ABSTRACT
Multiciliated cells produce over a hundred motile cilia anchored to the membrane by modified centrioles. Recent work has characterized an alternative cell cycle used by this post-mitotic cell type to generate additional centrioles without undergoing cell division.
Subject(s)
Cell Cycle , Cilia , Cilia/physiology , Cell Cycle/physiology , Animals , Centrioles/physiology , Centrioles/metabolismABSTRACT
Budding yeast, Saccharomyces cerevisiae, is widely used as a model organism to study the genetics underlying eukaryotic cellular processes and growth critical to cancer development, such as cell division and cell cycle progression. The budding yeast cell cycle is also one of the best-studied dynamical systems owing to its thoroughly resolved genetics. However, the dynamics underlying the crucial cell cycle decision point called the START transition, at which the cell commits to a new round of DNA replication and cell division, are under-studied. The START machinery involves a central cyclin-dependent kinase; cyclins responsible for starting the transition, bud formation, and initiating DNA synthesis; and their transcriptional regulators. However, evidence has shown that the mechanism is more complicated than a simple irreversible transition switch. Activating a key transcription regulator SBF requires the phosphorylation of its inhibitor, Whi5, or an SBF/MBF monomeric component, Swi6, but not necessarily both. Also, the timing and mechanism of the inhibitor Whi5's nuclear export, while important, are not critical for the timing and execution of START. Therefore, there is a need for a consolidated model for the budding yeast START transition, reconciling regulatory and spatial dynamics. We built a detailed mathematical model (START-BYCC) for the START transition in the budding yeast cell cycle based on established molecular interactions and experimental phenotypes. START-BYCC recapitulates the underlying dynamics and correctly emulates key phenotypic traits of ~150 known START mutants, including regulation of size control, localization of inhibitor/transcription factor complexes, and the nutritional effects on size control. Such a detailed mechanistic understanding of the underlying dynamics gets us closer towards deconvoluting the aberrant cellular development in cancer.
Subject(s)
Cell Cycle , Models, Biological , Saccharomyces cerevisiae , Cell Cycle/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , DNA Replication , Computational Biology , Saccharomycetales/genetics , Saccharomycetales/metabolism , Saccharomycetales/physiology , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics , Phosphorylation , Repressor ProteinsABSTRACT
This article reviews the current knowledge and recent advancements in computational modeling of the cell cycle. It offers a comparative analysis of various modeling paradigms, highlighting their unique strengths, limitations, and applications. Specifically, the article compares deterministic and stochastic models, single-cell versus population models, and mechanistic versus abstract models. This detailed analysis helps determine the most suitable modeling framework for various research needs. Additionally, the discussion extends to the utilization of these computational models to illuminate cell cycle dynamics, with a particular focus on cell cycle viability, crosstalk with signaling pathways, tumor microenvironment, DNA replication, and repair mechanisms, underscoring their critical roles in tumor progression and the optimization of cancer therapies. By applying these models to crucial aspects of cancer therapy planning for better outcomes, including drug efficacy quantification, drug discovery, drug resistance analysis, and dose optimization, the review highlights the significant potential of computational insights in enhancing the precision and effectiveness of cancer treatments. This emphasis on the intricate relationship between computational modeling and therapeutic strategy development underscores the pivotal role of advanced modeling techniques in navigating the complexities of cell cycle dynamics and their implications for cancer therapy.
Subject(s)
Cell Cycle , Computer Simulation , Models, Biological , Neoplasms , Humans , Neoplasms/therapy , Neoplasms/pathology , Cell Cycle/physiology , Signal Transduction , Tumor Microenvironment , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Computational Biology/methodsABSTRACT
Activation of cell-cycle machinery in Alzheimer's disease (AD) brain was reported by Mark Smith and colleagues and by other researchers. Among other biochemical processes underlying this activation, the notion that AD brain, under the onslaught of oxidative and nitrosative damage leading to neuronal loss, neurons would attempt to replenish their numbers by entering the cell cycle. However, being post-mitotic, neurons entering the cell cycle would become trapped therein, ultimately leading to death of these neurons. Yang and co-workers and the Butterfield laboratory first reported that similar activation of the cell cycle was present in the brains of individuals with amnestic mild cognitive impairment (MCI), arguably the earliest clinical stage of AD, but who demonstrate normal activities of daily living and no dementia. Activation of the cell cycle in MCI brain is consonant with the concept that this process is an early aspect in the progression of AD. This brief review article discusses these findings and recognizes the contribution of Dr. Mark Smith to the investigation of cell-cycle activation in AD brain and other aspects of AD neuropathology.
Subject(s)
Alzheimer Disease , Brain , Cell Cycle , Cognitive Dysfunction , Disease Progression , Neurons , Humans , Alzheimer Disease/pathology , Cognitive Dysfunction/pathology , Neurons/pathology , Brain/pathology , Cell Cycle/physiology , Amnesia/pathology , AnimalsABSTRACT
Many animals share a lifelong capacity to adapt their growth rates and body sizes to changing environmental food supplies. However, the cellular and molecular basis underlying this plasticity remains only poorly understood. We therefore studied how the sea anemones Nematostella vectensis and Aiptasia (Exaiptasia pallida) respond to feeding and starvation. Combining quantifications of body size and cell numbers with mathematical modelling, we observed that growth and shrinkage rates in Nematostella are exponential, stereotypic and accompanied by dramatic changes in cell numbers. Notably, shrinkage rates, but not growth rates, are independent of body size. In the facultatively symbiotic Aiptasia, we show that growth and cell proliferation rates are dependent on the symbiotic state. On a cellular level, we found that >7% of all cells in Nematostella juveniles reversibly shift between S/G2/M and G1/G0 cell cycle phases when fed or starved, respectively. Furthermore, we demonstrate that polyp growth and cell proliferation are dependent on TOR signalling during feeding. Altogether, we provide a benchmark and resource for further investigating the nutritional regulation of body plasticity on multiple scales using the genetic toolkit available for Nematostella.
Subject(s)
Body Size , Cell Proliferation , Sea Anemones , Animals , Sea Anemones/cytology , Sea Anemones/physiology , Cell Cycle/physiology , Feeding Behavior/physiology , Signal Transduction , Symbiosis , TOR Serine-Threonine Kinases/metabolismABSTRACT
Mitochondria play critical roles in neural stem/progenitor cell proliferation and fate decisions. The subcellular localization of mitochondria in neural stem/progenitor cells during mitosis potentially influences the distribution of mitochondria to the daughter cells and thus their fates. Therefore, understanding the spatial dynamics of mitochondria provides important knowledge about brain development. In this study, we analyzed the subcellular localization of mitochondria in the fetal human neocortex with a particular focus on the basal radial glial cells (bRGCs), a neural stem/progenitor cell subtype attributed to the evolutionary expansion of the human neocortex. During interphase, bRGCs exhibit a polarized localization of mitochondria that is localized at the base of the process or the proximal part of the process. Thereafter, mitochondria in bRGCs at metaphase show unpolarized distribution in which the mitochondria are randomly localized in the cytoplasm. During anaphase and telophase, mitochondria are still localized evenly, but mainly in the periphery of the cytoplasm. Mitochondria start to accumulate at the cleavage furrow during cytokinesis. These results suggest that the mitochondrial localization in bRGCs is tightly regulated during the cell cycle, which may ensure the proper distribution of mitochondria to the daughter cells and, thus in turn, influence their fates.
Subject(s)
Cell Cycle , Ependymoglial Cells , Mitochondria , Neocortex , Humans , Neocortex/cytology , Neocortex/metabolism , Mitochondria/metabolism , Cell Cycle/physiology , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/cytologyABSTRACT
Understanding mechanisms underlying various physiological and pathological processes often requires accurate and fully automated analysis of dense cell populations that collectively migrate. In such multicellular systems, there is a rising interest in the relations between biophysical and cell cycle progression aspects. A seminal tool that led to a leap in real-time study of cell cycle is the fluorescent ubiquitination-based cell cycle indicator (FUCCI). Here, we introduce ConfluentFUCCI, an open-source graphical user interface-based framework that is designed, unlike previous tools, for fully automated analysis of cell cycle progression, cellular dynamics, and cellular morphology, in highly dense migrating cell collectives. We integrated into ConfluentFUCCI's pipeline state-of-the-art tools such as Cellpose, TrackMate, and Napari, some of which incorporate deep learning, and we wrap the entire tool into an isolated computational environment termed container. This provides an easy installation and workflow that is independent of any specific operation system. ConfluentFUCCI offers accurate nuclear segmentation and tracking using FUCCI tags, enabling comprehensive investigation of cell cycle progression at both the tissue and single-cell levels. We compare ConfluentFUCCI to the most recent relevant tool, showcasing its accuracy and efficiency in handling large datasets. Furthermore, we demonstrate the ability of ConfluentFUCCI to monitor cell cycle transitions, dynamics, and morphology within densely packed epithelial cell populations, enabling insights into mechanotransductive regulation of cell cycle progression. The presented tool provides a robust approach for investigating cell cycle-related phenomena in complex biological systems, offering potential applications in cancer research and other fields.
Subject(s)
Cell Cycle , Cell Movement , Cell Cycle/physiology , Cell Movement/physiology , Humans , Software , Ubiquitination , Image Processing, Computer-Assisted/methodsABSTRACT
The exchangeable Zn2+ pool in cells is not static but responds to perturbations as well as fluctuates naturally through the cell cycle. Here, we present a protocol to carry out long-term live-cell imaging of cells expressing a cytosolic Zn2+ sensor. We then describe how to track cells using the published pipeline EllipTrack and how to analyze the single-cell traces to determine changes in labile Zn2+ in response to perturbation. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al.1.
Subject(s)
Biosensing Techniques , Cell Cycle , Fluorescence Resonance Energy Transfer , Zinc , Zinc/metabolism , Zinc/analysis , Biosensing Techniques/methods , Cell Cycle/physiology , Fluorescence Resonance Energy Transfer/methods , Humans , HeLa CellsABSTRACT
Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.
Subject(s)
Cell Differentiation , Cell Proliferation , Neural Stem Cells , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Animals , Mice , Cell Culture Techniques/methods , Cells, Cultured , Cell Cycle/physiology , AutophagyABSTRACT
CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.
Subject(s)
3T3-L1 Cells , Adipocytes , Adipogenesis , Cell Cycle , Hyaluronan Receptors , PPAR gamma , Adipogenesis/genetics , Adipogenesis/physiology , Hyaluronan Receptors/metabolism , Hyaluronan Receptors/genetics , Animals , PPAR gamma/metabolism , PPAR gamma/genetics , Mice , Cell Cycle/genetics , Cell Cycle/physiology , Humans , Adipocytes/metabolism , Gene Deletion , Cell Differentiation/genetics , Male , Adipose Tissue/metabolism , Adipose Tissue/cytology , Signal Transduction/physiologyABSTRACT
Long non-coding RNAs (lncRNAs) are a diverse class of RNA molecules that do not code for proteins but play critical roles in gene regulation. One such role involves the modulation of cell cycle progression and proliferation through interactions with cyclin-dependent kinases (CDKs), key regulators of cell division. Dysregulation of CDK activity is a hallmark of cancer, contributing to uncontrolled cell growth and tumor formation. These lncRNA-CDK interactions are part of a complex network of molecular mechanisms underlying cancer pathogenesis, involving various signaling pathways and regulatory circuits. Understanding the interplay between lncRNAs, CDKs, and cancer biology holds promise for developing novel therapeutic strategies targeting these molecular targets for more effective cancer treatment. Furthermore, targeting CDKs, key cell cycle progression and proliferation regulators, offers another avenue for disrupting cancer pathways and overcoming drug resistance. This can open new possibilities for individualized treatment plans and focused therapeutic interventions.
Subject(s)
Cyclin-Dependent Kinases , Disease Progression , Neoplasms , RNA, Long Noncoding , Humans , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/enzymology , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Gene Expression Regulation, Neoplastic , Animals , Signal Transduction/genetics , Cell Proliferation/genetics , Cell Cycle/genetics , Cell Cycle/physiologyABSTRACT
Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.
Subject(s)
Brain , Mice, Knockout , Neural Stem Cells , Nonsense Mediated mRNA Decay , Tumor Suppressor Protein p53 , Animals , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Mice , Brain/metabolism , Neural Stem Cells/metabolism , Nonsense Mediated mRNA Decay/genetics , Epistasis, Genetic , Microcephaly/genetics , Cell Cycle/physiology , Cell Cycle/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/geneticsABSTRACT
Aerobic glycolysis, or the Warburg effect, is used by cancer cells for proliferation while producing lactate. Although lactate production has wide implications for cancer progression, it is not known how this effect increases cell proliferation and relates to oxidative phosphorylation. Here, we elucidate that a negative feedback loop (NFL) is responsible for the Warburg effect. Further, we show that aerobic glycolysis works as an amplifier of oxidative phosphorylation. On the other hand, quiescence is an important property of cancer stem cells. Based on the NFL, we show that both aerobic glycolysis and oxidative phosphorylation, playing a synergistic role, are required to achieve cell quiescence. Further, our results suggest that the cells in their hypoxic niche are highly proliferative yet close to attaining quiescence by increasing their NADH/NAD+ ratio through the severity of hypoxia. The findings of this study can help in a better understanding of the link among metabolism, cell cycle, carcinogenesis, and stemness.
Subject(s)
Cell Proliferation , Feedback, Physiological , Glycolysis , Neoplastic Stem Cells , Oxidative Phosphorylation , Warburg Effect, Oncologic , Humans , Glycolysis/physiology , Feedback, Physiological/physiology , Neoplastic Stem Cells/metabolism , Cell Proliferation/physiology , Neoplasms/metabolism , NAD/metabolism , Lactic Acid/metabolism , Models, Biological , Cell Line, Tumor , Cell Cycle/physiologyABSTRACT
Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.
Subject(s)
Cell Cycle Proteins , Proteomics , Cell Cycle/physiology , Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Phosphorylation , Protein Stability , NIMA-Interacting Peptidylprolyl Isomerase/genetics , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , MitosisABSTRACT
In favorable conditions, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of their progenitor. The integrity of this process is maintained by "checkpoints" that hold a cell at particular transition points of the cycle until all requisite events are completed. The crucial functions of these checkpoints seem to depend on irreversible bistability of the underlying checkpoint control systems. Bistability of cell cycle transitions has been confirmed experimentally in frog egg extracts, budding yeast cells and mammalian cells. For fission yeast cells, a recent paper by Patterson et al. (2021) provides experimental evidence for an abrupt transition from G2 phase into mitosis, and we show that these data are consistent with a stochastic model of a bistable switch governing the G2/M checkpoint. Interestingly, our model suggests that their experimental data could also be explained by a reversible/sigmoidal switch, and stochastic simulations confirm this supposition. We propose a simple modification of their experimental protocol that could provide convincing evidence for (or against) bistability of the G2/M transition in fission yeast.
Subject(s)
Mitosis , Schizosaccharomyces , Schizosaccharomyces/metabolism , Mitosis/physiology , Cell Cycle/physiology , G2 Phase Cell Cycle Checkpoints , G2 Phase/physiology , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.
Subject(s)
Activating Transcription Factor 6 , Cell Cycle , Endoplasmic Reticulum , Eukaryotic Initiation Factor-2 , Protein Serine-Threonine Kinases , Signal Transduction , Unfolded Protein Response , eIF-2 Kinase , eIF-2 Kinase/metabolism , Humans , Cell Cycle/physiology , Endoplasmic Reticulum/metabolism , Phosphorylation , Eukaryotic Initiation Factor-2/metabolism , Activating Transcription Factor 6/metabolism , Protein Serine-Threonine Kinases/metabolism , Endoribonucleases/metabolism , Animals , HeLa Cells , Endoplasmic Reticulum Stress/physiologyABSTRACT
BACKGROUND: Cutaneous squamous cell carcinoma (CSCC) is one of the most common types of skin cancer worldwide. Therefore, the identification of biomarkers associated with CSCC progression could aid in the early detection of high-risk squamous cell carcinoma and the development of novel therapeutic strategies. OBJECTIVE: This study aimed to investigate the expression patterns of silent mating type Information Regulation 2 homolog 6 (SIRT6) in CSCC and its clinical significance. METHODS: The protein expression level of SIRT6 in tissues was detected by immunohistochemistry, and the correlation between SIRT6 expression and clinicopathological parameters in CSCC patients was analyzed. The relative expression of SIRT6 in CSCC cell lineage and tissue specimens was determined by western blotting and PCR. The effect of SIRT6 silencing on cell proliferation was evaluated using cell counting kit 8. Wound healing, transwell method, and flow cytometry were used to investigate the migration, invasion, and cell cycle distribution/apoptosis of CSCC cells after SIRT6 silencing, respectively. Western blot was used to detect the expression of EMT (Epithelial-Mesenchymal Transition), cycle, apoptosis, and other related proteins. RESULTS: The high expression of SIRT6 was correlated with the location of cancer tissue and Broder staging in CSCC patients. Knockdown of SIRT6 inhibited the proliferation, migration, invasion and EMT of CSCC cells, and promoted their apoptosis, with cells blocked in G1 phase. STUDY LIMITATIONS: No animal experiments were conducted to further verify the results. CONCLUSION: Decreased expression of SIRT6 can inhibit the occurrence and development of CSCC.
Subject(s)
Apoptosis , Carcinoma, Squamous Cell , Cell Proliferation , Sirtuins , Skin Neoplasms , Aged , Female , Humans , Male , Middle Aged , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/analysis , Blotting, Western , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/genetics , Cell Cycle/physiology , Cell Line, Tumor , Cell Movement , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Sirtuins/genetics , Sirtuins/metabolism , Skin Neoplasms/pathology , Skin Neoplasms/geneticsABSTRACT
Investigating cell-cycle progression has been challenging due to the complex interconnectivity of regulatory processes and inherent cell-to-cell heterogeneity, which often require synchronization procedures. However, recent advancements in cell-cycle sensors and single-cell imaging techniques have turned this heterogeneity into an advantage for investigating the molecular mechanisms underlying diverse responses. This has led to significant progress in our understanding of cell-cycle regulation. In this paper, we present a comprehensive live single-cell imaging workflow that leverages cutting-edge live-cell sensors. These advanced single-cell imaging procedures provide promising opportunities for elucidating the molecular mechanisms underpinnings of heterogeneous responses in cell-cycle progression.
Subject(s)
Cell Division , Cell Cycle/physiology , Cell Cycle CheckpointsABSTRACT
The development of technologies that allow measurement of the cell cycle at the single-cell level has revealed novel insights into the mechanisms that regulate cell cycle commitment and progression through DNA replication and cell division. These studies have also provided evidence of heterogeneity in cell cycle regulation among individual cells, even within a genetically identical population. Cell cycle mapping combines highly multiplexed imaging with manifold learning to visualize the diversity of "paths" that cells can take through the proliferative cell cycle or into various states of cell cycle arrest. In this chapter, we describe a general protocol of the experimental and computational components of cell cycle mapping. We also provide a comprehensive guide for the design and analysis of experiments, discussing key considerations in detail (e.g., antibody library preparation, analysis strategies, etc.) that may vary depending on the research question being addressed.