Visualization of Individual Proteins in a Neuron Using Atomic Force Microscopy

Abstract

Chapter Ⅰ. Force Mapping-based Protein Detection Method at Nanoscale Resolution Proteins are involved in most biological processes, performing a variety of functions in living organisms. Proteins act as biological catalysts in biochemical reactions, transport and store biologically essential materials, and interact with various biomolecules. In neurons, proteins play important roles in synaptic development, maturation, and neuroplasticity. Changes of protein distribution and expression within neurons have been assessed by various protein detection methods. Typical protein detection techniques are based on signal amplification and/or immunolabeling. In conventional signal amplification techniques, such as western blot and ELISA, target proteins can be identified and quantified, but these methods require experimental treatment including cell lysis to observe the targets in a cell, and suffer limited sensitivity. Imaging techniques, such as standard fluorescence microscopy and imaging-based immunoassay, can visualize proteins within the cell through specific labeling of a fluorophore. However, they target abundant proteins and cannot visualize proteins in a local area with high resolution due to the optical diffraction limit. Super-resolution fluorescence microscopy, a recently developed method overcoming the diffraction limit, has provided a useful tool to study nanoscale distribution of proteins in a cell. Despite these advances, fluorescence-based methods suffer from limitations: i) Target proteins need to be artificially expressed to fluoresce or tag a fluorophore. Such modification may interfere with native protein functions or experience false signals due to non-specific tagging. ii) The resolution of advanced optical microscopy is still not good enough to locate tiny proteins individually, in particular when proteins of interest are in high proximity. Atomic force microscopy (AFM) has been a powerful technique for studying structures and properties of materials at the nanometeric resolution without any signal amplification or fluorescence labeling. AFM can measure the interaction force within a single biomolecule or between biomolecules with picoNewton accuracy, thereby providing key information of biomolecules from single molecule perspective. Force mapping of AFM, a method combining the high spatial resolution and the ability to measure the biomolecular interaction, can directly locate individual proteins at a specific local area in a single cell. Force mapping can show the nanoscale distribution of a specific protein, and the lateral resolution of a few nanometers provides highly resolved maps that cannot be accessed by other methods. AFM approach can help us to understand structure and dynamics of biomolecules in cells further in depth, and such understanding will lead to crucial insight into complex biological systems.

Chapter ⅠⅠ. Visualization of LIMK1 in a Neuron Using Atomic Force Microscopy Neurons constantly respond to the external stimuli to adjust to given environment. The neuronal adaptation involves regulations of genes, noncoding DNA/RNAs, and proteins, which collectively influence structural and functional properties of the corresponding neurons. In particular, proteins in neurons regulate synaptic development, maturation, and plasticity. LIM kinase 1 (LIMK1) is a protein that plays important roles in regulation of cell motility, cytokinesis, and cellular morphology. LIMK1 is rich in hippocampal pyramidal neurons and stimulates growth of the neurons, especially of dendritic spines, and its expression is known to be regulated by miR-134. In this study, individual LIMK1 proteins in cultured neurons were directly located by AFM-based nanoscale force mapping method without any signal amplification or fluorescent labeling. Anti-LIMK1-tethered AFM probes were used to detect individual LIMK1 proteins through the force measurement, and high-resolution force maps were obtained in neurons, especially neuronal somas and dendritic spines. Because the distribution of miR-134 in neuronal somas and dendritic spines was previously revealed by AFM mapping, the distribution of LIMK1 can be compared with that of miR-134, and also the correlation of miR-134 to LIMK1 can be studied. The number density of LIMK1 was obtained in neuronal somas before and after the cells were depolarized, and the decreased density of LIMK1 was observed in the depolarized somas. Also, the spatial distribution of LIMK1 in single spine areas was investigated, and the results indicated that LIMK1 proteins predominantly locate at heads of spines rather than dendritic shafts. The distinct distributions of LIMK1 at different parts of dendritic spines present a model related to the site of synthesis of LIMK1 proteins. The AFM-based force mapping method enables unveiling of the abundance and spatial distribution of a protein of interest in neurons with minimal pre-treatment. Furthermore, this approach should facilitate the studies of protein expression phenomena in depth in a wide range of biological systems.