Biology subjects

Banzai, K.

Publications and source records attributed to Banzai, K..

3 recordsLinked to original sources

bioRxiv2025

Two-Dimensional Nonlinear Structured Illumination Microscopy with rsEGFP2

Superresolution microscopy enables imaging of subcellular structures and dynamics with nanoscale detail. Among the various superresolution techniques, structured illumination microscopy (SIM) stands out for its compatibility with live-cell imaging. Linear SIM is restricted to a resolution improvement of a factor of two, improving the resolution to about 100 nm. Nonlinear SIM (NSIM) utilizes reversibly switchable fluorescent proteins to generate a nonlinear response, allowing for the collection of higher spatial frequency information and theoretically extending the resolution without limit. By employing rsEGFP2 and patterned depletion illumination (PD) to generate the desired nonlinearity in the fluorescent response, we have successfully achieved 2D PD-NSIM imaging of actin in live U2OS cells with sub-80 nm resolution.

bioengineering↗

bioRxiv2024

Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in Drosophila

i.Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP1-10/11) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP1-10 and GFP11 fragments, creating a fluorescence signal. We insert the GFP11 fragment into a specific genomic locus while expressing its counterpart, GFP1-10, through an available Gal4 driver line. The unique aspect of this system is that neither GFP1-10 nor GFP11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP11 tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m (Ten-m) as a model, highlighting the generation and validation of GFP11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

cell biology↗

bioRxiv2021

Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori

Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, it is still inconclusive how cholinergic genes express only in cholinergic neurons in insects. In this study, we analyzed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. We found multiple enhancer regions that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. We found that dibutyryl-cAMP, an analog of cAMP, influences the expression of ChAT. Tissue-specific expression analysis of transcriptional factors showed potential candidates that control the cholinergic gene locus expression. Our investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.

molecular biology↗