Biology subjects

Suda, Y.

Publications and source records attributed to Suda, Y..

7 recordsLinked to original sources

bioRxiv2026

Contact of mobile cis-Golgi network with ER exit sites executes COPI-mediated receptor recycling

COPI-mediated retrograde transport is essential for maintaining homeostasis between the endoplasmic reticulum (ER) and the Golgi in eukaryotic cells. However, its spatiotemporal dynamics remain unclear. In budding yeast, ER export occurs through a hug-and-kiss mechanism, wherein mobile cis-Golgi network (CGN) elements transiently approach ER exit sites (ERES) to capture cargo before departing to deliver it to the Golgi. Here, using high-resolution 3D and 4D live imaging, we show that the COPI-mediated recycling of a cargo receptor from the CGN back to the ER occurs during the hug-and-kiss event. Furthermore, this recycling is complete before the CGN detaches from the ERES, suggesting that COPI-mediated retrograde transport is spatiotemporally coupled to ER export during a single and transient CGN-ERES association. Consistent with this model, when COPI function is impaired, the receptor fails to return efficiently to the ER and instead remains in the CGN after separation from the ERES. Additionally, COPI impairment prolonged CGN-ERES association, suggesting that COPI function also facilitates CGN detachment from ERES. Thus, we propose that rapid COPI-mediated recycling drives CGN maturation directly at the ERES interface, enabling the CGN to detach and proceed along the secretory pathway.

cell biology↗

bioRxiv2025

Deep Learning Pipeline for Segmentation and Quantification of Overlapping Membranes in 2D Image

Quantitative morphological analysis is crucial for understanding cellular processes. While 3D Z-stack imaging offers high-resolution data, the complexity of 3D structures makes direct interpretation and manual annotation challenging and time-consuming, especially for large datasets. Maximum Intensity Projection (MIP) is a common strategy to create more interpretable 2D representations, but this inevitably leads to artificial overlaps between structures, significantly hindering accurate automated segmentation of individual instances by conventional methods or standard deep learning tools. To address this critical challenge in 2D projection analysis, we developed DeMemSeg, a deep learning pipeline based on Mask R-CNN, specifically designed to segment overlapping membrane structures, called prospore membranes (PSMs) during yeast sporulation. DeMemSeg was trained on a custom-annotated dataset, leveraging a systematic image processing workflow. Our optimized model accurately identifies and delineates individual, overlapping PSMs, achieving segmentation performance and derived morphological measurements that are statistically indistinguishable from expert manual annotation. Notably, DeMemSeg successfully generalized to segment PSMs from unseen data acquired from gip1{Delta} mutant cells, capturing the distinct morphological defects in PSMs. DeMemSeg thus provides a robust, automated solution for objective quantitative analysis of complex, overlapping membrane morphologies directly from widely used 2D MIP images, offering a practical tool and adaptable workflow to advance cell biology research.

cell biology↗

bioRxiv2025

Molecular basis of the autoregulatory mechanism of motor neuron-related splicing factor 30

Motor neuron-related splicing factor 30 (SPF30, also known as SMNDC1) is a paralog of the survival motor neuron protein that regulates the expression of various genes by affecting mRNA splicing. SPF30 has an autoregulatory mechanism that controls its expression. However, the detailed molecular mechanisms determining cellular levels of SPF30 remain unclear. Here, we demonstrated that SPF30 expression was controlled via the negative autoregulatory feedback, whereby increased SPF30 expression caused the inclusion of cassette exon within intron 2 and/or the generation of a newly spliced variant with exon 4a (produced by splicing 17 bp upstream of the canonical intron 3 and exon 4 junctions). Altered transcripts with cassette exon or exon 4a were subjected to nonsense-mediated mRNA decay, leading to reduced SPF30 mRNA levels. Conversely, the loss of SPF30 protein resulted in a drastic reduction in exon 4a inclusion compared to cassette exon inclusion, suggesting that exon 4a inclusion contributes more to adjusting SPF30 expression levels. An in vivo splicing assay designed to reflect exon 4a inclusion levels demonstrated that a short stretch of sequence within exon 4 of SPF30 mRNA was required for exon 4a inclusion. Additionally, the C-terminal region of SPF30 was crucial for the autoregulatory mechanism. Specifically, the C-terminal region of SPF30, including the latter part of -helix and a kink-like structure, was required for binding to RNA containing exon 4a. Collectively, these results reveal the molecular basis of the autoregulatory mechanism underlying SPF30 gene expression.

molecular biology↗

bioRxiv2023

Presence of the ER-Golgi intermediate compartment that matures into the Golgi in yeast

Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated. Here, we use high-speed, high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term "yeast ERGIC". It occasionally attaches onto the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the cisternal maturation process from ERGIC to Golgi and further to the trans-Golgi network. SummaryTojima et al. perform spatiotemporal mapping of a variety of proteins residing in and around the Golgi apparatus in budding yeast using super-resolution live imaging microscopy. They identified the ER-Golgi intermediate compartment that matures into the Golgi.

cell biology↗

bioRxiv2023

Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis

Gametogenesis in budding yeast involves a large-scale rearrangement of membrane traffic to allow de novo formation of a membrane, called the prospore membrane (PSM). However, the mechanism underlying this event is not fully elucidated. Here, we show that the number of endoplasmic reticulum exit sites (ERES) per cell fluctuates and switches from decreasing to increasing upon the onset of PSM formation. Reduction in ERES number, presumably accompanying a transient stall in membrane traffic, resulting in the loss of preexisting Golgi apparatus from the cell, was followed by local ERES regeneration, leading to Golgi reassembly in nascent spores. We have revealed that protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1, promote ERES regeneration through Sec16 foci formation. Furthermore, sed4{Delta}, a mutant with impaired ERES formation, showed defects in PSM growth and spore formation. Thus, ERES regeneration in nascent spores facilitates the segregation of membrane traffic organelles, leading to PSM growth.

cell biology↗

bioRxiv2022

Automation of yeast spot assays using an affordable liquid handling robot

The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and environmental stresses on cell growth and survival. Automation of the spot assay experiments from preparing a dilution series to spotting to observing spots continuously has been implemented based on large laboratory automation devices and robots, especially for high-throughput functional screening assays. However, there has yet to be an affordable solution for the automated spot assays suited to researchers in average laboratories and with high customizability for end-users. To make reproducible spot assay experiments widely available, we have automated the plate-based yeast spot assay of budding yeast using Opentrons OT-2 (OT-2), an affordable liquid-handling robot, and a flatbed scanner. We prepared a 3D-printed mount for the Petri dish to allow for precise placement of the Petri dish inside the OT-2. To account for the uneven height of the agar plates, which were made by human hands, we devised a method to adjust the z-position of the pipette tips which is based on the weight of each agar plate. During the incubation of the agar plates, a flatbed scanner was used to automatically take images of the agar plates over time, allowing researchers to quantify and compare the cell density within the spots at optimal time points a posteriori. Furthermore, the accuracy of the newly developed automated spot assay was verified by performing spot assays with human experimenters and the OT-2 and quantifying the yeast-grown area of the spots. This study will contribute to the introduction of automated spot assays and the automated acquisition of growth processes in conventional laboratories that are not adapted for high-throughput laboratory automation.

bioengineering↗

bioRxiv2019

Physiological determinants of the increase in oxygen consumption during exercise in individuals with stroke

BackgroundUnderstanding the physiological limitations of the increase in oxygen consumption ([V]O2) during exercise is essential to improve cardiorespiratory fitness in individuals with stroke. However, the physiological determinants of the increase in [V]O2 during exercise have not been examined using multivariate analysis in individuals with stroke. This study aimed to identify the physiological determinants of the increase in [V]O2 during a graded exercise in terms of the respiratory function, cardiac function, and ability of skeletal muscles to extract oxygen.\n\nMethodsEighteen individuals with stroke (60.1 {+/-} 9.4 years of age, 67.1 {+/-} 30.8 days poststroke) underwent a graded exercise test for the assessment of cardiorespiratory response to exercise. The increase in [V]O2 from rest to ventilatory threshold and that from rest to peak exercise were measured as a dependent variable. The increases in respiratory rate, tidal volume, heart rate, stroke volume, and arterial-venous oxygen difference from rest to ventilatory threshold and those from rest to peak exercise were measured as independent variables.\n\nResultsFrom rest to ventilatory threshold, the increases in heart rate ({beta} = 0.546) and arterial-venous oxygen difference ({beta} = 0.398) were significant determinants of the increase in [V]O2 (adjusted R2 = 0.703, p < 0.001). From rest to peak exercise, the increases in tidal volume ({beta} = 0.611) and heart rate ({beta} = 0.353) were significant determinants of the increase in [V]O2 (adjusted R2 = 0.702, p < 0.001).\n\nConclusion[V]O2 is well-known to increase nearly linearly with increasing heart rate; however, our results suggest that arterial-venous oxygen difference and tidal volume are also significant physiological determinants of the increase in [V]O2 from rest to ventilatory threshold and that from rest to peak exercise, respectively. Our findings could potentially contribute to the development of appropriate therapies to improve cardiorespiratory fitness in individuals with stroke.

physiology↗