Cell-to-cell variability in inducible Caspase9-mediated cell death

AbstractiCasp9 suicide gene has been widely used as a promising killing strategy in various cell therapies. However, different cells show significant heterogeneity in response to apoptosis inducer, posing challenges in clinical applications of killing strategy. The cause of the heterogeneity remains elusive so far. Here, by simultaneously monitoring the dynamics of iCasp9 dimerization, Caspase3 activation, and cell fate in single cells, we found that the heterogeneity was mainly due to cell-to-cell variability in initial iCasp9 expression and XIAP/Caspase3 ratio. Moreover, multiple-round drugging cannot increase the killing efficiency. Instead, it will place selective pressure on protein levels, especially on the level of initial iCasp9, leading to drug resistance. We further show this resistance can be largely eliminated by combinatorial drugging with XIAP inhibitor at the end, but not at the beginning, of the multiple-round treatments. Our results unveil the source of cell fate heterogeneity and drug resistance in iCasp9-mediated cell death, which may enlighten better therapeutic strategies for optimized killing.

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Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

10.1101/427237 ◽

2018 ◽

Author(s):

Maximilian W. Fries ◽

Kalina T. Haas ◽

Suzan Ber ◽

John Saganty ◽

Emma K. Richardson ◽

...

Keyword(s):

Cell Death ◽

Single Cell ◽

Cell Fate ◽

Genetic Heterogeneity ◽

Cellular Response ◽

Single Cells ◽

Caspase Activation ◽

Cell Fate Decisions ◽

Network Activation ◽

Biochemical Imaging

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

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Heterogeneity of Sonic Hedgehog Response Dynamics and Fate Specification in Single Neural Progenitors

10.1101/412858 ◽

2018 ◽

Cited By ~ 3

Author(s):

Fengzhu Xiong ◽

Andrea R. Tentner ◽

Tom W. Hiscock ◽

Peng Huang ◽

Sean G. Megason

Keyword(s):

Cell Fate ◽

Sonic Hedgehog ◽

Cellular Response ◽

Single Cells ◽

Neural Progenitors ◽

Significant Heterogeneity ◽

Response Dynamics ◽

Output Analysis ◽

Response Level ◽

Fate Specification

SUMMARYDuring neural tube patterning, a gradient of Sonic hedgehog (Shh) signaling specifies ventral progenitor fates. The cellular response to Shh is processed through a genetic regulatory network (GRN) to code distinct fate decisions. This process integrates Shh response level, duration and other inputs and is affected by noise in signaling and cell position. How reliably a single cell’s Shh response profile predicts its fate choice is unclear. Here we use live imaging to track neural progenitors that carry both Shh and fate reporters in zebrafish embryos. We found that there is significant heterogeneity between Shh response and fate choice in single cells. We quantitatively modeled reporter intensities to obtain single cell response levels over time and systematically determined their correlation with multiple models of cell fate specification. Our input-output analysis shows that while no single metric perfectly predicts fate choices, the maximal Shh response level correlates best overall with progenitor fate choices across the anterior-posterior axis.

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Single cell protein analysis for systems biology

Essays in Biochemistry ◽

10.1042/ebc20180014 ◽

2018 ◽

Vol 62 (4) ◽

pp. 595-605 ◽

Cited By ~ 18

Author(s):

Ezra Levy ◽

Nikolai Slavov

Keyword(s):

Systems Biology ◽

Single Cell ◽

Cell Fate ◽

Fluorescent Proteins ◽

Single Cells ◽

Protein Analysis ◽

Single Cell Protein ◽

Cell Protein ◽

Protein Levels ◽

Trade Offs

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have regulatory roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review examples connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single-cell protein analysis, and we discuss their trade-offs, with an emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantitating the transcriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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FBXO45-MYCBP2 regulates mitotic cell fate by targeting FBXW7 for degradation

10.1101/493791 ◽

2018 ◽

Author(s):

Kai T. Richter ◽

Yvonne T. Kschonsak ◽

Barbara Vodicska ◽

Ingrid Hoffmann

Keyword(s):

Cell Death ◽

Cell Fate ◽

Chemotherapy Resistance ◽

Mitotic Cell ◽

Mitotic Arrest ◽

Protein Levels ◽

Mitotic Slippage ◽

Microtubule Targeting Agents ◽

Fate Decision ◽

F Box Protein

SUMMARYCell fate decision upon prolonged mitotic arrest induced by microtubule targeting agents depends on the activity of the tumor suppressor and F-box protein FBXW7. FBXW7 promotes mitotic cell death and prevents premature escape from mitosis through mitotic slippage. Mitotic slippage is a process that can cause chemoresistance and tumor relapse. Therefore, understanding the mechanisms that regulate the balance between mitotic cell death and mitotic slippage is an important task. Here we report that FBXW7 protein levels markedly decline during extended mitotic arrest. FBXO45 binds to a conserved acidic N-terminal motif of FBXW7 specifically under a prolonged delay in mitosis, leading to ubiquitylation and subsequent proteasomal degradation of FBXW7 by the FBXO45-MYCBP2 E3 ubiquitin ligase. Moreover, we find that FBXO45-MYCBP2 counteracts FBXW7 in that it promotes mitotic slippage and prevents cell death in mitosis. Targeting this interaction represents a promising strategy to prevent chemotherapy resistance.

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Single cell protein analysis for systems biology

10.7287/peerj.preprints.26965 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ezra Levy ◽

Nikolai Slavov

Keyword(s):

Systems Biology ◽

Single Cell ◽

Cell Fate ◽

Fluorescent Proteins ◽

Single Cells ◽

Protein Analysis ◽

Single Cell Protein ◽

Cell Protein ◽

Protein Levels ◽

Trade Offs

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have functional roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review such examples of connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single cell protein analysis, and we discuss their trade-offs, with emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantifying the trasncriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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Single cell protein analysis for systems biology

10.7287/peerj.preprints.26965v1 ◽

2018 ◽

Cited By ~ 2

Author(s):

Ezra Levy ◽

Nikolai Slavov

Keyword(s):

Systems Biology ◽

Single Cell ◽

Cell Fate ◽

Fluorescent Proteins ◽

Single Cells ◽

Protein Analysis ◽

Single Cell Protein ◽

Cell Protein ◽

Protein Levels ◽

Trade Offs

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have functional roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review such examples of connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single cell protein analysis, and we discuss their trade-offs, with emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantifying the trasncriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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Use of Flexible Materials with a Novel Pressure Driven Equibiaxial Cell Stretching Device for Mechanical Stimulation of Single Mammalian Cells

MRS Proceedings ◽

10.1557/proc-773-n5.6 ◽

2003 ◽

Vol 773 ◽

Author(s):

James D. Kubicek ◽

Stephanie Brelsford ◽

Philip R. LeDuc

Keyword(s):

Cell Fate ◽

Mechanical Stimulation ◽

Mammalian Cells ◽

Cellular Response ◽

Single Cells ◽

Complex Nature ◽

Cellular Behavior ◽

Cell Stretching ◽

Stimulation Of ◽

Pressure Driven

AbstractMechanical stimulation of single cells has been shown to affect cellular behavior from the molecular scale to ultimate cell fate including apoptosis and proliferation. In this, the ability to control the spatiotemporal application of force on cells through their extracellular matrix connections is critical to understand the cellular response of mechanotransduction. Here, we develop and utilize a novel pressure-driven equibiaxial cell stretching device (PECS) combined with an elastomeric material to control specifically the mechanical stimulation on single cells. Cells were cultured on silicone membranes coated with molecular matrices and then a uniform pressure was introduced to the opposite surface of the membrane to stretch single cells equibiaxially. This allowed us to apply mechanical deformation to investigate the complex nature of cell shape and structure. These results will enhance our knowledge of cellular and molecular function as well as provide insights into fields including biomechanics, tissue engineering, and drug discovery.

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Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

Nature Communications ◽

10.1038/s41467-020-20806-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joanna L. Fox ◽

Michelle A. Hughes ◽

Xin Meng ◽

Nikola A. Sarnowska ◽

Ian R. Powley ◽

...

Keyword(s):

Cell Death ◽

Structural Analysis ◽

Cell Fate ◽

Catalytic Domain ◽

Caspase 8 ◽

Type I ◽

Signaling Complex ◽

Catalytic Domains ◽

Regulated Cell Death ◽

Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

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Targeting BCL-2 in Cancer: Advances, Challenges, and Perspectives

Cancers ◽

10.3390/cancers13061292 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1292 ◽

Cited By ~ 1

Author(s):

Shirin Hafezi ◽

Mohamed Rahmani

Keyword(s):

Drug Resistance ◽

Cell Death ◽

Targeted Therapies ◽

Single Agent ◽

Therapeutic Agents ◽

Malignant Cells ◽

Future Perspectives ◽

Comprehensive Overview ◽

Antiapoptotic Proteins ◽

Preclinical And Clinical Studies

The major form of cell death in normal as well as malignant cells is apoptosis, which is a programmed process highly regulated by the BCL-2 family of proteins. This includes the antiapoptotic proteins (BCL-2, BCL-XL, MCL-1, BCLW, and BFL-1) and the proapoptotic proteins, which can be divided into two groups: the effectors (BAX, BAK, and BOK) and the BH3-only proteins (BIM, BAD, NOXA, PUMA, BID, BIK, HRK). Notably, the BCL-2 antiapoptotic proteins are often overexpressed in malignant cells. While this offers survival advantages to malignant cells and strengthens their drug resistance capacity, it also offers opportunities for novel targeted therapies that selectively kill such cells. This review provides a comprehensive overview of the extensive preclinical and clinical studies targeting BCL-2 proteins with various BCL-2 proteins inhibitors with emphasis on venetoclax as a single agent, as well as in combination with other therapeutic agents. This review also discusses recent advances, challenges focusing on drug resistance, and future perspectives for effective targeting the Bcl-2 family of proteins in cancer.

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