Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions. ARTICLE HISTORY

The cell cycle is canonically described as a series of four consecutive phases: G1, S, G2, and M. In single cells, the duration of each phase varies, but the quantitative laws that govern phase durations are not well understood. Using time‐lapse microscopy, we found that each phase duration follows an Erlang distribution and is statistically independent from other phases. We challenged this observation by perturbing phase durations through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Despite large changes in durations in cell populations, phase durations remained uncoupled in individual cells. These results suggested that the independence of phase durations may arise from a large number of molecular factors that each exerts a minor influence on the rate of cell cycle progression. We tested this model by experimentally forcing phase coupling through inhibition of cyclin‐dependent kinase 2 ( CDK 2) or overexpression of cyclin D. Our work provides an explanation for the historical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.The human protein Ser/Thr kinases Adaptor protein 2-Associated Kinase 1 (AAK1) and BMP-2-Inducible Kinase (BMP2K/BIKE) play critical roles in mediating endocytosis and other key signaling pathways. Both are broadly expressed and are members of the NAK family of human kinases, which also includes Cyclin G-Associated Kinase (GAK) and Myristoylated and Palmitoylated Serine/Threonine Kinase 1 (MPSK1/STK16). The family shares little homology outside of their kinase domains. 1 AAK1 and BMP2K are the most closely related, with overall sequence identity of 50% and kinase domain sequence identity of 74%. 2 A key function of AAK1 is regulation of receptor-mediated endocytosis via binding directly to clathrin and phosphorylating the medium subunit of AP2 (adaptor protein 2), which stimulates binding to cargo proteins. [3][4][5] AAK1 also modulates the Notch pathway, partially through its phosphorylation of Numb. 6, 7 BMP2K plays a role in osteoblast differentiation, is a clathrin-coated vesicle-associated protein, and, like AAK1, also associates with Numb. 8, 9 Due to their many functions, AAK1 and BMP2K have been implicated as potential drug targets for diverse conditions. AAK1 has been linked to diseases affecting the brain such as schizophrenia, Parkinson's disease and amyotrophic lateral sclerosis as well as implicated as a potential anti-viral target for the treatment of Hepatitis C. 5, 10, 11 BMP2K has been associated with myopia and evaluated as a potential treatment for HIV. 12, 13 A dual AAK1/BMP2K small molecule inhibitor was recently reported as a novel therapeutic to treat neuropathic pain. 14 X-ray crystal structures for the kinase domains of all NAK family members have been solved and reported. 2, 15, 16 Published and novel high-resolution crystal structures of AAK1 and BMP2K reveal target-specific structural features that have enabled our design of specific chemical probes and allowed further

Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

Successful genome duplication is required for cell proliferation and demands extraordinary precision and accuracy. The mechanisms by which cells enter, progress through, and exit S phase are intense areas of focus in the cell cycle and genome stability fields. Key molecular events in the G1 phase of the cell division cycle, especially origin licensing, are essential for pre‐establishing conditions for efficient DNA replication during the subsequent S phase. If G1 events are poorly regulated or disordered, then DNA replication can be compromised leading to genome instability, a hallmark of tumorigenesis. Upon entry into S phase, coordinated origin firing and replication progression ensure complete, timely, and precise chromosome replication. Both G1 and S phase progressions are controlled by master cell cycle protein kinases and ubiquitin ligases that govern the activity and abundance of DNA replication factors. In this short review, we describe current understanding and recent developments related to G1 progression and S phase entrance and exit with a particular focus on origin licensing regulation in vertebrates.

The onset of COVID-19, coupled with the finer lens placed on systemic racial disparities within our society, has resulted in increased discussions around mental health. Despite this, mental health struggles in research are still often viewed as individual weaknesses and not the result of a larger dysfunctional research culture. Mental health interventions in the science, technology, engineering, and mathematics (STEM) academic community often focus on what individuals can do to improve their mental health instead of focusing on improving the research environment. In this paper, we present four aspects of research that may heavily impact mental health based on our experiences as research scientists: bullying and harassment; precarity of contracts; diversity, inclusion, and accessibility; and the competitive research landscape. Based on these aspects, we propose systemic changes that institutions must adopt to ensure their research culture is supportive and allows everyone to thrive.

The cellular decision governing the transition between proliferative and arrested states is crucial to the development and function of every tissue. While the molecular mechanisms that regulate the proliferative cell cycle are well established, we know comparatively little about what happens to cells as they diverge into cell cycle arrest. We performed hyperplexed imaging of 47 cell cycle effectors to obtain a map of the molecular architecture that governs cell cycle exit and progression into reversible ("quiescent") and irreversible ("senescent") arrest states. Using this map, we found multiple points of divergence from the proliferative cell cycle; identified stress-specific states of arrest; and resolved the molecular mechanisms governing these fate decisions, which we validated by single-cell, time-lapse imaging. Notably, we found that cells can exit into senescence from either G1 or G2; however, both subpopulations converge onto a single senescent state with a G1-like molecular signature. Cells can escape from this "irreversible" arrest state through the upregulation of G1 cyclins. This map provides a more comprehensive understanding of the overall organization of cell proliferation and arrest.

BackgroundGlioblastoma (GBM) is the most common adult primary brain tumor. Multimodal treatment is empiric and prognosis remains poor. Recurrent PIK3CA missense mutations (PIK3CAmut) in GBM are restricted to three functional domains: adaptor binding (ABD), helical, and kinase. Defining how these mutations influence gliomagenesis and response to kinase inhibitors may aid in the clinical development of novel targeted therapies in biomarker-stratified patients.MethodsWe used normal human astrocytes immortalized via expression of hTERT, E6, and E7 (NHA). We selected two PIK3CAmut from each of 3 mutated domains and induced their expression in NHA with (NHARAS) and without mutant RAS using lentiviral vectors. We then examined the role of PIK3CAmut in gliomagenesis in vitro and in mice, as well as response to targeted PI3K (PI3Ki) and MEK (MEKi) inhibitors in vitro.ResultsPIK3CAmut, particularly helical and kinase domain mutations, potentiated proximal PI3K signaling and migration of NHA and NHARAS in vitro. Only kinase domain mutations promoted NHA colony formation, but both helical and kinase domain mutations promoted NHARAS tumorigenesis in vivo. PIK3CAmut status had minimal effects on PI3Ki and MEKi efficacy. However, PI3Ki/MEKi synergism was pronounced in NHA and NHARAS harboring ABD or helical mutations.ConclusionPIK3CAmut promoted differential gliomagenesis based on the mutated domain. While PIK3CAmut did not influence sensitivity to single agent PI3Ki, they did alter PI3Ki/MEKi synergism. Taken together, our results demonstrate that a subset of PIK3CAmut promote tumorigenesis and suggest that patients with helical domain mutations may be most sensitive to dual PI3Ki/MEKi treatment.