Biology subjects

Sakamaki, Y.

Publications and source records attributed to Sakamaki, Y..

12 recordsLinked to original sources

bioRxiv2026

Sarcoplasmic reticulum remodeling through autophagosome biogenesis during Drosophila metamorphosis

The sarcoplasmic reticulum (SR) is an elaborate membrane network and the primary intracellular Ca2+ store for muscle contraction. SR abundance, morphology, and spatial organization must be remodeled during muscle atrophy and hypertrophy, yet the mechanisms underlying this remodeling remain poorly understood. Autophagy has been implicated in SR homeostasis and may contribute to this process. We previously showed that a subset of Drosophila larval muscles remodels into adult dorsal internal oblique muscles through an autophagy-dependent process accompanied by extensive SR reorganization. Here, we show that autophagy is essential for maintaining SR abundance and architecture during muscle remodeling. Blocking autophagosome formation caused the accumulation of large aggregates of SR membranes, whereas blocking autophagosome-lysosome fusion led to the accumulation of autophagosomes with SR transmembrane proteins localized to their membranes. Moreover, disrupting LC3-interacting region (LIR)-dependent cargo recognition did not impair SR remodeling, arguing against a major contribution from selective autophagic degradation of the SR. Instead, our findings suggest that SR membrane components are incorporated into autophagosomal membranes during muscle remodeling. We propose that this membrane flow couples the removal of excess SR membrane to autophagosome biogenesis during atrophy, thereby facilitating large-scale SR reorganization during muscle remodeling.

cell biology↗

bioRxiv2025

Transfer of the synechan biosynthesis and regulatory pathway enables sulfated polysaccharide production in Synechococcus elongatus PCC 7942

Many sulfated polysaccharides (SPSs) possess useful physicochemical properties and biological activities and are widely used in industry. Currently, major SPSs are derived from livestock and marine organisms. To contribute both to addressing global environmental challenges and to the development of diverse SPSs, microbial production systems are therefore required. Nevertheless, bacteria capable of producing SPSs are limited. In contrast, many cyanobacteria synthesize diverse SPSs, making them promising hosts to produce valuable SPSs, sources for novel SPSs, and genetic resources for SPS biosynthesis. However, no study has introduced an entire heterologous SPS biosynthetic system into cyanobacteria to produce a heterologous SPS. We recently identified a novel SPS, synechan, and elucidated the genetic basis for the biosynthesis and regulation of cyanobacterial SPSs. Here, we heterologously expressed the gene set responsible for synechan biosynthesis and its regulation in the non-SPS-producing cyanobacterium Synechococcus elongatus PCC 7942 and successfully induced the production of an SPS. This work provides a foundation for engineering cyanobacteria to produce useful SPSs and, to our knowledge, the first functional heterologous reconstitution in cyanobacteria of a large gene cluster encoding a complex membrane-associated biosynthetic system and thus an important step in synthetic-biology-based engineering of cyanobacteria.

synthetic biology↗

bioRxiv2025

Protein kinase C inhibitor suppresses 2-cell stage development and perinuclear vesicle formation in mouse zygotes

Nuclear structure and nucleocytoplasmic interaction are closely linked to the regulation of cellular and higher-order biological processes. An alternative pathway for nucleocytoplasmic transport involving perinuclear vesicle formation and release, termed nuclear envelope budding (NEB), has been observed in diverse species and cell types. NEB-like events have also been reported in mammalian zygotes, including mice. However, their molecular basis remains unclear. Recent results suggest that protein kinase C (PKC) signaling regulates perinuclear vesicle biogenesis during NEB. While multiple PKC isoforms are expressed in mouse oocytes, their functions in zygotes are not fully understood. We investigated the effects of pharmacological PKC inhibition on zygotic development and NEB-like perinuclear vesicle formation. Our results suggest that NEB-like events in mouse zygotes may involve PKC-dependent mechanisms and that PKC activity might be critical for the 1-to 2-cell transition.

developmental biology↗

bioRxiv2025

Linear ubiquitination triggers Amph-mediated T-tubule biogenesis

T-tubules are specialized invaginations of the plasma membrane essential for muscle contraction. While their physiological importance is well established, the mechanisms underlying T-tubule formation remain elusive. Here, we identify LUBEL/RNF31, a ubiquitin E3 ligase responsible for linear (M1-linked) ubiquitination, as a key regulator of T-tubule biogenesis through proximity proteomics and RNAi screening in Drosophila. Loss of LUBEL leads to the formation of Amphiphysin (Amph)-positive membrane sheets instead of tubular networks in muscle cells. Mechanistically, the ubiquitin ligase activity of LUBEL, and direct interaction with Amph, a BAR domain protein involved in membrane tubule extension, are crucial for proper T-tubule morphology. LUBEL and M1-linked ubiquitin chains assemble into condensates on membranes, facilitating Amph-mediated membrane tubulation. Notably, the Amph-LUBEL/RNF31 interaction is evolutionarily conserved across a broad range of species, underscoring a fundamental role for linear ubiquitination in membrane remodeling. Our findings uncover a previously unrecognized role for linear ubiquitination in membrane deformation driven by BAR domain proteins.

cell biology↗

bioRxiv2024

Transcriptional Dynamics Uncover the Role of BNIP3 in Mitophagy during Muscle Remodeling in Drosophila

Differentiated muscle cells contain myofibrils and well-organized organelles, enabling powerful contractions. Muscle cell reorganization occurs in response to various physiological stimuli; however, the mechanisms behind this remodeling remain enigmatic due to the lack of a genetically trackable system. Previously, we reported that a subset of larval muscle cells is remodeled into adult abdominal muscle through an autophagy-dependent mechanism in Drosophila. To unveil the underlying mechanisms of this remodeling, we performed a comparative time-course RNA-seq analysis of isolated muscle cells with or without autophagy. It revealed both transcriptional dynamics independent of autophagy and highlighted the significance of BNIP3-mediated mitophagy in muscle remodeling. Mechanistically, we found that BNIP3 recruits autophagic machinery to mitochondria through its LC3-interacting (LIR) motif and minimal essential region (MER), which interact with Atg8a and Atg18a, respectively. Loss of BNIP3 leads to a substantial accumulation of larval mitochondria, ultimately impairing muscle remodeling. In summary, this study demonstrates that BNIP3-dependent mitophagy is critical for orchestrating the dynamic process of muscle remodeling. Impact statementA time-course RNA-seq analysis of muscle remodeling reveals transcriptional dynamics independent of autophagy and highlights the significance of BNIP3 in mitochondrial degradation in vivo.

cell biology↗

bioRxiv2023

Discovery of Novel Replication Proteins for Large Plasmids in Cyanobacteria and Their Potential Applications in Genetic Engineering

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid". Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

microbiology↗

bioRxiv2023

Intradermal Administration of Rescue Dose of Antiemetics in Chemotherapy-induced Nausea and Vomiting

Antiemetic therapy is provided to patients with cancer to prevent chemotherapy- induced nausea and vomiting (CINV), based on the emetic risk of anticancer agents. However, breakthrough CINV can occur despite appropriate antiemetic prophylaxis. It is preferable to administer the rescue dose of antiemetics in breakthrough CINV that occur at home, in a form that is immediately effective and that can be easily self- administered by the patient. In the present study, we examined and compared the blood pharmacokinetics of subcutaneous (SC) injection, a preferred route of self-injection, and intradermal (ID) injection, a less invasive route, with an aim of developing simple self- injectable rescue antiemetics. A single administration of four kinds of antiemetics via SC and ID routes in rats resulted in lower Tmax values compared with previously reported oral administration, indicating that the administration of antiemetics via ID and SC routes may be effective in providing faster antiemetic effects and to alleviate patient symptoms.

pharmacology and toxicology↗

bioRxiv2022

Exploration of the autonomous replication region and its utilization for expression vectors in cyanobacteria.

Due to their photosynthetic capabilities, cyanobacteria is expected to be an ecologically friendly host for the production of biomaterials. However, compared to other bacteria, there is little information of autonomous replication sequences, and tools for genetic engineering, especially expression vector systems, are limited. In this study, we established an effective screening method, namely AR-seq (Autonomous Replication sequencing), for finding autonomous replication regions in cyanobacteria and utilized the region for constructing expression vector. AR-seq using the genomic library of Synechocystis sp. PCC 6803 revealed that a certain region containing Rep-related protein (here named as Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. The reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in a wide range of multiple cyanobacterial species, not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, the GFP expression in pYS-based system can be tightly regulated by IPTG, achieving 10-fold higher levels than that of chromosome-based system. Furthermore, pYS can be used together with conventional vector pEX, which was constructed from an endogenous plasmid in 5. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

synthetic biology↗

bioRxiv2022

NCOA4 drives ferritin phase separation to facilitate macroferritinophagy and endosomal microferritinophagy

A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, the exact function of NCOA4 remains elusive. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).

cell biology↗

bioRxiv2021

TBC1D18, a novel Rab5-GAP, coordinates endosome maturation together with Mon1

Endosome maturation is essential for efficient degradation of internalized extracellular molecules and plasma membrane proteins. Two Rab GTPases, Rab5 and Rab7, are known to regulate endosome maturation, and a Rab5-to-Rab7 conversion mediated by a Rab7 activator, Mon1-Ccz1, is essential for progression of the maturation process. However, the importance and mechanism of Rab5 inactivation during endosome maturation is poorly understood. Here we report a novel Rab5 inactivator (Rab5-GTPase activating protein [Rab5-GAP]), TBC1D18, which is associated with Mon1 and mediates endosome maturation. We found that Rab5 hyperactivation in addition to Rab7 inactivation occurs in the absence of Mon1. We present evidence showing that the severe defects in endosome maturation observed in Mon1-KO cells are attributable to Rab5 hyperactivation rather than to Rab7 inactivation. We then identified TBC1D18 as a Rab5-GAP by comprehensive screening of TBC-domain-containing Rab-GAPs. Expression of TBC1D18 in Mon1-KO cells rescued the defects in endosome maturation, whereas its depletion attenuated endosome formation and degradation of endocytosed cargos. Moreover, TBC1D18 was found to be able to interact with Mon1, and it localized in close proximity to lysosomes in a Mon1-dependent manner. Thus, TBC1D18 is a crucial regulator of endosome maturation that functions together with Mon1.

cell biology↗

bioRxiv2020

ALIX and ceramide differentially control polarized exosome release from epithelial cells

Exosomes, new players in cell-cell communication, are extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods for studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of exosomes are differentially secreted from the apical and basolateral sides. We also identified GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical-exosome-specific protein. We further demonstrated that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome secretion. Thus, two independent machineries, the ALIX-Syntenin1- Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.

cell biology↗

bioRxiv2020

An Autophagy-Dependent Tubular Lysosomal Network Synchronizes Degradative Activity Required for Muscle Remodeling

Previously, we reported that autophagy is critical for Drosophila muscle remodeling during metamorphosis (Fujita et al., 2017). However, little is known about how lysosomes meet increased degradative demand upon cellular remodeling. Here, we found an extensive tubular autolysosomal network in remodeling muscle. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling. Impact StatementAnalysis of developmentally-regulated Drosophila muscle remodeling revealed autophagy-dependent formation of an extensive, Syntaxin 17-marked, tubular network that synchronizes the abundant degradative activity across a broad region of the remodeling muscle

cell biology↗