Biology subjects

Kawashima, T.

Publications and source records attributed to Kawashima, T..

4 recordsLinked to original sources

bioRxiv2018

Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, Voltron, that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

neuroscience↗

bioRxiv2018

A genetically encoded fluorescent sensor for in vivo imaging of GABA

Current techniques for monitoring GABA, the primary inhibitory neurotransmitter in vertebrates, cannot follow ephemeral transients in intact neural circuits. We applied the design principles used to create iGluSnFR, a fluorescent reporter of synaptic glutamate, to develop a GABA sensor using a protein derived from a previously unsequenced Pseudomonas fluorescens strain. Structure-guided mutagenesis and library screening led to a usable iGABASnFR ({Delta}F/Fmax ~ 2.5, Kd ~ 9 M, good specificity, adequate kinetics). iGABASnFR is genetically encoded, detects single action potential-evoked GABA release events in culture, and produces readily detectable fluorescence increases in vivo in mice and zebrafish. iGABASnFR enabled tracking of: (1) mitochondrial GABA content and its modulation by an anticonvulsant; (2) swimming-evoked GABAergic transmission in zebrafish cerebellum; (3) GABA release events during inter-ictal spikes and seizures in awake mice; and (4) GABAergic tone decreases during isoflurane anesthesia. iGABASnFR will permit high spatiotemporal resolution of GABA signaling in intact preparations.

neuroscience↗

bioRxiv2018

A shared cis-regulatory module activates transcription in the suspensor of plant embryos

The mechanisms controlling the transcription of gene sets in specific regions of a plant embryo shortly after fertilization remain unknown. Previously, we showed that G564 mRNA, encoding a protein of unknown function, accumulates to high levels in the giant suspensor of both Scarlet Runner Bean (SRB) and Common Bean embryos, and a cis-regulatory module containing three unique DNA sequences, designated as the 10-bp, Region 2, and Fifth motifs, is required for G564 suspensor-specific transcription [Henry, K. F. et al., Plant Mol. Biol. 88(3):207-217 (2015); Kawashima, T. et al., Proc. Natl. Acad. Sci USA 106(9):3627-3632 (2009)]. We tested the hypothesis that these motifs are also required for transcription of the SRB GA 20-oxidase gene, which encodes a gibberellic acid hormone biosynthesis enzyme and is co-expressed with G564 at a high level in giant bean suspensors. We used deletion and gain-of-function experiments in transgenic tobacco embryos to show that two GA 20-oxidase DNA regions are required for suspensor-specific transcription - one in the 5 untranslated region (UTR) (+119 to +205) and another in the 5 upstream region (-341 to -316). Mutagenesis of sequences in these two regions determined that the cis-regulatory motifs required for G564 suspensor transcription are also required for GA 20-oxidase transcription within the suspensor, although the motif arrangement differs. Our results demonstrate the flexibility of motif positioning within a cis-regulatory module that activates gene transcription within giant bean suspensors, and suggest that G564 and GA 20-oxidase comprise part of a suspensor gene regulatory network.\n\nSignificanceLittle is known about how genes are expressed in different plant embryo regions. We tested the hypothesis that shared cis-regulatory motifs control the transcription of genes specifically in the suspensor. We carried out functional studies with the Scarlet Runner Bean (SRB) GA 20-oxidase gene that encodes a gibberellic acid (GA) hormone biosynthesis enzyme, and is expressed specifically within the suspensor. We show that cis-regulatory motifs required for GA 20-oxidase transcription within the suspensor are the same as those required for suspensor-specific transcription of the SRB G564 gene, although motif number, spacing and order differ. These cis-elements constitute a control module that is required to activate genes in the SRB suspensor and may form part of a suspensor regulatory network.

plant biology↗

bioRxiv2017

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus ×- yedoensis 'Somei-yoshino' across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the \"Ohanami Project\", to obtain environmental DNA samples from petal surfaces of Cerasus x yedoensis Somei-yoshino across the Japanese archipelago during spring 2015. C. x yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained across a wide distribution; this DNA likely originated from pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

microbiology↗