Spectrin-ankyrin interaction mechanics: A key force balance factor in the red blood cell membrane skeleton.

As major components of red blood cell (RBC) cytoskeleton, spectrin and F-actin form a network that covers the entire cytoplasmic surface of the plasma membrane. The cross-linked two layered structure, called the membrane skeleton, keeps the structural integrity of RBC under drastically changing mechanical environment during circulation. We performed force spectroscopy experiments on the atomic force microscope (AFM) as a means to clarify the mechanical characteristics of spectrin-ankyrin interaction, a key factor in the force balance of the RBC cytoskeletal structure. An AFM tip was functionalized with ANK1-62k and used to probe spectrin crosslinked to mica surface. A force spectroscopy study gave a mean unbinding force of ~30 pN under our experimental conditions. Two energy barriers were identified in the unbinding process. The result was related to the well-known flexibility of spectrin tetramer and participation of ankyrin 1-spectrin interaction in the overall balance of membrane skeleton dynamics.

Interaction between pheromone and its receptor of the fission yeast Schizosaccharomyces pombe examined by a force spectroscopy study.

Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 µm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe.

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, the mam2 upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.

Nonlinear displacement of ventral stress fibers under externally applied lateral force by an atomic force microscope.

Actin-based stress fibers (SFs) have fundamental importance in the maintenance of mechanical stability of living cells. Several in vitro measurements of their elastic properties have therefore been made, but direct mechanical manipulation of individual SFs in vivo for the determination of their mechanical properties has not been attempted. No less important is a search for the possible formation of a global mechanical network involving SFs and other intracellular filamentous components. In this article, we present an application of atomic force microscopy to probe into a live cell and laterally push selected SFs in a fibroblast cells (VNOf 06 fibroblast-like cells derived from rat vomeronasal tissue) transfected with a green fluorescent protein-ß-actin gene. The transfected cells were transferred to a serum-depleted medium before the atomic force microscope manipulation. The lateral displacement of the SFs under a point loading condition recorded on a fluorescence microscope revealed both linear and nonlinear displacements against the contour distance from the point of force loading. The nonlinear displacements of the SFs were attributed to their association with a cortical actomyosin-cell membrane complex that effectively pulled them back as a 2D thin plate.

Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study.

Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the "leg" of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70pN. These findings strongly support the idea that Gli349 is the "leg" protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

Detection of mRNA in single living cells using AFM nanoprobes.

Examining messenger RNA (mRNA) expression is useful for the determination of cell and tissue conditions. Many methods of determining mRNA expression require total RNA extraction or cell fixation. These processes cause difficulties in examining mRNA expression in single living cells without causing cell death. We think that analysis of specific mRNA expression in single living cells will become important in cell biology. In this chapter, we present a method to examine mRNA expression of single living cells without killing the cells. The single-cell nanoprobe (SCN) method uses atomic force microscopy (AFM) to extract mRNA. We also present examples of beta-actin mRNA detection and multiple mRNA detection from single living cells.

Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity.

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales. IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.

High-sensitivity detection of proteins using gel electrophoresis and atomic force microscopy.

We have developed a method to detect specific proteins with a high sensitivity using a gel electrophoresis method and force measurement of atomic force microscopy (AFM). Biotinylated proteins were separated by electrophoresis and fixed with cross-linking chemicals on the gel, followed by direct force measurement between the biotinylated proteins on the gel and a streptavidin-modified tip of an AFM cantilever. We were able to achieve a high enough sensitivity to detect the picogram order of the biotinylated proteins by evaluating the frequency of the interaction force larger than 100pN in the force profile, which corresponds to the rupture force of interaction between streptavidin and biotin.

Distribution of olfactory marker protein on a tissue section of vomeronasal organ measured by AFM.

Distribution of olfactory marker protein (OMP) on a tissue section of vomeronasal organ (VNO) was successfully measured by atomic force microscopy (AFM). Anti-OMP antibodies were covalently crosslinked with the tip of the AFM and were used as a probe to observe the distribution of OMP on a tissue section. First, force measurements were performed using a glass surface on which OMP was covalently immobilized to verify the success of tip modification. Clear differences of interaction forces were observed between a specific pair and the control experiments, indicating that the tip preparation succeeded. Next, distributions of OMP on the tissue section were observed by AFM and were compared with immunohistochemical observations. For large scale observation, a microbead was used as a probe in the AFM measurements. The results of the AFM measurements were well overlapped with that of immunohistochemistry, confirming the reliability of our method. A mapping of the AFM measurement with high resolution was also successfully obtained, which showed an advantage of the application of the AFM measurement in analysis of proteins on the tissue section.

Exploring transferrin-receptor interactions at the single-molecule level.

Interaction between the iron transporter protein transferrin (Tf) and its receptor at the cell surface is fundamental for most living organisms. Tf receptor (TfR) binds iron-loaded Tf (holo-Tf) and transports it to endosomes, where acidic pH favors iron release. Iron-free Tf (apo-Tf) is then brought back to the cell surface and dissociates from TfR. Here we investigated the Tf-TfR interaction at the single-molecule level under different conditions encountered during the Tf cycle. An atomic force microscope tip functionalized with holo-Tf or apo-Tf was used to probe TfR. We tested both purified TfR anchored to a mica substrate and in situ TfR at the surface of living cells. Dynamic force measurements showed similar results for TfR on mica or at the cell surface but revealed striking differences between holo-Tf-TfR and apo-Tf-TfR interactions. First, the forces necessary to unbind holo-Tf and TfR are always stronger compared to the apo-Tf-TfR interaction. Second, dissociation of holo-Tf-TfR complex involves overcoming two energy barriers, whereas the apo-Tf-TfR unbinding pathway comprises only one energy barrier. These results agree with a model that proposes differences in the contact points between holo-Tf-TfR and apo-Tf-TfR interactions.

mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization.

BACKGROUND: The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells. RESULTS: In order to evaluate the SCN method, we compared the SCN method with in situ hybridization (ISH). First, we examined spatial beta-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for beta-actin mRNA. In the SCN method, quantity of beta-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of beta-actin mRNA. We showed that intensity of ISH is higher; quantity of beta-actin mRNA detected by the SCN method increased more. CONCLUSION: In this study, we compare the SCN method with the ISH. We examined beta-actin mRNA expression in single cells using both methods. We picked up beta-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM.

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons.

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.

Quantitative analysis of the number of antigens immobilized on a glass surface by AFM.

To develop force measurements using an atomic force microscope (AFM) in a quantitative manner, it is necessary to estimate the number density of target molecules on a sample surface, and for this, the sensitivity of detection should be known. In this study, the AFM was used as a mechanical detector and an antigen and its antibody were used as a model to evaluate the sensitivity of detection. Antigens were immobilized on a glass surface and number density was estimated by monitoring optical absorbance due to product formation by the reaction of crosslinkers. The concentration of antigen was controlled by mixing control peptides. A microbead was used as a probe and antibodies were immobilized on the bead. AFM force measurements were then made for a range of number densities in the order of 10-10(6) antigen molecules per square micrometer of surface and were compared to evaluate the sensitivity of detection. Our result establishes the reliability of estimating a number of molecules like receptors on the cell surface, and indicates that the AFM is useful as a mechanical detector with high sensitivity.

Quantitative measurement of mRNA at different loci within an individual living cell.

Asymmetric localizations of cellular proteins and mRNAs are important for cell functions such as division, differentiation and development. The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins and thereby restricting their locations to appropriate cellular regions. We have previously reported a novel method based on atomic force microscopy (AFM) for examining gene expression in a single living cell without killing or destroying it. An AFM tip was inserted into a living cell to extract mRNAs, which were analyzed after multiplication by RT-PCR and quantitative PCR. By applying this method, in this study we performed quantitative measurement of mRNA at different loci within individual living cells.

Fetal development of vomeronasal system in the goat.

Our previous study morphologically revealed that the adult goat vomeronasal (VN) system was different from the rodent and opossum one, and at least two types of VN systems exist in mammals. However, it remains unknown whether the developments in both types of VN systems are ontogenetically distinct and when the goat VN system is established. In this study, we morphologically observed the fetal development of the goat accessory olfactory bulb (AOB) and VN neuron. In the fetus, Gi2-expressing VN terminals terminated at glomeruli throughout the AOB, and no immunoreactivities for Go were detected in the nerve terminals reaching into AOB. The layer structure of AOB rapidly developed in the latter half of gestation. In the VN organ (VNO), at the middle stage of gestation, the dendritic processes of VN neuron were exposed in the VN lumen, and scattered and thin microvilli existed on the protrusion of the VN neuron. In the apical part of dendritic processes, no clear vesicle existed. However, the immunohistochemistry of an olfactory marker protein (OMP) revealed that a few VN neurons with OMP exist in VN sensory epithelium (VSE) before birth, although marked immunoreactivities were detected in adult VSE. Fetal VN neurons appeared to be underdeveloped. These results suggest that the goat VN system is ontogenetically distinct from the rodent and opossum VN systems, and is underdeveloped before birth. The goat VN system will develop and mature during the early postnatal period similar to the rodent and opossum VN systems.

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers.

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4-0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.

Mapping of the receptor-associated protein (RAP) binding proteins on living fibroblast cells using an atomic force microscope.

The distribution of the receptor-associated protein (RAP) binding protein and the adhesion forces between RAP and its binding protein on living fibroblast cells were examined using an atomic force microscope (AFM). The distribution of RAP binding protein was obtained on 256 (16x16) locations in 2x2 micro m sections over the surface of living cells. The adhesion forces between RAP and the binding protein were measured with an AFM tip functionalized with RAP. In the presence of RAP in the scanning solution, the number of force curves with large adhesion force decreased. These results indicate that the adhesive forces observed here represent specific binding between RAP and the binding protein. This method will be a useful application of AFM to examine receptors on cell surfaces in high resolution.

Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell.

Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM). First, the distribution of the integrin beta(5) subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell. To visualize the distribution of the receptor on a living cell by an independent and potentially a more quantitative method, the AFM was used with a microbead attached to the cantilever tip to increase the area of contact and VN was immobilized on the microbead. Force measurements were then performed over a large area of a living murine osteoblastic cell using the microbead covered with VN.