Biology subjects

Kubo, T.

Publications and source records attributed to Kubo, T..

25 records · Page 2Linked to original sources

bioRxiv2022

Classification of Extracellular Vesicles based on Surface Glycan Structures by Spongy-like Separation Media

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogenous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in-situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 m) of SPM. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling.

biochemistry↗

bioRxiv2022

Puromycin is incorporated into regenerating flagella of Chlamydomonas reinhardtii as an indicator of nascent flagellar proteins

The unicellular alga Chlamydomonas reinhardtii assembles flagella after pH shock-induced deflagellation, in which process the cell vigorously synthesizes flagellar proteins. Previously, newly synthesized flagellar proteins were chased using radioisotopes. Here, we have developed an alternative, non-radioactive method using the surface sensing of translation (SUnSET) assay, an assay that takes advantage of the incorporation of the antibiotic puromycin into newly synthesized peptides. Just after deflagellation, puromycin-labeled proteins increased dramatically in the cytoplasm and newly assembled flagella. Axonemal incorporation of newly synthesized proteins occurred from the distal tip of the flagellum. Cycloheximide, a protein synthesis inhibitor, almost completely prevented the cellular and flagellar incorporation of puromycin, although, as reported previously, it allowed reassembly of half-length flagella from the cytoplasmic "flagellar precursor" proteins. In contrast to wild-type cells, a cycloheximide-resistant mutant act2 produced nearly full-length flagella containing puromycin-labeled proteins even in the presence of cycloheximide. These and other results demonstrate that the SUnSET method serves as a powerful tool for visualizing flagellar incorporation of nascent proteins in Chlamydomonas.

cell biology↗

bioRxiv2022

Simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

O_LIPrevious research unveiled that the overexpression of catalytically promiscuous CYP81A cytochrome P450s underlies the multiple-herbicide resistance (MHR) in a Californian population of Echinochloa phyllopogon. However, it does not fully accommodate the resistance to diverse herbicides in MHR E. phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. C_LIO_LIWe investigated the high-level resistance to diclofop-methyl in MHR E. phyllopogon. Detailed diclofop-methyl metabolism was analyzed, followed by gene expression study and functional characterization of P450 genes. The generality of the MHR mechanism was investigated using another MHR line. C_LIO_LIThe MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81A12/21. Gene expression study identified the genetically linked overexpression of a novel gene CYP709C69 with CYP81A12/21 in the MHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast. The activity was observed in some CYP709C in plants. Unlike the broad substrate-specificity in CYP81As, CYP709C69 showed narrow substrate-specificity. The overexpression of the CYP81A and CYP709C69 was also observed in another MHR line. C_LIO_LIThe present findings establish a novel concept that genetically-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds. C_LI

plant biology↗

bioRxiv2020

Agonist and antagonist diverted twisting motions of single TRPV1 channel

Transient receptor potential vanilloid type 1 (TRPV1) channels are activated by heat, vanilloids, and extracellular protons. Cryo-EM has revealed various conformations of TRPV1, and these structures suggest an intramolecular twisting motion in response to ligand binding. However, limited experimental data support this observation. Here we analyzed the intramolecular motion of TRPV1 using diffracted X-ray tracking (DXT). DXT analyzes trajectories of Laue spots generated from attached gold nanocrystals, and provides picometer spatial and microsecond time scale information about intramolecular motion. We observed that both an agonist and a competitive antagonist evoked rotating bias in TRPV1, though these biases were in opposing directions. Furthermore, the rotational bias generated by capsaicin was reversed between the wild type and the capsaicin-insensitive Y511A mutant. Our findings bolster the understanding of the mechanisms used activation and modulation of TRP channels, and this knowledge can be exploited for pharmacological usage such as inhibitor design.

biophysics↗

bioRxiv2020

Wavefront-sensorless adaptive optics with a laser-free spinning disk confocal microscope

Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution.

cell biology↗

bioRxiv2020

Expert programmers have fine-tuned cortical representations of source code

Expertise enables humans to achieve outstanding performance on domain-specific tasks, and programming is no exception. Many have shown that expert programmers exhibit remarkable differences from novices in behavioral performance, knowledge structure, and selective attention. However, the underlying differences in the brain are still unclear. We here address this issue by associating the cortical representation of source code with individual programming expertise using a data-driven decoding approach. This approach enabled us to identify seven brain regions, widely distributed in the frontal, parietal, and temporal cortices, that have a tight relationship with programming expertise. In these brain regions, functional categories of source code could be decoded from brain activity and the decoding accuracies were significantly correlated with individual behavioral performances on source-code categorization. Our results suggest that programming expertise is built up on fine-tuned cortical representations specialized for the domain of programming.

neuroscience↗

bioRxiv2018

Diffusion rather than IFT provides most of the tubulin required for axonemal assembly

Tubulin enters the cilia by diffusion and motor-based intraflagellar transport (IFT). The respective contributions of each route in providing tubulin for axonemal assembly are unknown. To attenuate IFT-based transport, we expressed modified GFP-tubulins in strains possessing IFT81 and IFT74 with altered tubulin binding sites. E-hook deficient GFP-{beta}-tubulin normally incorporated into the axonemal microtubules; its transport frequency was reduced by ~90% in control cells and essentially abolished when expressed in a strain possessing IFT81 with an incapacitated tubulin-binding site. Despite the strong reduction in IFT, the share of E-hook deficient GFP-{beta}-tubulin in the axoneme was only moderately reduced indicating that most axonemal tubulin (~80%) enters cilia by diffusion. While not providing the bulk of axonemal tubulin, we propose that motor-based IFT is nevertheless critical for ciliogenesis because it ensures high concentrations of tubulin near the ciliary tip promoting axonemal elongation.

cell biology↗