Biology subjects

Iwasaki, K.

Publications and source records attributed to Iwasaki, K..

24 records · Page 2Linked to original sources

bioRxiv2021

The excitatory/inhibitory balance of synaptic transmission is impaired at higher extracellular Ca2+ concentrations in Scn1a+/- mouse model of Dravet syndrome

Dravet syndrome (DS) is an intractable form of childhood epilepsy that occurs in infancy. More than 80% of all patients have a heterozygous abnormality in the SCN1A gene, which encodes a subunit of Na+ channels in the brain. However, the detailed pathogenesis of DS remains unclear. This study investigated the synaptic pathogenesis of this disease in terms of excitatory/inhibitory balance using a mouse model of DS. We show that excitatory postsynaptic currents were similar between Scn1a knock-in neurons (Scn1a+/- neurons) and wild-type neurons, but inhibitory postsynaptic currents were significantly lower in Scn1a+/- neurons. Moreover, both the vesicular release probability and the number of inhibitory synapses were significantly lower in Scn1a+/- neurons compared with wild-type neurons. There was no proportional increase in inhibitory postsynaptic current amplitude in response to increased extracellular Ca2+ concentrations. Our study revealed that the number of inhibitory synapses is significantly reduced in Scn1a+/- neurons, while the sensitivity of inhibitory synapses to extracellular Ca2+ concentrations is markedly increased. These data suggest that Ca2+ tethering in inhibitory nerve terminals may be disturbed following the synaptic burst, likely leading to epileptic symptoms.

neuroscience↗

bioRxiv2020

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope tag insertion

Antibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We have already demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing a PA12 tag as an inserted epitope. Nevertheless, we also found that the complex formation through the inserted PA12 tag inevitably caused structural change around the insertion site of the target. Hence, we here attempted to improve the insertion method and consequently discovered that utilization of a PA14 tag significantly reduced the structural change in the target. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. Due to this structural property, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Molecular dynamics simulations also indicated that the folding of the target was rigid regardless of the PA14 insertion and the complex formation with the NZ-1 Fab. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.

biophysics↗

bioRxiv2020

Presynaptically silent synapses are modulated by the density of surrounding astrocytes

The astrocyte, a major glial cell type, is involved in formation and maturation of synapses, and thus contributes to sustainable synaptic transmission between neurons. Given that the animals in the higher phylogenetic tree have brains with higher density of glial cells with respect to neurons, there is a possibility that the relative astrocytic density directly influences synaptic transmission. However, the notion has not been tested thoroughly. Here we addressed it, by using a primary culture preparation where single hippocampal neurons are surrounded by a variable but countable number of cortical astrocytes in dot-patterned microislands, and recording synaptic transmission by patch-clamp electrophysiology. Neurons with a higher astrocytic density showed a higher amplitude of evoked excitatory postsynaptic current (EPSC) than that of neurons with a lower astrocytic density. The size of readily releasable pool of synaptic vesicles per neuron was significantly higher. The frequency of spontaneous synaptic transmission (miniature EPSC) was higher, but the amplitude was unchanged. The number of morphologically identified glutamatergic synapses was unchanged, but the number of functional ones was increased, indicating a lower ratio of presynaptically silent synapses. Taken together, the higher astrocytic density enhanced excitatory synaptic transmission by increasing the number of functional synapses through presynaptic un-silencing.

neuroscience↗

bioRxiv2020

Degradation of white birch shelterbelts by attack of white-spotted longicorn beetle in central Hokkaido, northern Japan.

Widespread decline of white birch shelterbelts was observed in central Hokkaido, northern Japan. Many exit holes bored by adults of the white-spotted longicorn beetle have been found at the bases of the trunks of trees in these stands. The number of adult longicorn beetle exit holes (Nholes) of dead standing trees tended to be greater than that of living trees. Nholes tended to increase with increasing DBH, and there was a negative relationship between Nholes and tree vigor. We found a size-dependent lethal threshold in Nholes. A resonance-measurement device (RMD) for diagnosing the level of wood defection inside the trunk was also tested. The RMD examination together with the lethal threshold in Nholes can be a useful tool for the diagnosis of white birch trees. We estimated Nholes of dead standing trees with a DBH of 25 cm in each plot (ND25) to compare the severity of infestation among plots. Logistic regression analysis revealed that 50% of stands will be degraded if ND25 = 25.0. Thus, the degradation could also be evaluated by Nholes.

ecology↗

bioRxiv2019

Dynamic rotation of the protruding domain enhances the infectivity of norovirus

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the latter conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two P domain conformations were also found in the human norovirus GII.3 capsid. These results provide new insights into the infection mechanisms of the non-envelope viruses that function in dramatic environmental changes such as the digestive tract. Significance StatementThe capsid structures of caliciviruses have been reported to be classified into two different types, according to species and genotype. One is the rising P domain type as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus, where the P domain rises from the S domain surface. The other is the resting P domain type as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus, where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine norovirus infectious particles changes reversibly between the rising and resting P domain types in response to aqueous conditions. Our findings provide new insights into the mechanisms of viral infection of caliciviruses.

pathology↗

bioRxiv2019

Translocational unfolding in clostridial binary iota toxin complex

Protein translocation across the membrane is critical for microbial pathogenesis and various cellular functions. Bacterial binary toxins such as anthrax toxin are composed of enzyme components and a translocation channel, which catalyses substrate unfolding and translocation. Here we report the structures of the clostridial binary toxin (iota toxin) translocation channel Ib-pore and its complex with ADP-ribosyltransferase Ia. The Ib-pore structure at atomic resolution provides a similar structural framework as observed for the catalytic {phi}-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure showed a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia N-terminal domain interacts with Ib via two other Ib-pore bottlenecks with multiple weak interactions. Furthermore, Ib-binding induces Ia N-terminal -helix tilting and partial unfolding, whereupon the unfolded N-terminus continues to the {phi}-clamp gate. This study reveals the novel mechanism of N-terminal unfolding, which is crucial for protein translocation.

biophysics↗