Atomic force microscope as a nano- and micrometer scale biological manipulator: A short review.

The amazing capacity of atomic force microscope to let us touch the molecular and cellular level samples with a sharp probe stimulated its application to bio-medical field among others. In addition to topographical imaging of the sample surface, a direct mechanical manipulation has attracted innovative minds to develop new methodologies aiming at direct handling of proteins, DNA/RNA, and cells. Measurement of their mechanical properties brought about a vivid picture of their physical nature. Direct handling of individual molecules and cells prompted development of nano-medical applications. This short review summarized recent application of AFM for measurement of mechanical properties of biological samples and attempts to perform direct manipulations of nano-medicine.

Membrane wound healing at single cellular level.

We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A2 was made to contact the cell membrane. A small dark hole (4-14 µm2 in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014µm2s-1. The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2).

[A long-term survivor of cT4 esophageal carcinoma treated via a multimodal approach - a case report].

The patient was a 53-year-old man whose chief complaint was dysphagia. Pretreatment examination revealed 2 types of locally advanced esophageal squamous cell carcinoma at the middle to lower thoracic esophagus. A computed tomography (CT) scan showed a bulky primary tumor suspicious of aortic invasion and cardiac lymph node metastasis. The pretreatment diagnosis was cT4N2M0, cStageIVa. After induction chemotherapy with 5-fluorouracil (5-FU) and cisplatin (CDDP) (the FP regimen) according to the JCOG9907 regimen, subtotal esophagectomy and 2-field lymphadenectomy with retrosternal stomach roll reconstruction were performed. Intraoperatively, the primary tumor showed extensive and firm adhesion to the aortic wall. The postoperative diagnosis was pT4N0M0, pStageIII, RM1. Postoperative chemoradiotherapy (65 Gy+FP) was performed for the residual tumor at the aortic wall. The patient is alive without recurrences 4 years and 6 months after the esophagectomy. Downstaging of the tumor with induction chemotherapy and effective local control with surgery and postoperative chemoradiotherapy may have contributed to the patient's long-term survival. For multimodal treatment of cT4 esophageal cancer, an effective combination of chemotherapy, surgery, and chemoradiotherapy is essential to improve the treatment outcome.

[Gastric cancer arising from gastric polyps in gardner syndrome - a case report].

The patient was a 48-year-old woman who was diagnosed with early gastric cancer during a long-term follow-up period for Gardner syndrome. Subtotal colectomy for colon leiomyoma was performed when the patient was 22 years old. Partial resection of the ileum was performed for ileum leiomyoma at the age of 27. Total resection of the remaining colon with ileostomy was performed for a pelvic desmoid tumor at the age of 40. In addition, resection of a desmoid tumor of the abdominal wall was performed 8 times in the 25 years since the first operation. During the follow-up for gastric polyps associated with Gardner syndrome, gastric cancer was detected from biopsy specimens of a wide range of the fundus polyps. Endoscopic resection was considered not to be applicable because of the extensive nature of the lesion. Total gastrectomy was also considered not to be applicable because of concerns about short bowel syndrome due to intestinal reconstruction. Therefore, proximal gastrectomy with esophagogastric anastomosis was performed. The pathological diagnosis was 0-IIa, 70 × 44 mm, tub1, m, ly0, v0, n0, PM (-), DM (-), stageIA. The postoperative course was uneventful, and the patient was discharged on postoperative day (POD) 16. We speculate that long-term survival of patients with Gardner syndrome without severe short bowel syndrome might result in carcinogenesis of gastric polyps.

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.

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy.

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.

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.

Direct manipulation of intracellular stress fibres using a hook-shaped AFM probe.

Atomic force microscopy (AFM) is a highly successful technique for imaging nanometre-sized samples and measuring pico- to nano-newton forces acting between atoms and molecules. When it comes to the manipulation of larger samples with forces of tens and hundreds of nano-newtons, however, the present chemistry-based modification protocols for functionalizing AFM cantilevers to achieve the formation of covalent/non-covalent linkages between the AFM probe and the sample surface do not produce strong enough bonds. For the purpose of measuring the fracture strength and other mechanical properties of stress fibres (SFs) in living as well as semi-intact fibroblast cells, we fabricated an AFM probe with a hooking function by focused ion beam technology and used the AFM probe hook to capture, pull and eventually sever a chosen SF labelled with green or red fluorescent protein.

Tensile mechanics of alanine-based helical polypeptide: force spectroscopy versus computer simulations.

In nature, an alpha-helix is commonly used to build thermodynamically stable and mechanically rigid protein conformations. In view of growing interest in the mechanical rigidity of proteins, we measured the tensile profile of an alanine-based alpha-helical polypeptide on an atomic-force microscope to investigate the basic mechanics of helix extension with minimal interference from side-chain interactions. The peptide was extended to its maximum contour length with much less force than in reported cases of poly-L-Glu or poly-L-Lys, indicating that chain stiffness strongly depended on the physicochemical properties of side chains, such as their bulkiness. The low tensile-force extension originated presumably in locally unfolded parts because of spontaneous structural fluctuations. In 50% trifluoroethanol, the well-known helix-promoting agent, the rigidity of the sample polypeptide was markedly increased. Computer simulations of the peptide-stretching process showed that a majority of constituent residues underwent a transition from an alpha-helical to an extended conformation by overcoming an energy barrier around psi approximately 0 degrees on the Ramachandran plot. The observed lability of an isolated helix signified the biological importance of the lateral bundling of helices to maintain a rigid protein structure.

Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole.

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNA's from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5-10 microm, a phospholipase A(2) coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5-10 min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope.

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.

Simultaneous resection of gastric and gallbladder metastasis from renal cell carcinoma treated by laparoscopic and endoscopic cooperative surgery: a case report.

BACKGROUND: Metastases to the stomach or gallbladder from any malignancy is rarely noted, and simultaneous metastases to both organs are atypical. We present a unique case of simultaneous multifocal metastases of the stomach and gallbladder from renal cell carcinoma (RCC). CASE PRESENTATION: The case involved a 60-year-old man, with a past history of RCC (clear cell type, G2, T1b N0 M0 Stage I) treated by a right nephrectomy. Three years after the nephrectomy, a routine gastrointestinal endoscopy found an ulcerative lesion in the greater curvature of the gastric body. The gastric tumor was pathologically proven to be a metastasis from RCC. Furthermore, computed tomography incidentally revealed a mass lesion in the fundus of the gallbladder, which was also diagnosed as a potential metastasis from RCC. As endoscopic ultrasonography of the gastric tumor suggested the tumor potentially invaded to the submucosal layer, gastric wedge resection via a laparoscopic and endoscopic cooperative surgery (LECS) technique was applied to the gastric tumor, and laparoscopic cholecystectomy to the gallbladder tumor was simultaneously performed. Histological examination confirmed that the tumors of the stomach and gallbladder were both metastatic RCC. The hospitalization period after surgery was not eventful, and the patient was discharged on postoperative day 7. Thereafter, the patient required examination every 3 months, did not use anticancer agents, and has survived without relapse to 9 months after the surgery. CONCLUSIONS: For patients with locally resectable RCC metastases, complete metastasectomy may bring long-term tumor control. Moreover, LECS for gastric metastasis is a reasonable approach for minimal invasiveness and an oncologically feasible outcome.

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.

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.

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.

Spontaneous Rupture of Renal Metastasis from Hepatocellular Carcinoma.

We report a rare life-threatening case of spontaneous rupture of renal metastasis from hepatocellular carcinoma (HCC) that was managed by emergent transcatheter arterial embolization (TAE). A 76-year-old woman diagnosed with HCC presented with acute back pain in her right side and was transferred to our hospital. Initial enhanced computed tomography revealed retroperitoneal hemorrhage from the right kidney, which was retrospectively diagnosed as a spontaneous rupture of the metastatic renal tumor from the primary HCC. Detailed examination identified an active retroperitoneal hemorrhage from the lesion and the patient's condition became hemodynamically unstable; hence emergent TAE was performed. The hospitalization period after the TAE was uneventful and sorafenib was subsequently administered. Unfortunately, two months after the TAE, the tumor locally progressed within the retroperitoneal space. Tumors were controlled by repeated TAE as the patient did not want to undergo a nephrectomy. Consequently, she survived for more than one year after emergent TAE, exhibiting low levels of tumor marker. After rupture of the metastatic renal HCC, tumors were expected to progress into the retroperitoneal space, and nephrectomy was the next possible radical treatment to offer the best chance of long-term disease control.

Increased levels of granular tau oligomers: an early sign of brain aging and Alzheimer's disease.

Development of neurofibrillary tangles (NFTs) is a pathological hallmark in various neurodegenerative disorders including Alzheimer's disease (AD). Recently, we identified a granular tau oligomer having a pre-filamentous structure. To determine the role of this oligomer in NFT formation, we quantified the amount of granular tau oligomer in 21 frontal cortex samples, each displaying varying degrees of Braak-staged NFT pathology. Here we report that granular tau oligomer levels in frontal cortex were significantly increased, even in brains displaying Braak-stage I neuropathology, a stage at which clinical symptoms of AD and NFTs in frontal cortex are believed to be absent. This suggests that increases in granular tau oligomer levels occur before NFTs form and before individuals manifest clinical symptoms of AD. Increased granular tau oligomer levels, therefore, may lead to NFT formation in frontal cortex, eventually leading to the development of AD. Thus, increases in granular tau oligomer levels may represent a very early sign of NFT formation and AD.

AFM observation of immobilized self-oscillating polymer.

Various types of nanocomponents have been developed to construct a nanodevice or nanomachine. Here, we add a new nanocomponent that has the function of self-oscillation. A thermoresponsive polymer carrying a Ru complex, a catalyst of the Belousov-Zhabotinsky reaction, was synthesized and immobilized on a glass plate. Periodic turbidity changes in the aqueous solution of the polymer were observed, and nanoscale self-oscillation of the immobilized polymer was observed by a scanning probe microscope.

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.

Phosphorylated retinoblastoma protein is a potential predictive marker of irinotecan efficacy for colorectal cancer.

Irinotecan has been used in the first-line treatment of metastatic colorectal cancer. However, no clear predictive marker of irinotecan efficacy has been identified. It is controversial whether the response to irinotecan could be predicted by the expression level of topoisomerase-I, a direct target of irinotecan. The present study aimed to identify a feasible predictive marker of irinotecan efficacy. We hypothesized that the efficacy of SN38 (an active metabolite of irinotecan) is related to the cell proliferation and the phosphorylation status of RB in colorectal cancer cells. Indeed, the IC50 of SN38 was positively correlated with the doubling time of each cell line (R2=0.9315). Moreover, the phosphorylation level of RB was related to SN38 sensitivity. Consistent with the in vitro data, colorectal cancer tissues of irinotecan responders showed a significantly higher rate of phosphorylated RB (serine 780) expression using immunohistochemistry (P=0.0006), although a generally used proliferative marker, Ki-67, showed no significance. Finally, we investigated whether the phosphorylation of RB plays a crucial role in the efficacy of irinotecan. To suppress the expression of phosphorylated RB, we performed the knockdown of CDKs, which are known to phosphorylate RB. Intriguingly, the knockdown of both CDK4 and CDK6, but not CDK2, allowed RB to become the most hypophosphorylated form and converted the SN38-sensitive cells to a resistant state. Taking together the above findings from in vitro and clinical research, the immunohistochemistry of phosphorylated RB protein might be feasible to predict the irinotecan efficacy of colorectal cancer in clinical practice.