Temporally multiplexed imaging of dynamic signaling networks in living cells

Cell, Год журнала: 2023, Номер 186(25), С. 5656 - 5672.e21

Опубликована: Ноя. 28, 2023

Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful image many at once, the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses proteins with different clocklike properties—such as reversibly photoswitchable switching kinetics—to represent cellular signals. We linearly decompose a brief (few-second-long) trace of fluorescence fluctuations, each point into weighted sum traces exhibited by fluorophore expressed cell. The weights then signal amplitudes. use TMI relationships between kinase activities individual cells, well cell-cycle signals, pointing toward broad utility throughout biology analysis transduction cascades systems.

Язык: Английский

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Mitogen signaling strength and duration can control cell cycle decisions

Science Advances, Год журнала: 2024, Номер 10(27)

Опубликована: Июль 5, 2024

Decades ago, mitogen-promoted signaling duration and strength were observed to be sensed by the cell critical for its decisions: proliferate or differentiate. Landmark publications established importance of mitogen not only in G 1 cycle phase but also through S 2 /M transition. Despite these early milestones, how signal strength, short strong weaker sustained, control fate has been largely unheeded. Here, we center on cardinal signaling-related questions, including (i) fluctuating mitogenic signals are converted into proliferation-differentiation decisions (ii) why extended weak is associated with differentiation, while bursts induce proliferation but, if too long, irreversible senescence. Our innovative broad outlook harnesses biology protein conformational ensembles, helping us define clarify decisions, thus fate.

Язык: Английский

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CDK2 and CDK4: Cell Cycle Functions Evolve Distinct, Catalysis-Competent Conformations, Offering Drug Targets

JACS Au, Год журнала: 2024, Номер 4(5), С. 1911 - 1927

Опубликована: Май 14, 2024

Cyclin-dependent kinases (CDKs), particularly CDK4 and CDK2, are crucial for cell cycle progression from the Gap 1 (G1) to Synthesis (S) phase by phosphorylating targets such as Retinoblastoma Protein (Rb). CDK4, paired with cyclin-D, operates in long G1 phase, while CDK2 cyclin-E, manages brief G1-to-S transition, enabling DNA replication. Aberrant CDK signaling leads uncontrolled proliferation, which is a hallmark of cancer. Exactly how they accomplish their catalytic phosphorylation actions distinct efficiencies poses fundamental, albeit overlooked question. Here we combined available experimental data modeling active complexes establish conformational functional landscapes explain two cyclin/CDK differentially populate catalytically competent states progression. Our premise that could be more important than cyclin-CDK biochemical binding specificity efficiency likely prime determinant We observe dynamic ATP site, regulatory spine, interaction its cyclin partner. The N-terminus cyclin-D acts an allosteric regulator activation loop ATP-binding site CDK4. Integrated suite data, suggest complex less capable remaining conformation, may have lower befitting time scales, point critical residues motifs drive differences. mechanistic landscape apply broadly kinases, propose drug design strategies: (i) Inhibition stabilization targeting regulation (ii) entropy-optimized leverages dynamic, entropic aspects optimize efficacy.

Язык: Английский

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Cell cycle plasticity underlies fractional resistance to palbociclib in ER+/HER2− breast tumor cells

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(7)

Опубликована: Фев. 7, 2024

The CDK4/6 inhibitor palbociclib blocks cell cycle progression in Estrogen receptor-positive, human epidermal growth factor 2 receptor-negative (ER+/HER2-) breast tumor cells. Despite the drug's success improving patient outcomes, a small percentage of cells continues to divide presence palbociclib-a phenomenon we refer as fractional resistance. It is critical understand cellular mechanisms underlying resistance because precise resistant tissue strong predictor clinical outcomes. Here, hypothesize that arises from cell-to-cell differences core regulators allow subset escape therapy. We used multiplex, single-cell imaging identify fractionally both cultured and primary samples resected patients. Resistant showed premature accumulation multiple G1 including E2F1, retinoblastoma protein, CDK2, well enhanced sensitivity pharmacological inhibition CDK2 activity. Using trajectory inference approaches, show how plasticity among gives rise alternate "paths" individual treatment. Understanding drivers plasticity, eliminate paths, could lead improved cancer therapies targeting improve

Язык: Английский

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Slower CDK4 and faster CDK2 activation in the cell cycle

Structure, Год журнала: 2024, Номер 32(8), С. 1269 - 1280.e2

Опубликована: Май 3, 2024

Язык: Английский

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Therapeutic benefits of maintaining CDK4/6 inhibitors and incorporating CDK2 inhibitors beyond progression in breast cancer

Опубликована: Янв. 28, 2025

The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy has revolutionized treatment for hormone receptor-positive (HR+) metastatic breast cancer. However, the emergence resistance in most patients often leads to discontinuation with no consensus on effective second-line therapies. therapeutic benefits maintaining CDK4/6i or incorporating CDK2 (CDK2i) after disease progression remain unclear. Here, we demonstrate that sustained therapy, either alone combined CDK2i, significantly suppresses growth drug-resistant HR + Continued induces a non-canonical pathway retinoblastoma protein (Rb) inactivation via post-translational degradation, resulting diminished E2F activity delayed G1 progression. Importantly, our data highlight CDK2i should be effectively suppress overcome resistance. We also identify cyclin E overexpression as key driver inhibition. These findings provide crucial insights into overcoming cancer, supporting continued use strategic incorporation improve outcomes.

Язык: Английский

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Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Ноя. 29, 2023

Abstract Cyclin-dependent kinases 4 and 6 (CDK4/6) are critical for initiating cell proliferation by inactivating the retinoblastoma (Rb) protein. However, mammalian cells can bypass CDK4/6 Rb inactivation. Here we show a non-canonical pathway inactivation its interplay with external signals. We find that non-phosphorylated protein in quiescent is intrinsically unstable, offering an alternative mechanism E2F activity. Nevertheless, this incompletely induces Rb-protein loss, resulting minimal To trigger proliferation, upregulation of mitogenic signaling required stabilizing c-Myc, thereby augmenting Concurrently, stress promotes Cip/Kip levels, competitively regulating signaling. In cancer, driver mutations elevate c-Myc facilitating adaptation to inhibitors. Differentiated cells, despite maintain quiescence through modulation levels. Our findings provide mechanistic insights into model cell-cycle entry maintenance quiescence.

Язык: Английский

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Sequential activation of E2F via Rb degradation and c-Myc drives resistance to CDK4/6 inhibitors in breast cancer

Cell Reports, Год журнала: 2023, Номер 42(11), С. 113198 - 113198

Опубликована: Окт. 21, 2023

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are key therapeutic agents in the management of metastatic hormone-receptor-positive breast cancer. However, emergence drug resistance limits their long-term efficacy. Here, we show that cancer cells develop CDK4/6i via a sequential two-step process E2F activation. This entails retinoblastoma (Rb)-protein degradation, followed by c-Myc-mediated amplification transcriptional activity. treatment halts cell proliferation an Rb-dependent manner but dramatically reduces Rb-protein levels. this reduction Rb levels insufficiently induces To resistance, upregulation or activating mutations mitogenic hormone signaling required to stabilize c-Myc levels, thereby augmenting Our analysis pre-treatment tumor samples reveals strong correlation between rather than poor outcomes after treatment. Moreover, propose proteasome can potentially reverse restoring

Язык: Английский

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CDK4/6 activity is required during G ₂ arrest to prevent stress-induced endoreplication

Science, Год журнала: 2024, Номер 384(6695)

Опубликована: Май 2, 2024

Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G ) synthesis (S) phase transition of responding mitogen signaling, promoting E2F transcription inhibition anaphase-promoting complex. We found that this mechanism was still required in G 2 -arrested cells prevent exit after S phase. This revealed a role for maintaining state, challenging notion is irreversible do not require mitogens passing restriction point. Exit from occurred during ribotoxic stress actively mediated stress-activated protein kinases. Upon relief stress, significant fraction underwent second round DNA replication led whole-genome doubling.

Язык: Английский

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An intermediate Rb–E2F activity state safeguards proliferation commitment

Nature, Год журнала: 2024, Номер 631(8020), С. 424 - 431

Опубликована: Июнь 26, 2024

Abstract Tissue repair, immune defence and cancer progression rely on a vital cellular decision between quiescence proliferation 1,2 . Mammalian cells proliferate by triggering positive feedback mechanism 3,4 The transcription factor E2F activates cyclin-dependent kinase 2 (CDK2), which in turn phosphorylates inactivates the inhibitor protein retinoblastoma (Rb). This action further increases activity to express genes needed for proliferation. Given that can inadvertently amplify small signals, understanding how keep this check remains puzzle. Here we measured CDK2 signal changes single found engages only late G1 phase. Cells spend variable often extended times reversible state of intermediate before committing proliferate. is proportional amount phosphorylation conserved T373 residue Rb mediated or CDK4/CDK6. Such T373-phosphorylated bound chromatin but dissociates from it once hyperphosphorylated at many sites, fully E2F. preferential initial be explained its relatively slower rate dephosphorylation. Together, our study identifies primed activation whereby sense external internal signals decide whether reverse exit trigger initiates cell

Язык: Английский

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