Dynamic changes in mitochondrial localization in human neocortical basal radial glial cells during cell cycle.

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.

A Reversible Neural Stem Cell Quiescence and Activation Culture System for Metabolic Study.

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.

A negative feedback loop underlies the Warburg effect.

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.

lncRNAs and cyclin-dependent kinases: Unveiling their critical roles in cancer progression.

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.

Deletion of CD44 promotes adipogenesis by regulating PPARγ and cell cycle-related pathways.

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.

The bistable mitotic switch in fission yeast.

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.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

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.

Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.

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.

Expression analysis and biological regulation of silencing regulatory protein 6 (SIRT6) in cutaneous squamous cell carcinoma.

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.

Pas de deux: the coordinated coupling of erythroid differentiation with the cell cycle.

PURPOSE OF REVIEW: Recent work reveals that cell cycle duration and structure are remodeled in lock-step with distinct stages of erythroid differentiation. These cell cycle features have regulatory roles in differentiation, beyond the generic function of increasing cell number. RECENT FINDINGS: Developmental progression through the early erythroid progenitor stage (known as colony-forming-erythroid, or 'CFU-e') is characterized by gradual shortening of G1 phase of the cycle. This process culminates in a key transcriptional switch to erythroid terminal differentiation (ETD) that is synchronized with, and dependent on, S phase progression. Further, the CFU-e/ETD switch takes place during an unusually short S phase, part of an exceptionally short cell cycle that is characterized by globally fast replication fork speeds. Cell cycle and S phase speed can alter developmental events during erythroid differentiation, through pathways that are targeted by glucocorticoid and erythropoietin signaling during the erythroid stress response. SUMMARY: There is close inter-dependence between cell cycle structure and duration, S phase and replication fork speeds, and erythroid differentiation stage. Further, modulation of cell cycle structure and speed cycle impacts developmental progression and cell fate decisions during erythroid differentiation. These pathways may offer novel mechanistic insights and potential therapeutic targets.

Investigating Heterogeneous Cell-Cycle Progression Using Single-Cell Imaging Approaches.

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.

Cell Cycle Mapping Using Multiplexed Immunofluorescence.

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.

Triaging between post-translational modification of cell cycle regulators and their therapeutics in neurodegenerative diseases.

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, present challenges in healthcare because of their complicated etiologies and absence of healing remedies. Lately, the emerging role of post-translational modifications (PTMs), in the context of cell cycle regulators, has garnered big interest as a potential avenue for therapeutic intervention. The review explores the problematic panorama of PTMs on cell cycle regulators and their implications in neurodegenerative diseases. We delve into the dynamic phosphorylation, acetylation, ubiquitination, SUMOylation, Glycation, and Neddylation that modulate the key cell cycle regulators, consisting of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors. The dysregulation of these PTMs is related to aberrant cell cycle in neurons, which is one of the factors involved in neurodegenerative pathologies. Moreover, the effect of exogenous activation of CDKs and CDK inhibitors through PTMs on the signaling cascade was studied in postmitotic conditions of NDDs. Furthermore, the therapeutic implications of CDK inhibitors and associated alteration in PTMs were discussed. Lastly, we explored the putative mechanism of PTMs to restore normal neuronal function that might reverse NDDs.

A simple Turing reaction-diffusion model explains how PLK4 breaks symmetry during centriole duplication and assembly.

Centrioles duplicate when a mother centriole gives birth to a daughter that grows from its side. Polo-like-kinase 4 (PLK4), the master regulator of centriole duplication, is recruited symmetrically around the mother centriole, but it then concentrates at a single focus that defines the daughter centriole assembly site. How PLK4 breaks symmetry is unclear. Here, we propose that phosphorylated and unphosphorylated species of PLK4 form the 2 components of a classical Turing reaction-diffusion system. These 2 components bind to/unbind from the surface of the mother centriole at different rates, allowing a slow-diffusing activator species of PLK4 to accumulate at a single site on the mother, while a fast-diffusing inhibitor species of PLK4 suppresses activator accumulation around the rest of the centriole. This "short-range activation/long-range inhibition," inherent to Turing systems, can drive PLK4 symmetry breaking on a either a continuous or compartmentalised Plk4-binding surface, with PLK4 overexpression producing multiple PLK4 foci and PLK4 kinase inhibition leading to a lack of symmetry-breaking and PLK4 accumulation-as observed experimentally.

Discovery of small molecule degraders for modulating cell cycle.

The cell cycle is a complex process that involves DNA replication, protein expression, and cell division. Dysregulation of the cell cycle is associated with various diseases. Cyclin-dependent kinases (CDKs) and their corresponding cyclins are major proteins that regulate the cell cycle. In contrast to inhibition, a new approach called proteolysis-targeting chimeras (PROTACs) and molecular glues can eliminate both enzymatic and scaffold functions of CDKs and cyclins, achieving targeted degradation. The field of PROTACs and molecular glues has developed rapidly in recent years. In this article, we aim to summarize the latest developments of CDKs and cyclin protein degraders. The selectivity, application, validation and the current state of each CDK degrader will be overviewed. Additionally, possible methods are discussed for the development of degraders for CDK members that still lack them. Overall, this article provides a comprehensive summary of the latest advancements in CDK and cyclin protein degraders, which will be helpful for researchers working on this topic.

A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway.

PTP1B plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify molecular targets of PTP1B that mediate its role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify Cdk3 as a novel PTP1B substrate. Substrate trapping experiments and docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at tyrosine residue 15 in vitro and interacts with it in human glioblastoma cells. Next, we found that pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with diminished activity of Cdk3, hypophosphorylation of Rb, and the downregulation of E2F target genes Cdk1, Cyclin A, and Cyclin E1. Finally, we observed that the expression of a constitutively active Cdk3 mutant bypasses the requirement of PTP1B for cell cycle progression and expression of E2F target genes. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells.

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

PLK4 self-phosphorylation drives the selection of a single site for procentriole assembly.

Polo-like kinase 4 (PLK4) is a key regulator of centriole biogenesis, but how PLK4 selects a single site for procentriole assembly remains unclear. Using ultrastructure expansion microscopy, we show that PLK4 localizes to discrete sites along the wall of parent centrioles. While there is variation in the number of sites PLK4 occupies on the parent centriole, most PLK4 localize at a dominant site that directs procentriole assembly. Inhibition of PLK4 activity leads to stable binding of PLK4 to the centriole and increases occupancy to a maximum of nine sites. We show that self-phosphorylation of an unstructured linker promotes the release of active PLK4 from the centriole to drive the selection of a single site for procentriole assembly. Preventing linker phosphorylation blocks PLK4 turnover, leading to supernumerary sites of PLK4 localization and centriole amplification. Therefore, self-phosphorylation is a major driver of the spatial patterning of PLK4 at the centriole and plays a critical role in selecting a single centriole duplication site.

Autophagy modulates the stability of Wee1 and cell cycle G2/M transition.

The mammalian cell cycle is divided into four sequential phases, namely G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). Wee1, whose turnover is tightly and finely regulated, is a well-known kinase serving as a gatekeeper for the G2/M transition. However, the mechanism underlying the turnover of Wee1 is not fully understood. Autophagy, a highly conserved cellular process, maintains cellular homeostasis by eliminating intracellular aggregations, damaged organelles, and individual proteins. In the present study, we found autophagy deficiency in mouse liver caused G2/M arrest in two mouse models, namely Fip200 and Atg7 liver-specific knockout mice. To uncover the link between autophagy deficiency and G2/M transition, we combined transcriptomic and proteomic analysis for liver samples from control and Atg7 liver-specific knockout mice. The data suggest that the inhibition of autophagy increases the protein level of Wee1 without any alteration of its mRNA abundance. Serum starvation, an autophagy stimulus, downregulates the protein level of Wee1 in vitro. In addition, the half-life of Wee1 is extended by the addition of chloroquine, an autophagy inhibitor. LC3, a central autophagic protein functioning in autophagy substrate selection and autophagosome biogenesis, interacts with Wee1 as assessed by co-immunoprecipitation assay. Furthermore, overexpression of Wee1 leads to G2/M arrest both in vitro and in vivo. Collectively, our data indicate that autophagy could degrade Wee1-a gatekeeper of the G2/M transition, whereas the inhibition of autophagy leads to the accumulation of Wee1 and causes G2/M arrest in mouse liver.