Using Standard Optical Flow Cytometry for Synchronizing Proliferating Cells in the G1 Phase

Vecsler, Manuela; Lazar, Itay; Tzur, Amit

doi:10.1371/journal.pone.0083935

Cited by 18 publications

(16 citation statements)

References 14 publications

(29 reference statements)

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“…forward scatter width (FSC-W) signal were isolated by FACSAria III (BD). This sorting protocol 573 yields a nearly pure G1 population without pre-synchronization (Vecsler, Lazar et al, 2013). 574…”

Section: Synchronization Of Ndb Cells In G1 Asynchronous Ndb Cells Smentioning

confidence: 99%

Cell cycle oscillators underlying orderly proteolysis of E2F8

Wasserman

Nachum

Cohen

et al. 2019

Preprint

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19E2F8 is a transcriptional repressor that antagonizes the canonical cell cycle 20 transcription factor E2F1. Despite the importance of this atypical E2F family member in cell 21 cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control 22 its dynamics. To address this question, we developed a complementary set of static and 23 dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, 24 and a full transition from pro-metaphase to G1. This revealed an interlocking molecular 25 switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 26 sites in E2F8 and the activation of APC/C Cdh1 , but not APC/C Cdc20 . Further, we revealed a 27 differential stability of E2F8, accounting for its accumulation in late G1 while APC/C Cdh1 is 28 still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we 29 identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our 30 data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating 31 an extensive coordination between phosphorylation, ubiquitination and transcription in 32 promoting orderly cell cycle progression. 33 34 35 36 37 38 39 40 41 42 Ran et al., 2008, Ouseph, Li et al., 2012). Despite being part of the 'repressive' branch of E2F 57proteins, E2F7 and E2F8 belong to the pro-proliferative gene network underlying cell 58 proliferation (Cohen, Vecsler et al., 2013). 59 E2F1, as well as E2F7 and E2F8, are regulated post-translationally via temporal 60 proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit cell 61 cycle ubiquitin ligase and core component of the cell cycle machinery (King, Peters et al., 62 1995, Sudakin, Ganoth et al., 1995. The APC/C uses two related co-activators termed Cdc20 63 and Cdh1, which bind substrates and recruit them to the APC/C for ubiquitination and 64 subsequent degradation (Kernan, Bonacci et al., 2018). We previously identified both E2F7 65 and E2F8 as targets of the Cdh1-bound form of APC/C (APC/C Cdh1 ) (Cohen et al., 2013). 66These findings, together with other supporting studies (Boekhout, Yuan et al., 2016), shifted 67 the model by which the E2F1-E2F7-E2F8 circuitry communicates with the cell cycle clock to 68 regulate the transition from the G1-phase of the cell cycle into S-phase. Nevertheless, the 69 exact inter-dynamics of E2F1-E2F7-E2F8 circuitry throughout G1 and the mechanism by 70 which they are achieved are not entirely resolved. No less obscure is the interplay between 71 E2F1 and atypical E2Fs during G2-phase and mitosis. Dissecting these complex signaling 72 circuits is important for understanding the decision making mechanisms at two critical 73 points in the life of a proliferating cell -commitment to DNA replication and division. 74Cell-free systems are known for their capacity to reproduce complex cellular 75 processes in vitro while maintaining a physiologically relevant context, bridging the gap 76...

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Section: Synchronization Of Ndb Cells In G1 Asynchronous Ndb Cells Smentioning

confidence: 99%

Cell cycle oscillators underlying orderly proteolysis of E2F8

Wasserman

Nachum

Cohen

et al. 2019

Preprint

Self Cite

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“…In fact, cCMP occurs in mammalian cells, and HEK-293 cells contain $30 pmol of cCMP per 10 6 cells under basal conditions [8]. Since the volume of a HEK-293 cell is $1000 femtoliters [24], this corresponds to a cCMP concentration around 30 lM. This calculation, however, disregards the fact that in many cell types a large part of the volume is occupied by the nucleus.…”

Section: Discussionmentioning

confidence: 99%

PDE7A1 hydrolyzes cCMP

et al. 2014

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a b s t r a c tThe degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3 0 ,5 0 -cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC-MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP K M value of 135 ± 19 lM. The V max for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3 0 ,5 0 -cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.

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“…7. Select a specific cell size, which is directly related to the cell cycle stage, by gating the cells on the forward-scatter width (Vecsler, Lazar, & Tzur, 2013).…”

Section: Additional Materials (Also See Basic Protocol 1)mentioning

confidence: 99%

Quantifying the Proteolytic Cleavage of Plasma Membrane Proteins in Living Cells

Calamai

Pavone

2018

CP Cell Biology

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The standard approach to study the activity of proteases consists of lysing cells and measuring the changes in the fluorescence properties of a synthetic substrate after cleavage in vitro. Here, a general protocol that uses a bi-fluorescent chimeric construct of a known substrate protein that follows the proteolytic processing in living cells is described. This approach is useful, in particular, to search for pharmacological conditions altering the cleavage rate of a certain protease, or to investigate the biological factors influencing a certain proteolytic mechanism. Three different methods (microscopy, flow cytometry, and spectroscopy) to detect fluorescence changes due to alteration in the processing are described. This approach was originally developed for studying conditions affecting the proteolytic activity of the β-secretase Bace1 on the amyloid precursor protein APP, but can in principle be applied to investigate any membrane protein undergoing ectodomain shedding by proteolytic cleavage. © 2018 by John Wiley & Sons, Inc.

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Using Standard Optical Flow Cytometry for Synchronizing Proliferating Cells in the G1 Phase

Cited by 18 publications

References 14 publications

Cell cycle oscillators underlying orderly proteolysis of E2F8

Cell cycle oscillators underlying orderly proteolysis of E2F8

PDE7A1 hydrolyzes cCMP

Quantifying the Proteolytic Cleavage of Plasma Membrane Proteins in Living Cells

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