Biology subjects

Nakayama, T.

Publications and source records attributed to Nakayama, T..

29 records · Page 2Linked to original sources

bioRxiv2023

Six3 acts independently of Pax6 to provide an essential contribution to lens development

The Six3 transcription factor is essential for forebrain and eye development, and SIX3 mutations cause the congenital disorder holoprosencephaly. We created a six3 mutant in Xenopus tropicalis with a mild holoprosencephaly phenotype, and unlike mouse Six3 mutants that are headless/eyeless, the Xenopus mutant forms some eye structures, allowing direct study of Six3 function in eye formation. We focus here on striking deficits in lens formation. Early lens induction occurs normally in the mutant, e.g., the essential eye gene pax6, is activated in lens ectoderm, persisting in the eye to a late developmental stage, but in many embryos the lens fails to form. We found that bmp4, bmp7.1, smad7, dll1, dlc, mab21l1 and/or mab21l2, previously unknown as six3 eye targets, are downregulated in the mutant. We show that six3 is required for lens formation, acting primarily in developing retina during neurulation through BMP and Notch signaling, and that mab21l1/mab21l2 regulate(s) this BMP activity. This work reveals previously unrecognized essential roles for six3 in eye development, identifying its key role in signaling needed for lens formation, and acting independently of pax6 activity. SUMMARY STATEMENTThis study identifies the six3 transcription factor as the mediator of key inductive signals driving lens formation, acting independently of pax6 in early phases of lens formation.

developmental biology↗

bioRxiv2021

The Brachypodium distachyon cold-acclimated plasma membrane proteome is primed for stress resistance

In order to survive sub-zero temperatures, some plants undergo cold acclimation where low, non-freezing temperatures and/or shortened day lengths allow cold hardening and survival during subsequent freeze events. Central to this response is the plasma membrane, where low-temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first plasma membrane proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time course experiment investigated cold acclimation-induced changes in the proteome following two-phase partitioning plasma membrane enrichment and label-free quantification by nano-liquid chromatography mass spectrophotometry. Two days of cold acclimation were sufficient for membrane protection as well as an initial increase in sugar levels, and coincided with a significant change in the abundance of 154 proteins. Prolonged cold acclimation resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained cold acclimation response elicited over several days. A meta-analysis revealed that the identified plasma membrane proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress and salt resistance suggesting crosstalk between stress responses, such that cold acclimation may prime plants for other abiotic and biotic stresses. The plasma membrane proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

plant biology↗

bioRxiv2021

Genome evolution of a non-parasitic secondary heterotroph, the diatom Nitzschia putrida

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a non-photosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms revealed that a combination of genes loss, the horizontal acquisition of genes involved in organic carbon degradation, a unique secretome and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a non-parasitic, free-living secondary heterotroph.

genomics↗

bioRxiv2020

The SYP123-VAMP727 SNARE complex is involved in the delivery of inner cell wall components to the root hair shank in Arabidopsis

A root hair is a long tubular protrusion from a root hair cell established via tip growth, which is accomplished by the polarized deposition of membranous and cell wall components at the root hair apex accompanied by simultaneous hardening of the shank. The polarized secretion of materials to the root hair apex is well investigated; however, little is known about the deposition of inner cell wall materials at the root hair shank. We have previously reported that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2)/ROP10 signaling is required for the regulation of cortical microtubule construction and the deposition of inner cell wall components at the root hair shank during hardening. To unravel the alternate secretion mechanism for delivery of the inner cell wall components to root hair shank, here, we demonstrate that root hair-specific Qa-SNARE, SYP123, localizes to the subapical zone and shank of elongating root hairs in Arabidopsis. SYP123-mediated root hair elongation was inhibited by the FAB1 inhibitor YM201636, and inhibition of PtdIns(3,5)P2 production impaired the plasma membrane localization of SYP123. We also showed that SYP123 forms a SNARE complex with VAMP727 on the plasma membrane, and syp123 and vamp727 mutants exhibited lower cell wall stiffness in the root hair shank because of impaired deposition of inner cell wall components. These results indicate that SYP123/VAMP727-mediated secretion is involved in the transport of inner cell wall components for hardening of the root hair shank.

plant biology↗

bioRxiv2020

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models

SARS-CoV-2 is the coronavirus that causes the respiratory disease COVID-19, which is now the third-leading cause of death in the United States. The FDA has recently approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though recent data from the WHO shows little to no benefit with use of this anti-viral agent. Here we report the discovery of ethacridine, a safe antiseptic use in humans, as a potent drug for use against SARS-CoV-2 (EC50 ~ 0.08 M). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Indeed, ethacridine is effective in various cell types, including primary human nasal epithelial cells. Taken together, these data identify a promising, potent, and new use of the old drug possessing a distinct mode of action for inhibiting SARS-CoV-2.

microbiology↗

bioRxiv2020

Conformational Tuning of Amylin by Charged SMA Copolymers

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene- maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are -helical, the ones formed with SMAEA are rich in {beta}-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed {pi}-{pi} interaction with Phe23, electrostatic {pi}-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylins C-terminus (residues 31-37) that belongs to one of the two {beta}-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.

biophysics↗

bioRxiv2019

Large DNA virus promoted the endosymbiotic evolution to make a photosynthetic eukaryote

Chloroplasts in photosynthetic eukaryotes originated from a cyanobacterial endosymbiosis far more than 1 billion years ago1-3. Due to this ancientness, it remains unclear how this evolutionary process proceeded. To unveil this mystery, we analysed the whole genome sequence of a photosynthetic rhizarian amoeba4, Paulinella micropora5,6, which has a chloroplast-like organelle that originated from another cyanobacterial endosymbiosis7-10 about 0.1 billion years ago11. Here we show that the predacious amoeba that engulfed cyanobacteria evolved into a photosynthetic organism very quickly in the evolutionary time scale, probably aided by the drastic genome reorganization activated by large DNA virus. In the endosymbiotic evolution of eukaryotic cells, gene transfer from the endosymbiont genome to the host nucleus is essential for the evolving host cell to control the endosymbiont-derived organelle12. In P. micropora, we found that the gene transfer from the free-living and endosymbiotic bacteria to the amoeba nucleus was rapidly activated but both simultaneously ceased within the initiation period of the endosymbiotic evolution, suggesting that the genome reorganization drastically proceeded and completed. During this period, large DNA virus appeared to have infected the amoeba, followed by the rapid amplification and diversification of virus-related genes. These findings led us to re-examine the conventional endosymbiotic evolutionary scenario that exclusively deals with the host and the symbiont, and to extend it by incorporating a third critical player, large DNA virus, which activates the drastic gene transfer and genome reorganization between them. This Paulinella version of the evolutionary hypothesis deserves further testing of its generality in evolutionary systems and could shed light on the unknown roles of large DNA viruses13 in the evolution of terrestrial life.

evolutionary biology↗

bioRxiv2019

Dinoflagellates with relic endosymbiont nuclei as novel models for elucidating organellogenesis

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process integrating an endosymbiont alga into be a host-governed plastid (organellogenesis). Nevertheless, past studies suggested that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which from-endosymbiont-to-host DNA transfer is on-going and of which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide the evidence for the presence of DNA in the two nucleomorphs and transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. Combined, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as new models for elucidating organellogenesis.

evolutionary biology↗

bioRxiv2019

Practical and safe method of cryopreservation for clinical application of human adipose-derived mesenchymal stem cells without a programmable freezer or serum

BackgroundAdipose-derived mesenchymal stem cells (ADSCs) have emerged as a promising therapeutic modality for cellular therapy because of their rapid proliferation and potent cellular activity compared to conventional bone marrow-derived mesenchymal stem cells (MSCs). Cosmetic lipoaspirates provide an easily obtainable source of ADSCs. Cryopreservation facilitates their clinical application due to increased transportability and pooling of sufficient numbers of cells. However, proper cryopreservation techniques have not been established yet.\n\nMethodsWe evaluated the post-thaw viability and ADSC functions after cryopreservation with three cryoprotectants (serum containing 10% dimethylsulfoxide (DMSO), serum-free: CP-1TM, DMSO-free: SCB-DFTM) at two temperature (-80{degrees}C, -150{degrees}C) and two cell densities: (1 x 106, 7 x 106 cells/mL) for up to 18 months using cryovials. After determining optimal conditions, we also tested if large quantities of ADSCs remained viable after 18 months of cryopreservation in a 100-mL cryobag. Rate-controlled freezing methods or liquid nitrogen storage were not exploited.\n\nResultsADSCs cryopreserved in serum containing 10% DMSO or CP-1TM at -150{degrees}C and 7 x 106 cells/mL were most viable (>85%) after 18 months without perturbation of MSC functions. Even suboptimal conditions (-80{degrees}C, 1 x 106 cells/mL, no DMSO) assured >80% viability when stored for up to 9 months. Large quantities of ADSCs in a cryobag were properly cryopreserved.\n\nConclusionsA programmable freezer or liquid nitrogen storage is not necessary. CP-1TM is preferable in terms of side effects. Simplified cryopreservation methods (-80{degrees}C and no DMSO) can be used for up to 9 months, resulting in reduced infusion toxicities and lower costs.

cell biology↗

bioRxiv2019

Phytolacca americana PaGT2 is an ambidextrous polyphenol glucosyltransferase

The health benefits of polyphenols have attracted their use as potential therapeutic agents, food additives, and cosmetics. However, low water solubility of polyphenols limits their cell absorbability, obscuring further exploration. Glycosylation is known to enhance the solubility of polyphenols preserving their pharmacological properties. Here, we show that a uridine diphosphate (UDP) glucosyltransferase from Phytolacca americana (PaGT2) regioselectively catalyzes the transfer of glucose from UDP-glucose to stilbenoids such as piceatannol and flavonoids such as kaempferol. To understand the structure-function relationship of PaGT2, we determined the crystal structure of PaGT2 as well as PaGT2 complexed with donor analogue UDP-2-fluoro glucose and stilbenoid acceptor analogues. While only one conserved histidine residue is recognized as a catalytic residue in known UGTs, the crystal structures of PaGT2 suggested the presence of two catalytically active residues (His18 and His81) at two sides of the catalytic pocket. Although the single catalytic residue mutant His18Ala or His81Ala did not completely impair the glucosylation activity of the enzyme, the double mutant His18Ala/His81Ala failed to form glucoside products. These results showed that both catalytic residues in PaGT2 actively and independently catalyze glucosylation, hence we called PaGT2 as an ambidextrous UGT. The information from PaGT2 will be advantageous for the engineering of efficient biocatalysts for production of therapeutic polyphenols.

biochemistry↗

bioRxiv2018

Microtubule dynamics is required for rapid coiling of haptonemata in haptophyte algae

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is \"coiling\", which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. The persistence lengths calculated from the curvature of the haptonematal microtubules indicated their unusual flexibility. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca2+, suggesting changes in the microtubule surface lattice. Addition of Ca2+ caused bend propagation toward the proximal region. These results indicate that switching microtubule conformation with the aid of Ca2+-binding microtubule-associated proteins is responsible for rapid haptonematal coiling.\n\nSummary StatementMicroscopy observations and pharmacological experiments revealed that the rapid coiling of a non-motor microtubule-based motile organelle, the haptonema, is explained by conformational changes of microtubules, including twisting and writhing.

cell biology↗