bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2021–12–05
fiveteen papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology
  1. Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling.
  2. Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation.
  3. Cell cycle regulation of ER membrane biogenesis protects against chromosome missegregation.
  4. Transcription destabilizes centromere function.
  5. The microtubule-associated protein She1 coordinates directional spindle positioning by spatially restricting dynein activity.
  6. PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle.
  7. Epigenetic control of centromere: what can we learn from neocentromere?
  8. The kinetochore protein NNF1 has a moonlighting role in the vegetative development of Arabidopsis thaliana.
  9. Centromere protein F promotes progression of hepatocellular carcinoma through ERK and cell cycle-associated pathways.
  10. Chromosome Missegregation as a Modulator of Radiation Sensitivity.
  11. Induction of Mitosis Delay and Apoptosis by CDDO-TFEA in Glioblastoma Multiforme.
  12. Chromosome segregation in Archaea: SegA- and SegB-DNA complex structures provide insights into segrosome assembly.
  13. Minichromosome maintenance proteins in eukaryotic chromosome segregation.
  14. Telmisartan-Induced Cytotoxicity via G2/M Phase Arrest in Renal Cell Carcinoma Cell Lines.
  15. Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET.
  1. Curr Biol. 2021 Nov 24. pii: S0960-9822(21)01493-7. [Epub ahead of print]
    Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling.
    Babhrubahan Roy, Simon J Y Han, Adrienne N Fontan, Soubhagyalaxmi Jema, Ajit P Joglekar.
      Accurate chromosome segregation during cell division requires amphitelic chromosome attachment to the spindle apparatus. It is ensured by the combined activity of the spindle assembly checkpoint (SAC),1 a signaling mechanism that delays anaphase onset in response to unattached chromosomes, and an error correction mechanism that eliminates syntelic attachments.2 The SAC becomes active when Mps1 kinase sequentially phosphorylates the kinetochore protein Spc105/KNL1 and the signaling proteins that Spc105/KNL1 recruits to facilitate the production of the mitotic checkpoint complex (MCC).3-8 The error correction mechanism is regulated by the Aurora B kinase, but Aurora B also promotes SAC signaling via indirect mechanisms.9-12 Here we present evidence that Aurora B kinase activity directly promotes MCC production by working downstream of Mps1 in budding yeast and human cells. Using the ectopic SAC activation (eSAC) system, we find that the conditional dimerization of Aurora B in budding yeast and an Aurora B recruitment domain in HeLa cells with either Bub1 or Mad1, but not the phosphodomain of Spc105/KNL1, leads to ectopic MCC production and mitotic arrest.13-16 Importantly, Bub1 must recruit both Mad1 and Cdc20 for this ectopic signaling activity. These and other data show that Aurora B cooperates with Bub1 to promote MCC production, but only after Mps1 licenses Bub1 recruitment to the kinetochore. This direct involvement of Aurora B in SAC signaling may maintain SAC signaling even after Mps1 activity in the kinetochore is lowered.
    Keywords:  cell division; kinetochore; mitosis; signaling; spindle assembly checkpoint
    DOI:  https://doi.org/10.1016/j.cub.2021.10.049
  2. Nat Commun. 2021 Dec 01. 12(1): 7010
    Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation.
    Xinhong Song, Duccio Conti, Roshan L Shrestha, Dominique Braun, Viji M Draviam.
      Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation.
    DOI:  https://doi.org/10.1038/s41467-021-27131-9
  3. Dev Cell. 2021 Nov 24. pii: S1534-5807(21)00894-7. [Epub ahead of print]
    Cell cycle regulation of ER membrane biogenesis protects against chromosome missegregation.
    Holly Merta, Jake W Carrasquillo Rodríguez, Maya I Anjur-Dietrich, Tevis Vitale, Mitchell E Granade, Thurl E Harris, Daniel J Needleman, Shirin Bahmanyar.
      Failure to reorganize the endoplasmic reticulum (ER) in mitosis results in chromosome missegregation. Here, we show that accurate chromosome segregation in human cells requires cell cycle-regulated ER membrane production. Excess ER membranes increase the viscosity of the mitotic cytoplasm to physically restrict chromosome movements, which impedes the correction of mitotic errors leading to the formation of micronuclei. Mechanistically, we demonstrate that the protein phosphatase CTDNEP1 counteracts mTOR kinase to establish a dephosphorylated pool of the phosphatidic acid phosphatase lipin 1 in interphase. CTDNEP1 control of lipin 1 limits the synthesis of fatty acids for ER membrane biogenesis in interphase that then protects against chromosome missegregation in mitosis. Thus, regulation of ER size can dictate the biophysical properties of mitotic cells, providing an explanation for why ER reorganization is necessary for mitotic fidelity. Our data further suggest that dysregulated lipid metabolism is a potential source of aneuploidy in cancer cells.
    Keywords:  CTDNEP1; aneuploidy; lipid homeostasis; lipin; mTOR; medulloblastoma; micronuclei; mitosis; nuclear assembly
    DOI:  https://doi.org/10.1016/j.devcel.2021.11.009
  4. Biochem Biophys Res Commun. 2021 Nov 25. pii: S0006-291X(21)01603-X. [Epub ahead of print]586 150-156
    Transcription destabilizes centromere function.
    Yu Nakabayashi, Masayuki Seki.
      Bi-oriented attachment of microtubules to the centromere is a pre-requisite for faithful chromosome segregation during mitosis. Budding yeast have point centromeres containing the cis-element proteins CDE-I, -II, and -III, which interact with trans-acting factors such as Cbf1, Cse4, and Ndc10. Our previous genetic screens, using a comprehensive library of histone point mutants, revealed that the TBS-I, -II, and -III regions of nucleosomes are required for faithful chromosome segregation. In TBS-III deficient cells, peri-centromeric nucleosomes containing the H2A.Z homolog Htz1 are lacking, however, it is unclear why chromosome segregation is defective in these cells. Here, we show that, in cells lacking TBS-III, both chromatin binding at the centromere and the total amount of some of the centromere proteins are reduced, and transcription through the centromere is up-regulated during M-phase. Moreover, the chromatin binding of Cse4, Mif2, Cbf1, Ndc10, and Scm3 was reduced upon ectopic transcription through the centromere in wild-type cells. These results suggest that transcription through the centromere displaces key centromere proteins and, consequently, destabilizes the interaction between centromeres and microtubules, leading to defective chromosome segregation. The identification of new roles for histone binding residues in TBS-III will shed new light on nucleosome function during chromosome segregation.
    Keywords:  CENP-A; CENP-C; Chromosome segregation; H2A.Z; Histone; Transcription
    DOI:  https://doi.org/10.1016/j.bbrc.2021.11.077
  5. J Cell Sci. 2021 Dec 01. pii: jcs258510. [Epub ahead of print]134(23):
    The microtubule-associated protein She1 coordinates directional spindle positioning by spatially restricting dynein activity.
    Kari H Ecklund, Megan E Bailey, Kelly A Kossen, Carsten K Dietvorst, Charles L Asbury, Steven M Markus.
      Dynein motors move the mitotic spindle to the cell division plane in many cell types, including in budding yeast, in which dynein is assisted by numerous factors including the microtubule-associated protein (MAP) She1. Evidence suggests that She1 plays a role in polarizing dynein-mediated spindle movements toward the daughter cell; however, how She1 performs this function is unknown. We find that She1 assists dynein in maintaining the spindle in close proximity to the bud neck, such that, at anaphase onset, the chromosomes are segregated to mother and daughter cells. She1 does so by attenuating the initiation of dynein-mediated spindle movements within the mother cell, thus ensuring such movements are polarized toward the daughter cell. Our data indicate that this activity relies on She1 binding to the microtubule-bound conformation of the dynein microtubule-binding domain, and to astral microtubules within mother cells. Our findings reveal how an asymmetrically localized MAP directionally tunes dynein activity by attenuating motor activity in a spatially confined manner.
    Keywords:  Dynactin; Dynein; Microtubule-associated proteins; She1; Spindle positioning
    DOI:  https://doi.org/10.1242/jcs.258510
  6. Sci Adv. 2021 Dec 03. 7(49): eabh1004
    PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle.
    Laura J Bailey, Rebecca Teague, Peter Kolesar, Lewis J Bainbridge, Howard D Lindsay, Aidan J Doherty.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abh1004
  7. Genes Genomics. 2021 Nov 29.
    Epigenetic control of centromere: what can we learn from neocentromere?
    Taekyung Kim.
       BACKGROUND: The centromere is the special region on a chromosome, which serves as the site for assembly of kinetochore complex and is essential for maintaining genomic integrity. Neocentromeres are new centromeres that form on the non-centromeric regions of the chromosome when the natural centromere is disrupted or inactivated. Although neocentromeres lack the typical features found in centromeres, cells with neocentromeres divide normally during mitosis and meiosis. Neocentromeres not only arise naturally but their formation can also be induced experimentally. Therefore, neocentromeres are a great tool for studying functions and formation of centromeres.
    OBJECTIVE: To study neocentromeres and use that knowledge to gain insights into the epigenetic regulation of canonical centromeres.
    DISCUSSION: Here, we review the characteristics of naturally occurring centromeres and neocentromeres and those of experimentally induced neocentromeres. We also discuss the mechanism of centromere formation and epigenetic regulation of centromere function, which we learned from studying the neocentromeres. Although neocentromeres lack main features of centromeres, such as presence of repetitive ⍺-satellite DNA and pericentric heterochromatin, they behave quite similar to the canonical centromere, indicating the epigenetic nature of the centromere. Still, further investigation will help to understand the formation and maintenance of the centromere, and the correlation to human diseases.
    CONCLUSION: Neocentromeres helped us to understand the formation of canonical centromeres. Also, since neocentromeres are associated with certain cancer types, knowledge about them could be helpful to treat cancer.
    Keywords:  CENP-A; Centromere; Chromosome; Epigenetics; Neocentromere
    DOI:  https://doi.org/10.1007/s13258-021-01193-x
  8. Plant J. 2021 Nov 30.
    The kinetochore protein NNF1 has a moonlighting role in the vegetative development of Arabidopsis thaliana.
    Allipra Sreejith, Krishnapriya Anirudhan, Siddharth Shivanandan, Abhishek Raghunathan, Ravi Maruthachalam.
      The kinetochore is a supramolecular protein complex assembled on the chromosomes, essential for faithful segregation of the genome during cell divisions. More than 100 proteins are known to constitute the eukaryotic kinetochore architecture, primarily identified using non-plant organisms. A majority of them are fast evolving and are under positive selection. Thus, functional characterization of the plant kinetochore proteins is limited as only a few conserved orthologs sharing sequence similarities with their animal counterparts have been examined. Here, we report the functional characterization of the Arabidopsis thaliana homolog of the yeast NNF1/human PMF1 outer kinetochore protein and show that it has both kinetochore and non-kinetochore functions in plant growth and development. Knockout of NNF1 causes embryo lethality implying its essential role in cell division. AtNNF1 interacts with MIS12 in a Y2H and co-immuno pull down assay, confirming it as one of the constituents of the plant MIS12 complex. GFP-NNF1 localizes to the kinetochore, rescuing the embryo lethal nnf1-1-/- phenotype, but the rescued plants (GFP-NNF1nnf1-/- ) are dwarf displaying hypomorphic phenotypes with no evidence of mitotic or meiotic segregation defects. GFP-NNF1nnf1-/- dwarf plants have reduced levels of endogenous polyamines, which are partially rescued to WT upon exogenous application of polyamines. Mutations in the putative leucine zipper binding motif of NNF1 gave rise to a dominant-negative tall plant phenotype reminiscent of constitutive gibberellin action. These contrasting hypomorphic dwarf and anti morphic tall phenotypes facilitated us to attribute a moonlighting role for Arabidopsis NNF1 affecting polyamine and gibberellin metabolism other than its primary role in kinetochores.
    Keywords:  Arabidopsis; MIS12 complex; NNF1; centromeres; gibberellic acid; kinetochore; mitosis; moonlighting; polyamines
    DOI:  https://doi.org/10.1111/tpj.15614
  9. Cancer Gene Ther. 2021 Dec 02.
    Centromere protein F promotes progression of hepatocellular carcinoma through ERK and cell cycle-associated pathways.
    Hongjin Chen, Fubing Wu, Haojun Xu, Guanqun Wei, Min Ding, Fanggui Xu, Amudha Deivasigamani, Guoren Zhou, Kam M Hui, Hongping Xia.
      Hepatocellular carcinoma (HCC) is one of the deadliest cancer types worldwide. The centromere proteins (CENPs) are critical for the mitosis-related protein complex and are involved in kinetochore assembly and spindle checkpoint signaling during mitosis. However, the clinical significance of CENPs in the recurrence and progression of HCC remains poorly understood. Here, we examined the expression of all CENPs and their association with recurrence and survival of HCC patients using the global gene expression profile dataset established in our laboratory. The effect of silencing CENPF on cell viability, migration, and epithelial-mesenchymal transition (EMT) were detected using CCK-8, transwell, and western blot, respectively. RT-qPCR and western blot were performed to confirm the silencing of CENPF and the relationship between STAT5A and CENPF, while tumorigenesis was tested using the HCC Huh7 xenograft mouse model. Most of the CENPs is overexpressed in HCC, and overexpression of CENPF was significantly associated with the poor survival of HCC patients. CENPF promoted HCC cell lines migration and EMT progression. Knockdown CENPF inhibited cell growth activity against human HCC cells in vitro and xenograft tumors in vivo. Bioinformatics analysis revealed that CENPF genes are enriched in the cell cycle. Silencing CENPF arrested cell cycle at the G2/M phase and inhibited Cyclin B1 and Cyclin E1 expressions. Meanwhile, silencing CENPF prohibited phosphorylation of ERK and the expression of NEK2. Additionally, we found that STAT5A down-regulated CENPF expression and inhibited cancer cell growth viability. In conclusion, our data suggested that CENPF could be potentially developed into a theranostic biomarker to tackle HCC progression.
    DOI:  https://doi.org/10.1038/s41417-021-00404-7
  10. Semin Radiat Oncol. 2022 Jan;pii: S1053-4296(21)00052-7. [Epub ahead of print]32(1): 54-63
    Chromosome Missegregation as a Modulator of Radiation Sensitivity.
    Pippa F Cosper, Sarah E Copeland, John B Tucker, Beth A Weaver.
      Chromosome missegregation over the course of multiple cell divisions, termed chromosomal instability (CIN), is a hallmark of cancer. Multiple causes of CIN have been identified, including defects in the mitotic checkpoint, altered kinetochore-microtubule dynamics, centrosome amplification, and ionizing radiation. Here we review the types, mechanisms, and cellular implications of CIN. We discuss the evidence that CIN can promote tumors, suppress them, or do neither, depending on the rates of chromosome missegregration and the cellular context. Very high rates of chromosome missegregation lead to cell death due to loss of essential chromosomes; thus elevating CIN above a tolerable threshold provides a mechanistic opportunity to promote cancer cell death. Lethal rates of CIN can be achieved by a single insult or through a combination of insults. Because ionizing radiation induces CIN, additional therapies that increase CIN may serve as useful modulators of radiation sensitivity. Ultimately, quantifying the intrinsic CIN in a tumor and modulating this level pharmacologically as well as with radiation may allow for a more rational, personalized radiation therapy prescription, thereby decreasing side effects and increasing local control.
    DOI:  https://doi.org/10.1016/j.semradonc.2021.09.002
  11. Front Pharmacol. 2021 ;12 756228
    Induction of Mitosis Delay and Apoptosis by CDDO-TFEA in Glioblastoma Multiforme.
    Tai-Hsin Tsai, Ann-Shung Lieu, Tzuu-Yuan Huang, Aij-Lie Kwan, Chih-Lung Lin, Yi-Chiang Hsu.
      Background: Glioblastoma multiforme (GBM) is the vicious malignant brain tumor in adults. Despite advances multi-disciplinary treatment, GBM constinues to have a poor overall survival. CDDO-trifluoroethyl-amide (CDDO-TEFA), a trifluoroethylamidederivative of CDDO, is an Nrf2/ARE pathway activator. CDDO-TEFEA is used to inhibit proliferation and induce apoptosis in glioma cells. However, it not clear what effect it may have on tumorigenesis in GBM. Methods: This in vitro study evaluated the effects of CDDO-TFEA on GBM cells. To do this, we treated GBM8401 cell lines with CDDO-TFEA and assessed apoptosis, cell cycle. DNA content and induction of apoptosis were analyzed by flow cytometry and protein expression by Western blot analysis. Results: CDDO-TFEA significantly inhibited the cell viability and induced cell apoptosis on GBM 8401 cell line. The annexin-FITC/PI assay revealed significant changes in the percentage of apoptotic cells. Treatment with CDDO-TFEA led to a significant reduction in the GBM8401 cells' mitochondrial membrane potential. A significant rise in the percentage of caspase-3 activity was detected in the treated cells. In addition, treatment with CDDO-TFEA led to an accumulation of G2/M-phase cells. In addition, these results suggest that regarding increased protein synthesis during mitosis in the MPM-2 staining, indicative of a delay in the G2 checkpoint. An analysis of Cyclin B1, CDK1, Cyclin B1/CDK1 complex and CHK1 and CHK2 expression suggested that cell cycle progression seems also to be regulated by CDDO-TFEA. Therefore, CDDO-TFEA may not only induce cell cycle G2/M arrest, it may also exert apoptosis in established GBM cells. Conclusion: CDDO-TFEA can inhibit proliferation, cell cycle progression and induce apoptosis in GBM cells in vitro, possibly though its inhibition of Cyclin B1, CDK1 expression, and Cyclin B1/CDK1 association and the promotion of CHK1 and CHK2 expression.
    Keywords:  CDDO-TFEA; GBM; RTA 404; apoptosis; cell cycle
    DOI:  https://doi.org/10.3389/fphar.2021.756228
  12. Nucleic Acids Res. 2021 Nov 29. pii: gkab1155. [Epub ahead of print]
    Chromosome segregation in Archaea: SegA- and SegB-DNA complex structures provide insights into segrosome assembly.
    Cheng-Yi Yen, Min-Guan Lin, Bo-Wei Chen, Irene W Ng, Nicholas Read, Azhar F Kabli, Che-Ting Wu, Yo-You Shen, Chen-Hao Chen, Daniela Barillà, Yuh-Ju Sun, Chwan-Deng Hsiao.
      Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.
    DOI:  https://doi.org/10.1093/nar/gkab1155
  13. Bioessays. 2021 Nov 28. e2100218
    Minichromosome maintenance proteins in eukaryotic chromosome segregation.
    Gunjan Mehta, Kaustuv Sanyal, Suman Abhishek, Eerappa Rajakumara, Santanu K Ghosh.
      Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms.
    Keywords:  MCM; Minichromosome maintenance; budding yeast; centromere; chromosome segregation; kinetochore
    DOI:  https://doi.org/10.1002/bies.202100218
  14. Biol Pharm Bull. 2021 ;44(12): 1878-1885
    Telmisartan-Induced Cytotoxicity via G2/M Phase Arrest in Renal Cell Carcinoma Cell Lines.
    Yoshie Tsujiya, Ai Hasegawa, Motohiro Yamamori, Noboru Okamura.
      Renal cell carcinoma (RCC) is the most common type of kidney cancer. Given that stage IV RCC is intractable, there is a need for a novel treatment strategy. We investigated the antitumor effects of telmisartan (TEL) and their underlying mechanisms in RCC, including their impact on apoptosis, Akt/mammalian target of rapamycin (mTOR) pathways, and the cell cycle using two human RCC cell lines: 786-O and Caki-2. Cell viability was detected via fluorescence-based assays. Cells were stained with Hoechst 33342 to observe chromatin condensation, and Western blotting was performed to analyze protein expression. The cell cycle was assessed using flow cytometry. Invasion and migration assays were performed using 24-well chambers. TEL induced cell death in a dose-dependent manner and increased the percentage of cells with high chromatin condensation and Bax/Bcl-2 ratio in both cell lines. TEL-induced cell death was attenuated by neither peroxisome proliferator-activated receptor-γ nor -δ inhibitors. Although TEL elevated c-Jun N-terminal kinase levels and p38 phosphorylation rates in Caki-2 cells, as well as extracellular signal-regulated kinase phosphorylation rates in 786-O cells, their inhibitors did not suppress TEL-induced cell death. TEL decreased Akt phosphorylation in 786-O cells and mTOR phosphorylation in both cell lines, increased the population of cells in the G2/M phase, and altered G2/M-related proteins in both cell lines. TEL moderately suppressed cell invasion and migration in 786-O and Caki-2 cells, respectively, and increased cell invasion in Caki-2 cells, suggesting a potential therapeutic role of TEL in RCC.
    Keywords:  angiotensin II receptor blocker; apoptosis; cell cycle arrest; renal cell carcinoma; telmisartan
    DOI:  https://doi.org/10.1248/bpb.b21-00654
  15. Curr Top Membr. 2021 ;pii: S1063-5823(21)00010-7. [Epub ahead of print]88 75-118
    Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET.
    Sarah Lecinski, Jack W Shepherd, Lewis Frame, Imogen Hayton, Chris MacDonald, Mark C Leake.
      Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding while simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronized cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.
    Keywords:  Cellular stress; Confocal; FRET; Molecular crowding sensor; Slimfield; Vacuolar inheritance; Yeast
    DOI:  https://doi.org/10.1016/bs.ctm.2021.09.001
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