Assays for mitotic chromosome condensation in live yeast and mammalian cells

The dynamic reorganization of chromatin into rigid and compact mitotic chromosomes is of fundamental importance for faithful chromosome segregation. Owing to the difficulty of investigating this process under physiological conditions, the exact morphological transitions and the molecular machinery driving chromosome condensation remain poorly defined. Here, we review how imaging-based methods can be used to quantitate chromosome condensation in vivo, focusing on yeast and animal tissue culture cells as widely used model systems. We discuss approaches how to address structural dynamics of condensing chromosomes and chromosome segments, as well as to probe for mechanical properties of mitotic chromosomes. Application of such methods to systematic perturbation studies will provide a means to reveal the molecular networks underlying the regulation of mitotic chromosome condensatio

The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved

A midzone-based ruler adjusts chromosome compaction to analphase spindle length

Partitioning of chromatids during mitosis requires that chromosome compaction and spindle length scale appropriately with each other. However, it is not clear whether chromosome condensation and spindle elongation are linked. Here we have used chromosome fusions to examine the impact of increased chromosome length during yeast mitosis. We find that yeast cells could cope with a >50% increase in the length of their longest chromosome arm by decreasing the physical length of the mitotic chromosome arm through 1) reducing the number of copies of the repetitive rDNA array and 2) by increasing the level of mitotic condensation. Consistently, cells carrying the fused chromosomes became more sensitive to loss of condensin- and its regulator polo kinase/Cdc5. Length-dependent stimulation of condensation took place during anaphase and depended on aurora/Ipl1 activity, its localization to the spindle midzone, and phosphorylation of histone H3 on Ser10, a known Ipl1 substrate. The anaphase spindle therefore may function as a ruler to adapt the condensation of chromosomes to spindle length. Consistent with this, chromosome condensation levels correlate with the length of anaphase spindles

Assays for mitotic chromosome condensation in live yeast and mammalian cells

ISSN:0967-3849ISSN:1573-684

Too big not to fail: emerging evidence for size-induced senescence

Cellular senescence refers to a permanent and stable state of cell cycle exit. This process plays an important role in many cellular functions, including tumor suppression. It was first noted that senescence is associated with increased cell size in the early 1960s; however, how this contributes to permanent cell cycle exit was poorly understood until recently. In this review, we discuss new findings that identify increased cell size as not only a consequence but also a cause of permanent cell cycle exit. We highlight recent insights into how increased cell size alters normal cellular physiology and creates homeostatic imbalances that contribute to senescence induction. Finally, we focus on the potential clinical implications of these findings in the context of cell cycle arrest-causing cancer therapeutics and speculate on how tumor cell size changes may impact outcomes in patients treated with these drugs.ISSN:1742-464XISSN:1742-465

Hybrid machine-learning framework for volumetric segmentation and quantification of vacuoles in individual yeast cells using holotomography

The precise, quantitative evaluation of intracellular organelles in three-dimensional (3D) imaging data poses a significant challenge due to the inherent constraints of traditional microscopy techniques, the requirements of the use of exogenous labeling agents, and existing computational methods. To counter these challenges, we present a hybrid machine-learning framework exploiting correlative imaging of 3D quantitative phase imaging with 3D fluorescence imaging of labeled cells. The algorithm, which synergistically integrates a random-forest classifier with a deep neural network, is trained using the correlative imaging data set, and the trained network is then applied to 3D quantitative phase imaging of cell data. We applied this method to live budding yeast cells. The results revealed precise segmentation of vacuoles inside individual yeast cells, and also provided quantitative evaluations of biophysical parameters, including volumes, concentration, and dry masses of automatically segmented vacuoles.ISSN:2156-708

Deregulation of the G1/S-phase transition is the proximal cause of mortality in old yeast mother cells

Budding yeast cells produce a finite number of daughter cells before they die. Why old yeast cells stop dividing and die is unclear. We found that age-induced accumulation of the G1/S-phase inhibitor Whi5 and defects in G1/S cyclin transcription cause cell cycle delays and genomic instability that result in cell death. We further identified extrachromosomal rDNA (ribosomal DNA) circles (ERCs) to cause the G1/S cyclin expression defect in old cells. Spontaneous segregation of Whi5 and ERCs into daughter cells rejuvenates old mothers, but daughters that inherit these aging factors die rapidly. Our results identify deregulation of the G1/S-phase transition as the proximal cause of age-induced proliferation decline and cell death in budding yeast

Genome homeostasis defects drive enlarged cells into senescence

Cellular senescence refers to an irreversible state of cell-cycle arrest and plays important roles in aging and cancer biology. Because senescence is associated with increased cell size, we used reversible cell-cycle arrests combined with growth rate modulation to study how excessive growth affects proliferation. We find that enlarged cells upregulate p21, which limits cell-cycle progression. Cells that re-enter the cell cycle encounter replication stress that is well tolerated in physiologically sized cells but causes severe DNA damage in enlarged cells, ultimately resulting in mitotic failure and permanent cell-cycle withdrawal. We demonstrate that enlarged cells fail to recruit 53BP1 and other non-homologous end joining (NHEJ) machinery to DNA damage sites and fail to robustly initiate DNA damage-dependent p53 signaling, rendering them highly sensitive to genotoxic stress. We propose that an impaired DNA damage response primes enlarged cells for persistent replication-acquired damage, ultimately leading to cell division failure and permanent cell-cycle exit.ISSN:1097-2765ISSN:1097-416

DataSheet1_The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.pdf

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved. </p

Table2_The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.xlsx

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved. </p