Search

At the initial stage of carcinogenesis, cell competition often occurs between newly emerging transformed cells and the neighboring normal cells, leading to the elimination of transformed cells from the epithelial layer. For instance, when RasV12-transformed cells are surrounded by normal cells, RasV12 cells are apically extruded from the epithelium. However, the underlying mechanisms of this tumor-suppressive process still remain enigmatic. We first show by electron microscopic analysis that characteristic finger-like membrane protrusions are projected from both normal and RasV12 cells at their interface. In addition, FBP17, a member of the F-BAR proteins, accumulates in RasV12 cells, as well as surrounding normal cells, which plays a positive role in the formation of finger-like protrusions and apical elimination of RasV12 cells. Furthermore, cdc42 acts upstream of these processes. These results suggest that the cdc42/FBP17 pathway is a crucial trigger of cell competition, inducing “protrusion to protrusion response” between normal and RasV12-transformed cells.

We developed adaptive optical (AO) two-photon excitation microscopy by introducing a spatial light modulator (SLM) in a commercially available microscopy system. For correcting optical aberrations caused by refractive index (RI) interfaces at a specimen's surface, spatial phase distributions of the incident excitation laser light were calculated using 3D coordination of the RI interface with a 3D ray-tracing method. Based on the calculation, we applied a 2D phase-shift distribution to a SLM and achieved the proper point spread function. AO two-photon microscopy improved the fluorescence image contrast in optical phantom mimicking biological specimens. Furthermore, it enhanced the fluorescence intensity from tubulin-labeling dyes in living multicellular tumor spheroids and allowed successful visualization of dendritic spines in the cortical layer V of living mouse brains in the secondary motor region with a curved surface. The AO approach is useful for observing dynamic physiological activities in deep regions of various living biological specimens with curved surfaces.

We developed two-photon excitation stimulated emission depletion (STED) nanoscopy using high-peak-power sub-nanosecond 655-nm pulses. The STED pulse exhibited ideal optical properties and sufficient pulse energy to realize a 70-nm spatial resolution in the compact setup With electrically controllable components. For biological applications, we screened suitable fluorescent dyes or proteins and realized the sub-100 nm spatial resolution imaging of presynaptic protein clusters in fixed primary cultured neurons Without severe photobleaching. We expect this method to enable visualization of ultrastructures and the cluster dynamics of biomolecules representing physiological functions in living cells and tissue. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 mu m, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage. (C) 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and (c) two types of transient interactions between FMBP-1 and chromosomal DNA, and (d) stable binding of FMBP-1 to chromosomal DNA.