Biology subjects

Tojima, T.

Publications and source records attributed to Tojima, T..

4 recordsLinked to original sources

bioRxiv2023

RudLOV-a new optically synchronized cargo transport method reveals unexpected effect of dynasore

Live imaging of secretory cargoes is a powerful method for understanding the mechanisms of membrane trafficking. Inducing the synchronous release of cargoes from an organelle is a key for enhancing microscopic observation. We developed an optical cargo-releasing method named as retention using dark state of LOV2 (RudLOV), which enables exceptional spatial, temporal, and quantity control during cargo release. A limited amount of cargo-release using RudLOV successfully visualized cargo cisternal-movement and cargo-specific exit sites on the Golgi/trans-Golgi network. Moreover, by controlling the timing of cargo-release using RudLOV, we revealed the canonical and non-canonical effects of the well-known dynamin inhibitor dynasore, which inhibits early-Golgi but not late-Golgi transport and exit from the trans-Golgi network where dynamin-2 is active. Accumulation of COPI vesicles at the cis-side of the Golgi stacks in dynasore-treated cells suggests that dynasore targets COPI-uncoating/tethering/fusion machinery in the early-Golgi cisternae or endoplasmic reticulum but not in the late-Golgi cisternae. These results provide insight into the cisternal maturation of Golgi stacks.

cell 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↗

bioRxiv2022

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in GIST cells

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes KITs Golgi retention. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT is unable to trigger downstream activation. In the Golgi area, KIT activates the PKD2-phosphatidylinositol 4-kinaseIII{beta} (PKD2-PI4KIII{beta}) pathway through phospholipase {gamma}2 (PLC{gamma}2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in KIT release from the Golgi region, indicating that these PKD2-related pathways are responsible for the Golgi retention of KIT. Our findings unveil the molecular mechanisms underlying KIT mislocalization and provide evidence for a new strategy for inhibition of oncogenic signaling.

cancer biology↗

bioRxiv2022

The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment, and function independently of the Sec7p-residing region within the TGN.

cell biology↗