Biology subjects

Murakawa, T.

Publications and source records attributed to Murakawa, T..

8 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↗

bioRxiv2026

Two Glu/Asp Residues Cooperatively Mediate an Early Step of ATP Hydrolysis in GHKL ATPases MutL and GyrB

GHKL ATPases share a unique Bergerat ATP-binding fold and regulate diverse biological processes through ATP-dependent conformational changes. An early step of ATP hydrolysis in this family has been attributed to a single highly conserved glutamate residue proposed to function as the general base. However, mutations of this residue impair both the ATPase activity and ATP binding, complicating interpretation of its catalytic role. Re-examination of the high-resolution crystal structures revealed a second conserved acidic residue positioned within a hydrogen-bonding distance from the nucleophilic water molecule. Using Aquifex aeolicus MutL and GyrB as model enzymes, we combined systematic mutagenesis, ATPase and ATP-binding assays, and X-ray crystallography to dissect the roles of these residues. We show that alignment of the nucleophilic water can be maintained as long as the conserved glutamate retains hydrogen-bonding capability, whereas efficient ATP hydrolysis requires proton-accepting capacity at least one of the two acidic residues. These results indicate that the conserved glutamate primarily governs positioning of the nucleophilic water, while activation of this water for catalysis is achieved through cooperative general base function of the two acidic residues. Extending this framework to human MutL homologs, PMS2 and MLH1, we showed that clinically reported variants of uncertain significance in these DNA mismatch repair proteins substantially reduced the ATPase activity, indicating functional impairment. Together, our findings refine the catalytic mechanism of GHKL ATPases and provide a structural and functional framework for interpreting disease-associated variants in GHKL ATPases. Phylogenetic and ancestral state analysis further indicated that the second acidic residue was likely to be present in the common ancestor of major GHKL ATPase lineages but was later modified in a branch including Hsp90, suggesting evolutionary remodeling of the catalytic mechanism in the branch.

biochemistry↗

bioRxiv2026

Tsunahiki task: A newly developed group-based operant task for mice

A group of social animals, including humans, distributes work and rewards and establishes inter-individual relationships through shared experiences of working together. Understanding the neural mechanisms underlying group work is essential for elucidating how social relationships are formed. Thus, a behavioral paradigm for studying group work in laboratory rodents is required. Here, we developed a Tsunahiki task, a novel group-based operant task for mice. In this task, three mice jointly pulled three ropes, and once all ropes were pulled out, all members gained access to a reward area, regardless of who performed the work. The mice acquired the task within a few days. Importantly, repeated experience with the Tsunahiki task led to a shift in workload toward subordinate individuals and induced rank consolidation. These findings suggest that group work induces consolidation of intra-group disparities based on the dominance hierarchy. The Tsunahiki task provides a useful framework for investigating the neurobiological mechanisms underlying collaborative work, group formation, and social inequality in rodents. TeaserA group-based operant task for mice was newly developed, in which hierarchy affected work and reward distributions.

animal behavior and cognition↗

bioRxiv2025

Fast-spiking neurons in monkey orbitofrontal cortex underlie economic value computation

Inhibitory interneurons are fundamental constituents of cortical circuits that process information to shape economic behaviors. However, the role of inhibitory interneurons in this process in the core cortical reward region, the orbitofrontal cortex (OFC), remains elusive. Here, we show that presumed parvalbumin-containing GABAergic interneurons (fast-spiking neurons, FSNs) differ from regular-spiking pyramidal neurons (RSNs) in computing economic value. While monkeys perceived a visual lottery for probability and magnitude of rewards, identified FSNs constituted 12% of OFC neurons and showed high-frequency firing rates and wide dynamic ranges, which are both key intrinsic cellular characteristics regulating cortical computation. We found that FSNs showed stronger neural modulation compared to RSNs in signaling probability and magnitude information. Unambiguously, both neural populations signaled the expected values, but FSNs processed the reward information more strongly under the influence of dynamic range. Thus, stronger FSN signals may appear with wider dynamic range for expected value computation.

neuroscience↗

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↗

bioRxiv2021

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: "HA Frankenbody". This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

cell biology↗

bioRxiv2021

Distinct roles and actions of PDI family enzymes in catalysis of nascent-chain disulfide formation

The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains (RNC) of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with short segments exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Similarly to ERp46, a PDI mutant with an occluded substrate-binding pocket displayed shorter-time RNC binding and higher efficiency in disulfide introduction than wild-type PDI. Altogether, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation in RNC when longer nascent chains emerge out from ribosome.

biochemistry↗

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↗