Single-Molecule Visualization of MicroRNA in a Neuronal Cell Using Atomic Force Microscopy

Abstract

Chapter Ⅰ. Multiparametric Nanoscale Imaging of Biological and Materials Science using Atomic Force Microscopy In order to study of single molecule detection in biology and materials science, over the past three decades there have been remarkable developments and refinements in single molecule force spectroscopy tools. Atomic force microscopy (AFM) has established a new insight to analyze multiparametric nanoscale imaging and bio-molecular recognition in single molecule level. AFM force spectroscopy studies have provided a specific mapping of surface molecules with picoNewton level, and it can be operated in liquid and under physiological conditions. In summary, AFM provides evidence indicating localization and distribution of individual target molecules for the single molecule level.

Chapter Ⅱ. Visualization of MicroRNA in a Single Neuronal Cell Using Atomic Force Microscopy MicroRNAs (miRNAs) play key roles in wide variety of cellular processes, including development, differentiation, apoptosis, and cell proliferation. The dysregulation of individual miRNAs is linked to various human diseases, such as cancers, cardiovascular, autoimmune and monogenic. Especially, specific miRNAs of brain have important roles in neuronal differentiation, development, plasticity and induced neurological disorders. Therefore, accurate visualization and quantification of miRNA at the single cell level will lead to a better understanding of miRNA function. Here, we present a new direct and sensitive analytical method for miRNA detection using atomic force microscopy (AFM). A hybrid binding domain (HBD)-tethered tip specifically recognized miRNA/DNA duplexes, and individual miRNAs in the scan area could be counted on an adhesion force map. Moreover, we visualized individual miR-134s on fixed neurons after membrane removal and observed 2−4 miR-134s in the area of 1.0 × 1.0 m2 of soma. We also observed the number increased to 8−14 in stimulated neurons, and this change matches the ensemble-averaged increase in copy number. These results suggest that miRNAs distribution can be mapped at nanoscale visualization without modification or amplification. Furthermore, it is expected that corresponding mRNAs and proteins in the same region can be visualized by mapping with different AFM functionalized tips sequentially.

Chapter Ⅲ. Nanoscale imaging reveals miRNA-mediated control of functional states of dendritic spines Dendritic spines are major loci of excitatory inputs and undergo activity-dependent structural changes that contribute to synaptic plasticity and memory formation. Despite the existence of various classification types of spines, how they arise and which molecular components trigger their structural plasticity remain elusive. microRNAs (miRNAs) have emerged as critical regulators of synapse development and plasticity via their control of gene expression. Brain-specific miR-134s likely regulate the morphological maturation of spines, but their subcellular distributions and functional impacts have rarely been assessed yet. Here, we exploited atomic force microscopy to visualize in situ miR-134s, which indicated that they are mainly distributed at nearby dendritic shafts and necks of spines. The abundance of miR-134s varied between morphologically and functionally distinct spine types, and their amounts were inversely correlated with their postulated maturation stages. Moreover, spines exhibited reduced contents of miR-134s when selectively stimulated with beads containing brain-derived neurotrophic factor (BDNF). Taken together, in situ visualizations of miRNAs provided unprecedented insights into the “inverse synaptic-tagging” roles of miR-134s that are selective to inactive/irrelevant synapses and potentially a molecular means for modifying synaptic connectivity via structural alteration.