Biology subjects

Saeki, Y.

Publications and source records attributed to Saeki, Y..

11 recordsLinked to original sources

bioRxiv2025

Caveolin-1 controls lineage fidelity of adult neural stem cells via lysosomal degradation of PDGFRα

Caveolae are cell-surface signaling hubs involved in stem cell proliferation and differentiation. However, the molecular details of how they influence stem cell quiescence remain poorly understood. In this study, we show that most caveolae components, particularly caveolin-1 (Cav-1), are specifically upregulated in quiescent neural stem cells (NSCs). Furthermore, we demonstrate that Cav-1 restricts platelet-derived growth factor receptor (PDGFR) signaling, a key regulator for NSC proliferation and oligodendrocyte differentiation in the adult brain, through lysosomal degradation. Genetic ablation of Cav-1 leads to aberrant NSC activation, reduced neurogenesis, and enhanced ectopic oligodendrocyte differentiation on the ventricular surface in the ventral side of the ventricular-subventricular zone (V-SVZ), a key NSC niche responsible for neuroblast differentiation. Mechanistically, we show that Cav-1 deficiency suppresses the endo-lysosomal degradation of PDGFR, leading to PDGFR accumulation and excessive signaling for oligodendrocyte differentiation. Taken together, our results uncover a crucial homeostatic mechanism wherein caveolae function as a safeguard to maintain adult NSC quiescence and lineage fidelity by controlling lysosomal turnover of PDGFR in a niche-specific manner.

neuroscience↗

bioRxiv2025

Intrinsic and TDP-43 dysfunction-induced catabolic stress elicit neuroprotective cellular degradation in ALS-vulnerable motor neurons

Selective neuronal vulnerability is a defining feature of neurodegenerative disorders, exemplified by motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The nature of motor neurons underlying this selectivity remains unresolved. Here, by monitoring autophagy at single-cell resolution across the translucent zebrafish spinal cord, we identify motor neurons as the cell population with the highest autophagic flux. Large spinal motor neurons (SMNs), most susceptible to ALS, exhibit higher flux compared to smaller SMNs and ALS-resistant ocular motor neurons. Notably, large SMNs accelerates both autophagy and proteasome-mediated degradation, which are further augmented by TDP-43 loss. Additionally, acceleration of multiple unfolded protein response pathways indicates their innate tendency to accumulate misfolded proteins. Enhanced cellular degradation in large SMNs is neuroprotective as its inhibition halts axon outgrowth. These findings propose that cell size-associated degradation load underlies selective neuronal vulnerability in ALS, highlighting the alleviation of catabolic stress as a target of therapy and prevention.

neuroscience↗

bioRxiv2025

Condensation of Sidekick at tricellular junctions organizes mechanical forces for cell-cell adhesion remodeling

Epithelial morphogenesis relies on coordinated multicellular movements regulated by adhesion and force-generating molecules. It remains elusive how cells ensure organization of these molecules in space and time. Combining in vitro reconstitution and fly genetics, we demonstrate that condensation of an adhesive transmembrane protein shapes molecular dynamics at tricellular junctions to facilitate cell-cell junction remodeling. Specifically, we identify that the intracellular domain of Sidekick, known to localize to tricellular junctions and recruit mechanical force-generating myosin II, undergoes phase separation into condensates. This condensation controls the physiological localization and concentration of Sidekick on lipid membranes. Altering the molecular dynamics of these condensates disrupts Sidekicks localization against cellular movements, misdirecting myosin II and leading to delayed junction formation. These findings suggest that cells exploit the mechano-resistant properties of tricellular junction-anchored condensates to spatially allocate force-regulating molecules. This study underscores the importance of condensation to organize tricellular junctions for robust multicellular movement.

cell biology↗

bioRxiv2025

Editing of the polyubiquitin architecture on the collided ribosome maintains persistent RQC activity

In Ribosome-associated Quality Control (RQC), K63-linked polyubiquitination of uS10 on the stalled ribosome is crucial for recruiting the RQC-trigger (RQT) complex. However, the mechanisms governing the maintenance and recycling of polyubiquitin architecture on colliding ribosomes remain unclear. Here we demonstrate that two deubiquitinating enzymes (DUBs), Ubp2 and Ubp3, play key roles in editing and recycling polyubiquitin chains on uS10, thereby contributing to the promotion of RQC activity. Specifically, Ubp2 eliminates the K63-linked polyubiquitin chain from uS10 on the free 40S subunit for recycling, while Ubp3 predominantly cleaves the K48-linked di-ubiquitin and the K48/K63-mixed-linkage poly-ubiquitin chain from uS10 on the translating ribosomes. Notably, we further demonstrate that the ubiquitin chains containing K48-linkage of the uS10 on the colliding ribosome act as a negative signal for the RQT-mediated ribosome dissociation process. Collectively, our findings clarify the ubiquitin code in RQC and define the positive functions of these two DUBs in maintaining the persistent RQC activity.

molecular biology↗

bioRxiv2025

Development of CRISPR/Cas13-based analytical tools to study RNA-Protein Interactions

RNA-protein interactions (RPIs) are as important as protein-protein interactions (PPIs) for the formation of membraneless organelles (MLOs) and play a vital role in various biological processes. Despite remarkable advances in PPI analysis technologies in recent years, the development of RPI analysis tools has lagged behind. To advance RPI analysis, we integrated three established PPI tools--bimolecular fluorescence complementation (BiFC), NanoBiT, and split-TurboID--with the RNA-targeting CRISPR/Cas13. We applied these tools to analyze paraspeckles, one of the best known MLOs formed by interactions between the long non-coding RNA NEAT1 and the RNA-binding protein NONO. The optimized BiFC-dCas13 allows live cell imaging and quantitative detection of the NEAT1-NONO interaction. The NanoBiT-dCas13 detects dynamic changes in the NEAT1-NONO interaction in an immediate and reversible manner. As a proximity labeling tool, the Split-TurboID-dCas13 induces biotinylation of proteins surrounding paraspeckles, leading to the identification of the N6-methyladenosine reader protein YTHDC1 as a novel paraspeckles-associated protein. The BiFC-dCas13, NanoBiT-dCas13, and Split-TurboID-dCas13 systems have a broad utility for the analysis of RPIs and MLOs.

bioengineering↗

bioRxiv2022

An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants

As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA. 1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.

microbiology↗

bioRxiv2020

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells

In eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC-CB- HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC-HLBs with CBs, and nucleation of PCs near preformed CB- HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing, and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs. (187 words)

cell biology↗

bioRxiv2020

Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

eIF2 phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

molecular biology↗

bioRxiv2020

TGN/EE SNARE protein SYP61 is ubiquitinated and required for carbon/nitrogen-nutrient responses in Arabidopsis

Ubiquitination is a post-translational modification with reversible attachment of the small protein ubiquitin, which is involved in numerous cellular processes including membrane trafficking. For example, ubiquitination of cargo proteins is known to regulate their subcellular dynamics, and plays important roles in plant growth and stress adaptation. However, the regulatory mechanism of the trafficking machinery components remains elusive. Here, we report Arabidopsis trans-Golgi network/early endosome (TGN/EE) localized soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein SYP61 as a novel ubiquitination target of a membrane localized ubiquitin ligase ATL31. SYP61 is a key component of membrane trafficking in Arabidopsis. SYP61 was ubiquitinated with K63-linked chain by ATL31 in vitro and in plants. The knockdown mutants of SYP61 were hypersensitive to the disrupted carbon (C)/nitrogen (N)-nutrient stress, suggesting its critical role in plant homeostasis in response to nutrients. We also found the ubiquitination status of SYP61 is affected by C/N-nutrient availability. These results provided possibility that ubiquitination of SNARE protein has important role in plant physiology.

plant biology↗

bioRxiv2020

α-Synuclein strains that cause distinct pathologies differentially inhibit proteasome

Abnormal -synuclein aggregation has been implicated in several diseases and is known to spread in a prion-like manner. There is a relationship between protein aggregate structure (strain) and clinical phenotype in prion diseases, however, whether differences in the strains of -synuclein aggregates account for the different pathologies remained unclear. Here, we generated two types of -synuclein fibrils from identical monomer and investigated their seeding and propagation ability in mice and primary-cultured neurons. One -synuclein fibril induced marked accumulation of phosphorylated -synuclein and ubiquitinated protein aggregates, while the other did not, indicating the formation of -synuclein two strains. Notably, the former -synuclein strain inhibited proteasome activity and co-precipitated with 26S proteasome complex. Further examination indicated that structural differences in the C-terminal region of -synuclein strains lead to different effects on proteasome activity. These results provide a possible molecular mechanism to account for the different pathologies induced by different -synuclein strains.

neuroscience↗

bioRxiv2018

HERC2 promotes BLM and WRN to suppress G-quadruplex DNA

BLM and WRN are RecQ DNA helicases essential for genomic stability. Here we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacts with BLM, WRN, and replication protein A (RPA) complexes during S-phase of the cell cycle. Depletion of HERC2 dissociates RPA from BLM and WRN complexes and significantly increases G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4- suppressing function. In vitro, HERC2 releases RPA onto single-stranded DNA (ssDNA), rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 causes RPA accumulation in the helicase complexes and increases G4, indicating an essential role for E3 activity in G4 suppression. Both HERC2 depletion and E3 inactivation sensitize cells to the G4-interacting compounds, telomestatin and pyridostatin. Overall, HERC2 is a master regulator of G4 suppression and affects the sensitivity of cells to G4 stabilizers.

molecular biology↗