Determination of Cell Cycle Stage and Mitotic Exit Through the Quantification of the Protein Levels of Known Mitotic Regulators

Cepeda-García, Cristina

doi:10.1007/978-1-4939-6502-1_4

Cited by 6 publications

(5 citation statements)

References 16 publications

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“…Analysis of cell cycle progression demonstrated that, after their release from a G1 arrest, cells expressing Bfa1-eGFP and Nup159-GBP showed a short but consistent 15 min delay at the metaphase-to-anaphase transition, which likely reflects problems at this cell cycle stage (Fig 4D -4G). This delay, similar to that caused by lack of the FEAR network-dependent Cdc14 release [49][50][51], was maintained up to anaphase, with spindle disassembly and mitotic exit PLOS BIOLOGY taking place more slowly and gradually in cells expressing Bfa1-eGFP Nup159-GBP than in the wild type (Fig 4D -4G).…”

Section: Nup159 Association With Bfa1 Is Reduced In the Absence Of Dy...mentioning

confidence: 67%

“…Protein extracts were prepared using a trichloroacetic acid (TCA) precipitation method detailed in [50]. For TCA precipitation, 10 ml cells from liquid culture were incubated for 10 min in 5% TCA.…”

Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

“…Finally, 62.5 μl 3× Laemmli sample buffer was added, and protein extracts were boiled for 5 min at 100˚C before being loaded in a polyacrylamide gel. Western blot analysis of protein levels was performed as described in [50], using specific antibodies at the concentrations indicated in S2 Table . The protein expression levels were detected and quantified using WesternBright ECL reagents (Advansta), a ChemiDoc MP system, and Image Lab software (Bio-Rad).…”

Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

“…Both input and immunoprecipitated were warmed for 5 min before undergoing SDS-PAGE. Western blot analysis of the protein levels was performed as detailed in [50], using specific antibodies at the concentrations indicated in S2 Table . The protein expression levels were detected and quantified using WesternBright ECL reagents (Advansta) and a ChemiDoc MP system (Bio-Rad) or the ImageJ (http://rsbweb.nih.gov/ij/) software.…”

Section: Protein Co-immunoprecipitationmentioning

confidence: 99%

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Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy

de Oya,

Manzano-López,

Álvarez-Llamas

et al. 2023

PLoS Biol

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Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.

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Section: Nup159 Association With Bfa1 Is Reduced In the Absence Of Dy...mentioning

confidence: 67%

Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

Section: Protein Co-immunoprecipitationmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy

de Oya,

Manzano-López,

Álvarez-Llamas

et al. 2023

PLoS Biol

Self Cite

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show abstract

“…1B , P~Kss1, 0 min to 80 min, G1, S and G2). When the cyclin Clb2p-Myc levels dropped in M phase (R ichardson et al 1992; I rniger et al 1995; W asch and C ross 2002; C ross et al 2005; E luere et al 2007; K uczera et al 2010; C epeda-garcia 2017), P~Kss1p levels increased ( Fig. 1B , 100 min).…”

Section: Resultsmentioning

confidence: 99%

Spatiotemporal Control of Pathway Sensors and Cross-Pathway Feedback Regulate a Cell Differentiation MAPK Pathway in Yeast

Prabhakar

González²,

Dionne³

et al. 2020

Preprint

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Mitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. MAPK pathways can share components with other pathways yet induce specific responses through mechanisms that remain unclear. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the MAPK pathway that regulates the response to osmotic stress (HOG). By exploring temporal regulation of MAPK signaling, we show here that the two pathways exhibited different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding the mucin sensors (MSB2 for fMAPK and HKR1 for HOG). We also show that positive feedback through the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. By exploring spatial regulation of MAPK signaling, we found that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway feedback, regulate a MAPK pathway that controls a cell differentiation response in yeast.

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Regulation of Bud Emergence by a MAPK Pathway

Prabhakar¹,

Chow²,

Siegel³

et al. 2019

Preprint

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All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in yeast, where the Rho GTPase Cdc42p regulates symmetry breaking at bud sites and the establishment of polarity by interacting with effector proteins. The prevailing view of bud emergence does not account for regulation by extrinsic cues or signal transduction pathways. Here, we show that the MAPK pathway that controls filamentous growth (fMAPK pathway), which also requires Cdc42p and the effector p21 activated kinase (PAK) Ste20p, regulates bud emergence under nutrient-limiting conditions that favor filamentous/invasive growth. The fMAPK pathway regulated the expression of polarity targets that included the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and highest at a period in the cell cycle that coincided with bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced growth at multiple sites that resulted from multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

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Determination of Cell Cycle Stage and Mitotic Exit Through the Quantification of the Protein Levels of Known Mitotic Regulators

Cited by 6 publications

References 16 publications

Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy

Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy

Spatiotemporal Control of Pathway Sensors and Cross-Pathway Feedback Regulate a Cell Differentiation MAPK Pathway in Yeast

Regulation of Bud Emergence by a MAPK Pathway

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