Biology subjects

Matsumoto, A.

Publications and source records attributed to Matsumoto, A..

27 records · Page 2Linked to original sources

bioRxiv2022

Mycelial differentiation linked avermectin production in studied with Raman imaging

Streptomyces avermitilis is a gram-positive bacterium that undergoes complex physiological and morphological differentiation during its life cycle, which has implications in secondary metabolites production. Avermectin, produced by S. avermitilis, is widely used as an anthelmintic and insecticidal agent. In this study, we have applied Raman microspectroscopic imaging to elucidate the correlation between production of avermectin and the morphological differentiation in S. avermitilis. We demonstrate distinctive variations in the localization of avermectin at various morphological stages, such as, substrate mycelium, spore-bearing mycelium, spiral spore chains under solid culture conditions. Under liquid culture condition, however, avermectin is detected only in mycelia after early MII stage of differentiation. Morphological differentiation was observed in liquid and solid cultures, but the chemical profiles of the mycelia were substantially different. Spherical bodies containing avermectin with characteristically different chemical composition to that of spores were also observed under solid culture, which suggests possible release of extracellular vesicles (EVs). Key pointsO_LIAvermectin production is regulated during mycelial differentiation C_LIO_LILiquid and solid culture conditions affects mycelial differentiation C_LIO_LIRaman microspectroscopic analysis reveals localization profiles of avermectin C_LI

microbiology↗

bioRxiv2022

Spatiotemporal properties of glutamate input support direction selectivity in the dendrites of retinal starburst amacrine cells

AO_SCPLOWBSTRACTC_SCPLOWThe asymmetric summation of kinetically distinct glutamate inputs across the dendrites of retinal "starburst" amacrine cells is proposed to underlie their direction selective properties, but experimentally verifying input kinetics has been a challenge. Here, we used two-photon glutamate sensor (iGluSnFR) imaging to directly measure the input kinetics across individual starburst dendrites. We found that signals measured from proximal dendrites were relatively sustained compared to those measured from distal dendrites. These differences were observed across a range of stimulus sizes and appeared to be shaped mainly by excitatory rather than inhibitory network interactions. Temporal deconvolution analysis suggests that the steady-state vesicle release rate was [~] 3 times larger at proximal sites compared to distal sites. Using a connectomics-inspired computational model, we demonstrate that input kinetics play an important role in shaping direction selectivity at low stimulus velocities. Together, these results provide direct support for the space-time wiring model for direction selectivity.

neuroscience↗

bioRxiv2021

A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of colony counting assays. However, colony counting assays are cumbersome and time-consuming, and are unable to detect spatial patterns of bacterial colonization in plants. Here, to overcome these shortcomings, we devised a broadly applicable genetic engineering tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed plasmid vectors that have broad host ranges and enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location ubiquitously found across bacterial phyla. These vectors allowed for generation of bioluminescent transformants of various plant pathogenic bacteria belonging to the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. The bioluminescent transformant of Pseudomonas syringae pv. tomato DC3000 (Pto-lux) was as virulent in Arabidopsis thaliana as its parental strain. Direct luminescence measurements of Pto-lux-inoculated plant tissues reported bacterial titers in A. thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha as accurately as conventional colony counting assays. We further showed the utility of our vectors for converting the previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth with a dynamic range of 4 orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated spatial colonization patterns of the Pto-lux in/on inoculated plant tissues. Taken together, our vectors offer untapped opportunities for developing bioluminescence-based quantitative and spatial analysis of bacterial growth in a variety of plant-bacteria interactions. SIGNIFICANCE STATEMENTWe developed broad-host-range plasmid vectors that integrate the luciferase operon, luxCDABE, into a specific genomic location ubiquitously found across bacterial phyla. Using these vectors, we established a high-throughput method for bioluminescence-based quantitative assays of in planta bacterial growth with a dynamic range of 4 orders of magnitude and visualized spatiotemporal patterns of bacterial colonization in/on inoculated plant tissues.

plant biology↗

bioRxiv2020

Binocular integration of retinal motion information underlies optic flow processing by the cortex

Locomotion creates various patterns of optic flow on the retina, which provide the observer with information about their movement relative to the environment. However, it is unclear how these optic flow patterns are encoded by the cortex. Here we use two-photon calcium imaging in awake mice to systematically map monocular and binocular responses to horizontal motion in four areas of the visual cortex. We find that neurons selective to translational or rotational optic flow are abundant in higher visual areas, whereas neurons suppressed by binocular motion are more common in the primary visual cortex. Disruption of retinal direction selectivity in Frmd7 mutant mice reduces the number of translation-selective neurons in the primary visual cortex, and translation- and rotation-selective neurons as well as binocular direction-selective neurons in the rostrolateral and anterior visual cortex, blurring the functional distinction between primary and higher visual areas. Thus, optic flow representations in specific areas of the visual cortex rely on binocular integration of motion information from the retina.

neuroscience↗

bioRxiv2020

Synapse-specific direction selectivity in retinal bipolar cell axon terminals

The ability to encode the direction of image motion is fundamental to our sense of vision. Direction selectivity along the four cardinal directions is thought to originate in direction-selective ganglion cells (DSGCs), due to directionally-tuned GABAergic suppression by starburst cells. Here, by utilizing two-photon glutamate imaging to measure synaptic release, we reveal that direction selectivity along all four directions arises earlier than expected, at bipolar cell outputs. Thus, DSGCs receive directionally-aligned glutamatergic inputs from bipolar cell boutons. We further show that this bouton-specific tuning relies on cholinergic excitation and GABAergic inhibition from starburst cells. In this way, starburst cells are able to refine directional tuning in the excitatory visual pathway by modulating the activity of DSGC dendrites and their axonal inputs using two different neurotransmitters.

neuroscience↗

bioRxiv2020

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

ObjectiveDue to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used for the production of a pollen-free cultivar. In order to reduce the time and cost needed for the production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica. The expected function of this gene, its two dysfunctional mutations, and genetic diversity were described recently in a related study. ResultsWe have developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. At field stations, where PCR machines are not available, LAMP markers were developed. LAMP only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be easily seen by eye. Because the causative mutations were deletions, two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers, were developed. These assays can be carried out by capillary or agarose gel electrophoresis.

genetics↗

bioRxiv2020

Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don)

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

genetics↗

bioRxiv2020

Rapid 'multi-directed' cholinergic transmission at central synapses

Acetylcholine (ACh) is a key neurotransmitter that plays diverse roles in many parts of the central nervous system, including the retina. However, assessing the precise spatiotemporal dynamics of ACh is technically challenging and whether ACh transmits signals via rapid, point-to-point synaptic mechanisms, or broader-scale non-synaptic mechanisms has been difficult to ascertain. Here, we examined the properties of cholinergic transmission at individual contacts made between direction-selective starburst amacrine cells and downstream ganglion cells in the retina. Using a combination of electrophysiology, serial block-face electron microscopy, and two-photon ACh imaging, we demonstrate that ACh signaling bears the hallmarks of both non-synaptic and synaptic forms of transmission. ACh co-activates nicotinic ACh receptors located on the intersecting dendrites of pairs of ganglion cells, with equal efficiency (non-synaptic)-- and yet retains the ability to generate rapid miniature currents ([~]1 ms rise times: synaptic). Fast cholinergic signals do not appear to depend on anatomically well-defined synaptic structures. We estimate that ACh spread is limited to [~]1-2 {micro}m from its sites of release, which may help starbursts drive local direction-selective cholinergic responses in ganglion cell dendrites. Together, our results establish the functional architecture for cholinergic signaling at a central synapse and propose a novel motif whereby single presynaptic sites can co-transmit information to multiple neurons on a millisecond timescale.

neuroscience↗

bioRxiv2020

Detection of Penicillin G Produced by Penicillium chrysogenum KF 425 in Vivo with Raman Microspectroscopy and Multivariate Curve Resolution-Alternating Least Squares Methods

Raman microspectroscopy is a minimally invasive technique that can identify molecular structure without labeling. In this study, we demonstrate in vivo detection of the bioactive compound penicillin G inside Penicillium chrysogenum KF425 fungus cells. Highly overlapped spectroscopic signatures acquired using Raman microspectroscopic imaging are analyzed using a multivariate curve resolution-alternating least squares (MCR-ALS) method to extract the pure spectra of individual molecular constituents. In addition to detecting multiple constituents such as proteins and lipids, we observe the subcellular localization of penicillin G like granule particle inside the fungus body. To date, there have been no reports of direct visualization of intracellular localization of penicillin G. The methodology we present in this article is expected to be applied as a screening tool for the production of bioactive compounds by microorganisms.

microbiology↗