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.