. Metaphase spread and induced premature condensed chromosomes (PCCs) were observed under the AFM. DMs were detected to be composed of two compact spheres linked by fibers. The fibers of DMs directly connected with metaphase chromosomes were observed. Many single-minutes and few DMs were detected in G1 PCCs, while more DMs were detected in S PCCs than in G1 PCCs. Besides, all of the DMs in G2 PCCs were coupled. Our present results suggested that DMs might divide into single-minutes during or before G1-phase, followed by duplication of the single-minutes in S-phase. Moreover, we introduced a new powerful tool to study DMs and got some ideal results.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations

We present high-resolution atomic force microscopy (AFM) imaging of the single-ring mutant of the chaperonin GroEL (SR-EL) from Escherichia coli in buffer solution. The native GroEL is generally unsuitable for AFM scanning as it is easily being bisected by forces exerted by the AFM tip. The single-ring mutant of GroEL with its simplified composition, but unaltered capability of binding substrates and the co-chaperone GroES, is a more suited system for AFM studies. We worked out a scheme to systematically investigate both the apical and the equatorial faces of SR-EL, as it binds in a preferred orientation to hydrophilic mica and hydrophobic highly ordered pyrolytic graphite. High-resolution topographical imaging and the interaction of the co-chaperone GroES were used to assign the orientations of SR-EL in comparison with the physically bisected GroEL. The usage of SR-EL facilitates single molecule studies on the folding cycle of the GroE system using AFM.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.     

Atomic force microscopy studies provide direct evidence for dimerization of the HIV restriction factor APOBEC3G

APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg(2+), but dimers still predominated when Mg(2+) was depleted. A His-248/His-250 Zn(2+)-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.     

Deformability and adhesive force of artificial platelets measured by atomic force microscopy

Platelet glycoprotein GPIaIIa is an adhesive protein that recognizes collagen. We have investigated polymerized albumin particles conjugated with recombinant GPIaIIa (rGPIaIIa-poly Alb) for their platelet-like function. To evaluate the feasibility of these particles to achieve the hemostatic process, we measured the deformability (Young’s modulus and spring constant) and the adhesive force of the particles using atomic force microscopy, which can measure the mechanical properties of individual cells. Our results showed that the Young’s modulus of these particles was 2.3-fold larger than that of natural platelets and 12-fold larger than that of human red blood cells. The Young’s modulus of the particles may have been determined by the properties of the polymerized albumin particle, although the glycoprotein of the platelet surface also contributed to the higher modulus. Our results also showed that the adhesive force of the rGPIaIIa-poly Alb with the collagen ligand was 52% of that of natural platelets. These two mechanical properties (deformability and adhesive force) of cells or particles, such as rGPIaIIa-poly Alb, are important specifications for the optimum design of platelet substitutes.