Force Mapping Reveals the Spatial Distribution of Individual Proteins in a Neuron.

Conventional methods for studying the spatial distribution and expression level of proteins within neurons have primarily relied on immunolabeling and/or signal amplification. Here, we present an atomic force microscopy (AFM)-based nanoscale force mapping method, where Anti-LIMK1-tethered AFM probes were used to visualize individual LIMK1 proteins in cultured neurons directly through force measurements. We observed that the number density of LIMK1 decreased in neuronal somas after the cells were depolarized. We also elucidated the spatial distribution of LIMK1 in single spine areas and found that the protein predominantly locates at heads of spines rather than dendritic shafts. The study demonstrates that our method enables unveiling of the abundance and spatial distribution of a protein of interest in neurons without signal amplification or labeling. We expected that this approach should facilitate the studies of protein expression phenomena in depth in a wide range of biological systems.

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. 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 neurotropic 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.

Visualization and Quantification of MicroRNA in a Single Cell Using Atomic Force Microscopy.

MicroRNAs (miRNAs) play critical roles in controlling various cellular processes, and the expression levels of individual miRNAs can be considerably altered in pathological conditions such as cancer. Accurate quantification of miRNA at the single-cell level will lead to a better understanding of miRNA function. Here, we present a direct and sensitive method for miRNA detection using atomic force microscopy (AFM). A hybrid binding domain (HBD)-tethered tip enabled mature miRNAs, but not premature miRNAs, to be located individually on an adhesion force map. By scanning several sections of a micrometer-sized DNA spot, we were able to quantify the copy number of miR-134 in a single neuron and demonstrate that the expression was increased upon cell activation. 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 µm(2) of soma. The number increased to 8-14 in stimulated neurons, and this change matches the ensemble-averaged increase in copy number. These findings indicate that miRNAs can be reliably quantified at the single cell level with AFM and that their distribution can be mapped at nanometric lateral resolution without modification or amplification. Furthermore, the analysis of miRNAs, mRNAs, and proteins in the same sample or region by scanning sequentially with different AFM tips would let us accurately understand the post-transcriptional regulation of biological processes.

AFM Imaging Reveals MicroRNA-132 to be a Positive Regulator of Synaptic Functions.

The modification of synaptic and neural connections in adults, including the formation and removal of synapses, depends on activity-dependent synaptic and structural plasticity. MicroRNAs (miRNAs) play crucial roles in regulating these changes by targeting specific genes and regulating their expression. The fact that somatic and dendritic activity in neurons often occurs asynchronously highlights the need for spatial and dynamic regulation of protein synthesis in specific milieu and cellular loci. MicroRNAs, which can show distinct patterns of enrichment, help to establish the localized distribution of plasticity-related proteins. The recent study using atomic force microscopy (AFM)-based nanoscale imaging reveals that the abundance of miRNA(miR)-134 is inversely correlated with the functional activity of dendritic spine structures. However, the miRNAs that are selectively upregulated in potentiated synapses, and which can thereby support prospective changes in synaptic efficacy, remain largely unknown. Using AFM force imaging, significant increases in miR-132 in the dendritic regions abutting functionally-active spines is discovered. This study provides evidence for miR-132 as a novel positive miRNA regulator residing in dendritic shafts, and also suggests that activity-dependent miRNAs localized in distinct sub-compartments of neurons play bi-directional roles in controlling synaptic transmission and synaptic plasticity.

Quantification of a Neurological Protein in a Single Cell Without Amplification.

Proteins are key biomolecules that not only play various roles in the living body but also are used as biomarkers. If these proteins can be quantified at the level of a single cell, understanding the role of proteins will be deepened and diagnosing diseases and abnormality will be further upgraded. In this study, we quantified a neurological protein in a single cell using atomic force microscopy (AFM). After capturing specifically disrupted-in-schizophrenia 1 (DISC1) in a single cell onto a microspot immobilizing the corresponding antibody on the surface, force mapping with AFM was followed to visualize individual DISC1. Although a large variation of the number of DISC1 in a cell was observed, the average number is 4.38 × 103, and the number agrees with the ensemble-averaged value. The current AFM approach for the quantitative analysis of proteins in a single cell should be useful to study molecular behavior of proteins in depth and to follow physiological change of individual cells in response to external stimuli.