Biology subjects

Nakazawa, H.

Publications and source records attributed to Nakazawa, H..

8 recordsLinked to original sources

bioRxiv2026

Extensive loss of HoxA/D genes does not disrupt anterior vertebral patterning in zebrafish

Hox genes play central roles in specifying positional identities along the vertebrate anterior-posterior axis. In mice, genetic analyses have demonstrated that Hox genes distributed among the four Hox clusters contribute to vertebral patterning, with extensive functional redundancy among paralogous genes. Our previous genetic analysis in zebrafish identified important roles for HoxB- and HoxC-related genes in specifying anterior vertebral identities, whereas the contributions of HoxA- and HoxD-related genes remained unresolved. Here, we examined adult zebrafish carrying extensive combinations of hoxaa, hoxab, and hoxda cluster deletions and generated five-gene homozygous mutants carrying frameshift mutations in hoxa3a, hoxa4a, hoxa5a, hoxd3a, and hoxd4a. X-ray micro-CT analysis revealed no obvious alterations in anterior vertebral morphology in either the compound cluster mutants or the five-gene mutants. These results indicate that HoxA/D genes make only a limited detectable contribution to anterior vertebral patterning in zebrafish. Together with our previous findings, they suggest that vertebral patterning functions are distributed unevenly among zebrafish Hox clusters, with a predominant contribution from HoxB/C-related genes.

developmental biology↗

bioRxiv2026

Exploring diverse routes to high-affinity-antibody variable domains through deep-sequencing-informed machine learning

The integration of in vitro selection, deep sequencing, and machine learning (ML) has recently been developed as a powerful strategy for discovering functional antibodies. However, how training data composition and ML search space design influence the identification of high-affinity variants remains unclear. Here, we aimed to optimize ML-integrated directed evolution for functional antibody discovery by selecting training data from deep sequencing analysis. By performing phage display selection using camelid heavy-chain antibodies (VHHs), we demonstrated that early-round data, retaining more binding-negative variants, can be superior for training models to identify high-performance VHHs. We also investigated a lead-independent ML search space design by focusing on conserved residues in final rounds, successfully identifying variants with higher affinities than those from lead-based maturation (KD = 7.9 nM). These findings demonstrate that training data selection and search space design are critical for successful ML-guided antibody engineering and provide diverse pathways for discovering high-affinity VHH variants.

bioengineering↗

bioRxiv2024

Physical constraints on the positions and dimensions of the zebrafish swim bladder by surrounding bones

Precise regulation of organ size and position is crucial for optimal organ function. Since the swim bladder is primarily responsible for buoyancy in teleosts, early development and subsequent inflation of the swim bladder should be appropriately controlled with the body growth. However, the underlying mechanism remains unclear. In this study, we show that the size and position of the swim bladder are physically constrained by the surrounding bones in zebrafish. Non-invasive micro-CT scanning revealed that the anterior edge of the swim bladder is largely attached to the os suspensorium, which is an ossicle extending medioventrally from the 4th vertebra. Additionally, we observed that hoxc6a mutants, which lack the os suspensorium, exhibited an anterior projection of the swim bladder beyond the 4th vertebra. During the swim bladder development, we found that the counterclockwise rotation of the os suspensorium correlates with posterior regression of the swim bladder, suggesting that the os suspensorium pushes the swim bladder posteriorly into its proper position. Furthermore, our results revealed a close association between the posterior region of the swim bladder and the pleural ribs. In hoxaa cluster mutants with additional ribs, the swim bladder expanded posteriorly, accompanied by an enlarged body cavity. Taken together, our results demonstrate the importance of the surrounding bones in the robust regulation of swim bladder size and position in zebrafish.

developmental biology↗

bioRxiv2024

The functional roles of zebrafish HoxA- and HoxD-related clusters in the pectoral fin development

The paralogs 9-13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa-/-;hoxab-/-;hoxda-/- larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

developmental biology↗

bioRxiv2024

PIKFYVE inhibition induces endosome- and lysosome-derived vacuole enlargement via ammonium accumulation

FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), that is essential for PtdIns(3,5)P2 production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles by PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome-lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH4+, which is protonated NH3 in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study showed that PIKfyve inhibition disrupts lysosomal homeostasis via ammonium accumulation. Summary statementInhibition of the phosphoinositide kinase PIKFYVE results in endosome/lysosome enlargement and impaired lysosomal function. This study showed that the accumulation of glutamine-derived ammonium is the cause of these events.

cell biology↗

bioRxiv2023

Low-flux electron diffraction study on the intercellular lipid organization in the human lip stratum corneum

OBJECTIVEChapped lips, characterized by drying and cracking, remain a prevalent concern. Identifying the root causes of lip chapping is crucial for developing effective treatments. We examined the lateral packing structure of intercellular lipids (ICL) in the lip stratum corneum (SC) by low-flux electron diffraction (LFED) to obtain new insights into the causes of high transepidermal water loss (TEWL) and low water retention, which may increase the vulnerability of the lip to chapping. METHODSTwenty-one healthy subjects participated in this study. After water content and TEWL measurements, a layer of corneocytes was collected from each lip vermilion surface by the grid-stripping technique. The lateral packing structure of ICL on the collected corneocytes was analyzed by LFED. RESULTSSimilar to skin SC-ICLs, we found coexistence of orthorhombic and hexagonal phases in lip SC-ICLs. We also found that electron diffraction (ED) images with no sharp peaks and a relatively small broad peak at around 2.2 nm-1 appeared frequently, unlike skin SC-ICLs. This suggests that a large fraction of corneocytes in the lip SC is surrounded by thin ICL layers in the fluid phase. Such structural features of lip SC-ICLs can explain its inferior barrier function. Furthermore, we calculated the frequency of appearance of ED images with no sharp peaks, Af, and quantitatively analyzed its correlation with water content and TEWL. The analysis showed a negative correlation between Af and water content when Af > 50%. CONCLUSIONThis is the first report on the detailed analysis of lipid organization in lip SC-ICLs. We showed that the LFED method in combination with quasi-noninvasive sample collection by the grid-stripping technique is useful for statistical study of the fine structures in lip SC. We also found that the proportion of ICLs in a fluid phase was much higher in lip SC than in skin SC, which may be related to lower water content and vulnerability of lip to chapping. Our findings provide a promising approach for obtaining clues to the structural factors regulating the water content and TEWL in lip SC, leading to more effective lip care products.

biophysics↗

bioRxiv2021

Machine-learning-guided library design cycle for directed evolution of enzymes: the effects of training data composition on sequence space exploration

Machine learning (ML) is becoming an attractive tool in mutagenesis-based protein engineering because of its ability to design a variant library containing proteins with a desired function. However, it remains unclear how ML guides directed evolution in sequence space depending on the composition of training data. Here, we present a ML-guided directed evolution study of an enzyme to investigate the effects of a known "highly positive" variant (i.e., variant known to have high enzyme activity) in training data. We performed two separate series of ML-guided directed evolution of Sortase A with and without a known highly positive variant called 5M in training data. In each series, two rounds of ML were conducted: variants predicted by the first round were experimentally evaluated, and used as additional training data for the second-round prediction. The improvements in enzyme activity were comparable between the two series, both achieving enzyme activity 2.2-2.5 times higher than 5M. Intriguingly, the sequences of the improved variants were largely different between the two series, indicating that ML guided the directed evolution to the distinct regions of sequence space depending on the presence/absence of the highly positive variant in the training data. This suggests that the sequence diversity of improved variants can be expanded not only by conventional ML using the whole training data, but also by ML using a subset of the training data even when it lacks highly positive variants. In summary, this study demonstrates the importance of regulating the composition of training data in ML-guided directed evolution.

bioengineering↗

bioRxiv2018

A machine-learning-guided mutagenesis platform for accelerated discovery of novel functional proteins

Molecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space that requires high costs for screening experiments. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed to yellow while improving its fluorescence intensity. Using 155 and 78 variants for the initial and the second-round libraries, respectively, we successfully obtained a number of proteins showing yellow fluorescence, 12 of which had better fluorescence performance than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful platform for accelerated discovery of functional proteins.

bioengineering↗