Application of single-molecule analysis to singularity phenomenon of cells

Abstract

Single-molecule imaging in living cells is an effective tool for elucidating the mechanisms of cellular phenomena at the molecular level. However, the analysis was not designed for throughput and requires high expertise, preventing it from reaching large scale, which is necessary when searching for rare cells that induce singularity phenomena. To overcome this limitation, we have automated the imaging procedures by combining our own focusing device, artificial intelligence, and robotics. The apparatus, called automated in-cell single-molecule imaging system (AiSIS), achieves a throughput that is a hundred-fold higher than conventional manual imaging operations, enabling the analysis of molecular events by individual cells across a large population. Here, using AiSIS, we demonstrate the single-molecule imaging of molecular behaviors and reactions related to tau protein aggregation, which is considered a singularity phenomenon in neurological disorders. Changes in the dynamics and kinetics of molecular events were observed inside and on the basal membrane of cells after the induction of aggregation. Additionally, to detect rare cells based on the molecular behavior, we developed a method to identify the state of individual cells defined by the quantitative distribution of molecular mobility and clustering. Using this method, cellular variations in receptor behavior were shown to decrease following ligand stimulation. This cell state analysis based on large-scale single-molecule imaging by AiSIS will advance the study of molecular mechanisms causing singularity phenomena.

Inducing aggresome and insoluble tau aggregation using an optogenetic tool

“…OptoTau is a protein fused to CRY2Olig [21], which forms multimeric complexes when exposed to blue light, with human 2N4R tau carrying the P301L mutation (Figure 1A). We have previously revealed that transient overexpression of OptoTau into Neuro2a and blue light exposure induced tau clustering [25] [26]. This study established Neuro2a cells that stably express OptoTau and investigated the effects of blue light illumination over a longer period than in previous studies.…”

“…Recently, a series of studies revealed that transient overexpression of chimeric proteins combining tau with Cry2 or its derivative CRY2Olig, i.e., families of OptoTau, results in the blue light-dependent formation of LLPS droplets and tau oligomers [23] [24] [25] [26]. The present study manipulated tau by blue light in OptoTauKI cells, in which OptoTau was inserted into the genome by genome editing.…”

“…CRY2, which is a photoreceptor in Arabidopsis , and its derivative CRY2Olig are optogenetic modules that reversibly form oligomers upon blue light exposure [21]. Recently, researchers have introduced CRY2 to induce protein aggregation in neurodegenerative diseases, including tau [22] [23] [24] [25] [26]. CRY2 and tau fusion proteins accumulated on microtubules by blue light illumination-induced liquid–liquid phase separation (LLPS) [23].…”

“…Blue light illumination forms tau droplets and oligomers in primary cortical culture neurons of mice that overexpress tau fused with CRY2Olig, which is a higher capacity for homo-oligomer formation [24]. We have recently revealed that "OptoTau," which is a fusion of human tau with the P301L mutation and CRY2Olig, is transiently overexpressed in Neuo2a cells and induced LLPS droplets and oligomers of tau by illuminating blue light for 24 h [25] [26]. The present study manipulated intracellular tau by blue light illumination in Neuro2a cells that stably express OptoTau.…”