Sung-Woong Han

High-efficiency DNA injection into a single human mesenchymal stem cell using a nanoneedle and atomic force microscopy

We describe a low-invasive gene delivery method that uses an etched atomic force microscopy (AFM) tip or nanoneedle that can be inserted into a cell nucleus without causing cellular damage. The nanoneedle is 200 nm in diameter and 6 mum in length and is operated using an AFM system. The probabilities of insertion of the nanoneedle into human mesenchymal stem cells (MSCs) and human embryonic kidney cells (HEK293) were higher than those of typical microinjection capillaries. A plasmid containing the green fluorescent protein (GFP) gene was adsorbed on a poly-L-lysine-modified nanoneedle surface, which was then inserted into primary cultured single human MSCs. A highly efficient gene delivery of over 70% was achieved in human MSCs, which compared very favorably with other major nonviral gene delivery methods (lipofection approximately 50%, microinjection approximately 10 %). The single cells expressing GFP were collected and the amount of delivered DNA in each cell was analyzed. The highest rate of expressed GFP per delivered DNA was achieved using the nanoneedle, because the nanoneedle could be inserted into the nucleus directly without causing significant cell damage.

Development of a method to evaluate caspase-3 activity in a single cell using a nanoneedle and a fluorescent probe.

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).

Mechano-adaptive sensory mechanism of α-catenin under tension

The contractile forces in individual cells drive the tissue processes, such as morphogenesis and wound healing, and maintain tissue integrity. In these processes, α-catenin molecule acts as a tension sensor at cadherin-based adherens junctions (AJs), accelerating the positive feedback of intercellular tension. Under tension, α-catenin is activated to recruit vinculin, which recruits actin filaments to AJs. In this study, we revealed how α-catenin retains its activated state while avoiding unfolding under tension. Using single-molecule force spectroscopy employing atomic force microscopy (AFM), we found that mechanically activated α-catenin fragment had higher mechanical stability than a non-activated fragment. The results of our experiments using mutated and segmented fragments showed that the key intramolecular interactions acted as a conformational switch. We also found that the conformation of α-catenin was reinforced by vinculin binding. We demonstrate that α-catenin adaptively changes its conformation under tension to a stable intermediate state, binds to vinculin, and finally settles into a more stable state reinforced by vinculin binding. Our data suggest that the plastic characteristics of α-catenin, revealed in response to both mechanical and biochemical cues, enable the functional-structural dynamics at the cellular and tissue levels.

The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation

Human alpha-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinsons disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of alpha-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of alpha-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in alpha-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form alpha-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.

Direct insertion of proteins into a living cell using an atomic force microscope with a nanoneedle

We have developed a tool for directly inserting proteins into living cells by using atomic force microscopy (AFM) and an ultrathin needle, termed a nanoneedle. The surface of the nanoneedle was modified with His-tagged proteins using nickel chelating nitrilotriaceticacid (NTA). The fluorescent proteins, DsRed2-His6 and EGFP-His6, could be attached to and detached from the surface of the nanoneedle. These results suggest that the Ni-NTA modified nanoneedle can successfully be used for specific delivery of proteins. The nanoneedle modified with DsRed2-His6 was able to penetrate the surface of a living HeLa cell, as confirmed by laser scanning fluorescence microscopy and monitoring an exerting force on the nanoneedle using AFM. Force curves using the nanoneedle indicated that the needle was able to penetrate at displacement speeds of 0.10–10 µm/s. These results suggest that this technique can be used to directly insert proteins into living cells and is applicable for modulation or regulation of single cell activity.

Monitoring of hormonal drug effect in a single breast cancer cell using an estrogen responsive GFP reporter vector delivered by a nanoneedle.

In this study, we have evaluated a sensor system for a hormonal drug effect in a single cell level using a novel low invasive single cell DNA delivery technology using a nanoneedle. An estrogen responsive GFP reporter vector (pEREGFP9) was constructed and its estrogenic response activity was confirmed in breast cancer cells (MCF-7) using lipofection as the means of transferring the vector to the cells. The pEREGFP9 vector was delivered to a single MCF-7 using a nanoneedle and the effect of ICI 182,780, which is an antagonist of estrogen, was observed using the GFP expression level. By ICI 182,780 treatment, the fluorescence intensity of the GFP was decreased by 30-50% within 24h. This technology is the very first trial of single cell diagnosis and we are looking forward to applying it to precious single cell diagnosis in medical fields.

Successive detection of insulin-like growth factor-II bound to receptors on a living cell surface using an AFM.

In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin‐like growth factor‐II (IGF‐II) and activation of the IGF‐I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5‐azacytidine (5‐aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm2 was in contact with a cell surface. The forces required to rupture the interactions of IGF‐IIs on a cell and anti mouse IGF‐II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody–ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5‐aza to the medium. This method could be used in non‐invasive and successive evaluation of a living cells behavior. Copyright

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276–634; the mechano-sensitive M1-M3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.

Probing Amyloid β and the Antibody Interaction Using Atomic Force Microscopy

Amyloid β (Aβ) peptide is considered to be the critical causative factor in the pathogenesis of Alzheimers disease (AD) because the hydrophilic molecules accumulated outside of the neural cells and results in the formation of highly toxicity amyloid plaque. In this study, we probed the interaction between Aβ and the antibody using atomic force microscopy (AFM). We compared two kinds of antibodies which are the antibody for Aβ 1-42 (antibody42) and the antibody for Aβ 1-16 (antibody16). To detect the interaction between Aβ and the antibodies, the single molecular force spectroscopy was carried out using Aβ modified glass substrate and the antibodies modified AFM probes. In the results, the single Aβ-antibody42 dissociation constant was estimated to be 5.2 × 10-3 s-1 and the single Aβ-antibody16 dissociation constant was 2.8×10-2 s-1. The Aβ-antibody42 showed 5.3 times longer bond life time compare with Aβ-antibody16. It suggested that antibody42 is better choice for the Aβ sensor development.

Detection of Actin Filament and Cofilin Interaction Change by Actin Filament Curvature Decreasing

Actin filament senses mechanical forces and it is transduced into biochemical signals during many cellular processes. In the disassembling process of actin filaments, cofilin plays a central role as the actin filament depolymerization. In this study, we evaluated a quantitative analysis of the actin filament-cofilin interaction change dependent upon the actin filament curvature decrease using atomic force microscopy (AFM) and a fabricated wave-like substrate. A wave-like substrate was fabricated by a maskless photo-lithography of a spin coated film on a glass substrate, and graphene oxide sheet was used for the decreasing of non-specific interaction between protein and the substrate. By single-molecule force spectroscopy, we determined rupture force of actin filament-cofilin binding on the wave-like substrate and a flat substrate. The rupture force of actin filament-cofilin binding at the curvature of -1.35 μm-1 showed a value approximately 4 times higher than the rupture force at the curvature of -0.15 μm-1. The present study will provide the possibility and quantitative evidence that mechanical stress on cytoskeletal filaments can modulate how they interact with their binding proteins.