Detection of Phosphorylation Status of Cytokinetic Components

Meitinger, Franz; Palani, Saravanan; Pereira, Gislene

doi:10.1007/978-1-4939-3145-3_16

Cited by 7 publications

(4 citation statements)

References 34 publications

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“…The total yeast cell protein precipitation, sample preparation, and western blotting were performed as previously described 52 . Primary antibodies used were rabbit anti-GFP (Abcam, ab290) and rabbit anti-Tubulin (Abcam, EPR13799).…”

Section: Protein Methodsmentioning

confidence: 99%

PI(3,5)P₂asymmetry during mitosis is essential for asymmetric vacuolar inheritance

Huda,

Koyuncu,

Dilege

et al. 2024

Preprint

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Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is a low-abundance signaling lipid that plays crucial roles in various cellular processes, including endolysosomal system structure/function, stress response, and cell cycle regulation. PI(3,5)P2synthesis increases in response to environmental stimuli, yet how it changes in cycling cells under basal conditions remained elusive. Here, using in vivo biosensors and live cell imaging, we analyzed spatiotemporal changes in PI(3,5)P2levels during the cell cycle of the budding yeastS. cerevisiae. We established that PI(3,5)P2accumulates on the vacuole in the daughter cell while it disappears from the vacuole in the mother cell during mitosis. Employing a ratiometric in vivo pH sensor, we showed that the daughter vacuole is acidified while the mother vacuole gets alkalinized concomitant with the changes in PI(3,5)P2distribution during mitosis. Our results further suggest that the asymmetry of PI(3,5)P2and the PI(3,5)P2effector Atg18 determine the asymmetry of vacuolar pH, providing insights into how the mother cell ages while the daughter cell is rejuvenated.

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Section: Protein Methodsmentioning

confidence: 99%

PI(3,5)P₂asymmetry during mitosis is essential for asymmetric vacuolar inheritance

Huda,

Koyuncu,

Dilege

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…The total yeast cell protein precipitation, sample preparation and western blotting were performed as described [ 111 ]. Primary antibodies used in this study are mouse-anti-HA (gift from Gislene Pereira), rabbit-anti-tubulin (Abcam EPR13799), mouse-anti-myc (gift from Gislene Pereira), rabbit-anti-Clb2 (gift from Gislene Pereira) and rabbit-anti-Kin4 (gift from Gislene Pereira).…”

Section: Methodsmentioning

confidence: 99%

The signalling lipid PI3,5P ₂ is essential for timely mitotic exit

Huda,

Bektas,

Bekdas

et al. 2023

Open Biol.

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Coordination of mitotic exit with chromosome segregation is key for successful mitosis. Mitotic exit in budding yeast is executed by the mitotic exit network (MEN), which is negatively regulated by the spindle position checkpoint (SPOC). SPOC kinase Kin4 is crucial for SPOC activation in response to spindle positioning defects. Here, we report that the lysosomal signalling lipid phosphatidylinositol-3,5-bisphosphate (PI3,5P 2 ) has an unanticipated role in the timely execution of mitotic exit. We show that the lack of PI3,5P 2 causes a delay in mitotic exit, whereas elevated levels of PI3,5P 2 accelerates mitotic exit in mitotic exit defective cells. Our data indicate that PI3,5P 2 promotes mitotic exit in part through impairment of Kin4. This process is largely dependent on the known PI3,5P 2 effector protein Atg18. Our work thus uncovers a novel link between PI3,5P 2 and mitotic exit.

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“…Total yeast protein precipitation, sample preparation, and Western blotting following Laemmli SDS-PAGE were performed as previously described (Meitinger et al, 2016). Total cell native protein extracts were prepared by lysis of cells using acid washed glass beads (Sigma) in a cooling bead beater homogenizer (Analytik Jena, SpeedMill Plus).…”

Section: Protein Methodsmentioning

confidence: 99%

Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

Kocakaplan

Karabürk

Dilege

et al. 2021

eLife

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Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the Spindle Position Checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5 and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.

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Detection of Phosphorylation Status of Cytokinetic Components

Cited by 7 publications

References 34 publications

PI(3,5)P₂asymmetry during mitosis is essential for asymmetric vacuolar inheritance

PI(3,5)P₂asymmetry during mitosis is essential for asymmetric vacuolar inheritance

The signalling lipid PI3,5P ₂ is essential for timely mitotic exit

Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

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Detection of Phosphorylation Status of Cytokinetic Components

Cited by 7 publications

References 34 publications

PI(3,5)P2asymmetry during mitosis is essential for asymmetric vacuolar inheritance

PI(3,5)P2asymmetry during mitosis is essential for asymmetric vacuolar inheritance

The signalling lipid PI3,5P 2 is essential for timely mitotic exit

Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

Contact Info

Product

Resources

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PI(3,5)P₂asymmetry during mitosis is essential for asymmetric vacuolar inheritance

PI(3,5)P₂asymmetry during mitosis is essential for asymmetric vacuolar inheritance

The signalling lipid PI3,5P ₂ is essential for timely mitotic exit