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.

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.

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.

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.

Relevance and Regulation of Cell Density.

Cell density shows very little variation within a given cell type. For example, in humans variability in cell density among cells of a given cell type is 100 times smaller than variation in cell mass. This tight control indicates that maintenance of a cell type-specific cell density is important for cell function. Indeed, pathological conditions such as cellular senescence are accompanied by changes in cell density. Despite the apparent importance of cell-type-specific density, we know little about how cell density affects cell function, how it is controlled, and how it sometimes changes as part of a developmental process or in response to changes in the environment. The recent development of new technologies to accurately measure the cell density of single cells in suspension and in tissues is likely to provide answers to these important questions.

Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence.

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.

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.

Cdc14 Localization as a Marker for Mitotic Exit: In Vivo Quantitative Analysis of Cdc14 Release.

To complete cell division and to exit from mitosis into the next G1 phase, eukaryotic cells need to inactivate the cyclin-dependent kinase (Cdk) and reverse Cdk-phosphorylation events. In budding yeast mitotic exit depends on the phosphatase Cdc14. During the majority of the cell cycle Cdc14 is sequestered and kept inactive in the nucleolus. Activation of Cdc14 at anaphase onset coincides with its release from the nucleolus into the nucleus and subsequently into the cytoplasm. Here we describe a microscopy method, originally developed in the laboratory of Frederick Cross (Lu and Cross, Cell 141:268-279, 2010), that allows quantifying Cdc14 release in live cells using the open source software FIJI. We adapted this method and show that it is able to distinguish between Cdc14 activation defects caused by mutations in the "cdcFourteen Early Anaphase Release"-(FEAR) and the mitotic exit network (MEN) using slk19∆ and cdc15-1 mutant strains.

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress.

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.

A midzone-based ruler adjusts chromosome compaction to anaphase 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 find that yeast cells could cope with a 45% increase in the length of their longest chromosome arm by increasing its condensation. The spindle midzone, aurora/Ipl1 activity, and Ser10 of histone H3 mediated this response. Thus, the anaphase spindle may function as a ruler to adapt the condensation of chromatids, promoting their segregation regardless of chromosome or spindle length.

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 condensation.