Biology subjects

Ikeda, H.

Publications and source records attributed to Ikeda, H..

3 recordsLinked to original sources

bioRxiv2022

Histology-associated transcriptomic heterogeneity in ovarian folliculogenesis revealed by quantitative single-cell RNA-sequencing for tissue sections with DRaqL

High-quality single-cell RNA-sequencing (RNA-seq) with spatial resolution remains challenging. Laser capture microdissection (LCM) is a widely used, potent approach to isolate arbitrarily targeted cells from tissue sections for comprehensive transcriptomics. Here, we developed DRaqL (direct RNA recovery and quenching for LCM), an experimental approach for efficient lysis of single cells isolated by LCM from alcohol- and formalin-fixed sections without RNA purification. Single-cell RNA-seq combined with DRaqL allowed transcriptomic profiling from alcohol-fixed sections with efficiency comparable to that of profiling from freshly dissociated cells, together with effective exon- exon junction profiling. Furthermore, the combination of DRaqL and protease treatment enabled robust and efficient single-cell transcriptome analysis from tissue sections strongly fixed with formalin. Applying this method to mouse ovarian sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with oocyte size, and detected oocyte-specific splice isoforms. In addition, our statistical model revealed heterogeneity of the relationship between the transcriptome of oocytes and their size, resulting in identification of a size-transcriptome relationship anomaly in a subset of oocytes. Finally, we identified genes that were differentially expressed in granulosa cells in association with the histological affiliations of granulosa cells to the oocytes, suggesting distinct epigenetic regulations and cell-cycle activities governing the germ-soma relationship. Thus, we developed a versatile, efficient approach for robust single-cell cDNA amplification from tissue sections and provided an experimental platform conducive to high-quality transcriptomics, thereby revealing histology-associated transcriptomic heterogeneity in folliculogenesis in ovarian tissues.

developmental biology↗

bioRxiv2022

Structural basis for ion selectivity in potassium-selective channelrhodopsins

The KCR channelrhodopsins are recently-discovered light-gated ion channels with high K+ selectivity, a property that has attracted broad attention among biologists- due to intense interest in creating novel inhibitory tools for optogenetics leveraging this K+ selectivity, and due to the mystery of how this selectivity is achieved in the first place. Indeed, the molecular and structural mechanism for K+ selectivity in KCRs has remained especially puzzling since these 7-transmembrane retinal-binding proteins completely lack structural similarity with known K+ channels, which generally coordinate K+ in a precisely symmetric conduction pathway formed by a tight interface among multiple small monomeric channel subunits (presumably not an accessible mechanism for the large KCR rhodopsin proteins). Here we present the cryo-electron microscopy structures of two KCRs from Hyphochytrium catenoides with distinct spectral properties for light absorption and channel actuation, HcKCR1, and HcKCR2, at resolutions of 2.6 and 2.5 [A], respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal in HcKCR2, explaining the large spectral difference between HcKCR1 and 2. Next, our combined structural, electrophysiological, computational, and spectroscopic analyses revealed a new solution to the challenging problem of K+-selective transport. KCRs indeed do not exhibit the canonical tetrameric K+ selectivity filter that specifically coordinates dehydrated K+; instead, single KCR monomers form a size exclusion filter using aromatic residues at the extracellular side of the pore which inhibits passage of bulky hydrated ions. This unique feature allows KCRs to function as K+ channels under relevant physiological conditions, providing not only a novel mechanism for achieving high K+ permeability ratios in biological ion channels, but also a framework for designing the next generation of inhibitory optogenetic tools. In BriefThe first structures of K+-selective channelrhodopsins (HcKCR1 and 2) are determined, revealing a K+ selectivity mechanism distinctly different from canonical K+ channels. HighlightsO_LIThe cryo-EM structures of K+-selective channelrhodopsins, HcKCR1 and 2, in nanodisc C_LIO_LIConditions under which naturally-occurring microbial rhodopsins have a 6-s-cis retinal C_LIO_LIIdentification of key residues for high K+ permeability ratios C_LIO_LIThe unique K+ selectivity mechanism of KCRs C_LI

biophysics↗

bioRxiv2020

Lipooligosaccharide, Vag8, and pertussis toxin of Bordetella pertussis cooperatively cause coughing in mice

Whooping cough, a contagious respiratory disease caused by Bordetella pertussis, is characterized by paroxysmal coughing; however, the mechanism has not been studied because of the lack of versatile animal models that reproduce the cough. Here, we present a mouse model that reproduces coughing after intranasal inoculation with the bacteria or its components and demonstrate that lipooligosaccharide (LOS), pertussis toxin (PTx), and Vag8 of the bacteria cooperatively function to cause coughing. LOS-induced bradykinin sensitized a transient receptor potential ion channel, TRPV1, which acts as a sensor to evoke the cough reflex. Vag8 further increased bradykinin levels by inhibiting the C1 esterase inhibitor, the major downregulator of the contact system, which generates bradykinin. PTx inhibits intrinsic negative regulation systems for TRPV1 through inactivation of Gi GTPases. Our findings provide a basis for answering long-standing questions on the pathophysiology of the pertussis cough.

microbiology↗