Biology subjects

Akopyan, K.

Publications and source records attributed to Akopyan, K..

4 recordsLinked to original sources

bioRxiv2026

CRISPR-PTM and CRISPR-VEIS: Multiplexed platforms for quantitative functional analysis of endogenous phosphosites

Connecting protein post-translational modifications (PTMs) to phenotypic outcomes is a central challenge. Although phosphoproteomics has richly catalogued specific sites, reliable methods to measure the endogenous effects of individual phosphosites on cellular fitness and signaling are still lacking. Here, we introduce CRISPR-PTM and CRISPR-VEIS as complementary platforms for quantitative, endogenous phosphosite interrogation at both individual and clustered phosphorylation events. CRISPR-PTM is a multiplexed knock-in framework generating defined phosphosite variants with internal allelic markers, enabling precise relative fitness effects in pooled populations. CRISPR-VEIS (Visualisation of Edits In Situ) is an in situ mRNA-genotyping approach that directly links endogenous allelic edits to single-cell phenotypes, addressing needs for subclonal isolation or exogenous reporters. We applied these methods to the WEE1-CDK1 regulatory pathway, where canonical CDK1-Y15 phosphorylation alone cannot explain WEE1 loss or inhibition phenotypes. CRISPR-PTM systematically quantified fitness consequences of CDK1 phosphosite variants and identified Y19 as a previously unrecognized WEE1-dependent inhibitory site. Single non-phosphorylatable substitutions at Y15 or Y19 had minimal impact, but combined CDK1-Y15F/Y19F editing caused pronounced fitness defects, phenocopying WEE1 inactivation and showing epistasis to WEE1 inhibitors. CRISPR-VEIS further demonstrated that acute endogenous editing of both sites correlated with elevated CDK activity at the single-cell level. Together, CRISPR-PTM and CRISPR-VEIS provide broadly applicable approaches for quantitative analysis of PTM function, enabling direct linkage of endogenous phosphosite variation to cellular fitness and signaling phenotypes.

cell biology↗

bioRxiv2025

Cumulative microscopy reveals cellular states in fibroblasts from patients with genetic disorders

Analysis of cellular states and signaling trajectories can provide insights into causes of disease. We developed cumulative microscopy, a method to perform cyclical imaging without elution or quenching steps. Cumulative microscopy computationally extracts individual signals from accumulating fluorescence during sequential imaging. We used cumulative microscopy to quantitatively assess cell cycle and stress markers in individual primary fibroblasts from patients with rare genetic proliferative disorders with increased cancer risk. Neural network-based analysis of cumulative microscopy data suggested that cells from patients with Cartilage-hair hypoplasia (CHH), but not Mulibrey Nanism (MUL), showed replication stress. We analyzed cell states and cell trajectories and found that a subset of cells from patients with CHH showed spontaneous replication stress, followed by cell cycle exit in both G1 and G2 phase. We note that replication stress potentially could underlie both proliferative defects and increased cancer risk in CHH patients and conclude that cumulative microscopy is an efficient, quantitative, and generalizable approach to multiplex microscopy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/668398v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@4bcd0dorg.highwire.dtl.DTLVardef@83262corg.highwire.dtl.DTLVardef@3f3f13org.highwire.dtl.DTLVardef@46174b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Wee1 inhibition decouples Cdk1 and Plk1 activities: a role for gradual Cdk1 activation throughout G2 phase.

At completion of DNA replication, the mitotic kinases CDK1 and PLK1 are activated. Their activities increase slowly through early G2 phase, but the reason for this low-level activity before mitotic entry is not clear. Using a combination of experiments and mathematical modelling, we find that gradual CDK1 activation through G2 phase stimulates production of mitotic factors and coordinates activation of CDK1 and PLK1. We find that inhibition of CDK1 during G2 phase limits transcription of mitotic factors. Conversely, the duration of premature mitosis by forced activation of CDK1 is inversely related to the time-point in G2 when mitosis is triggered. Forced CDK1 activation not only leads to a lack of mitotic factors, but also decouples CDK1 and PLK1 activation. Accordingly, we find that duration of forced mitosis by WEE1 inhibition can be partially rescued by expression of constitutively active PLK1. Our results show a function for slow mitotic kinase activation through G2 phase and suggests a mechanism for how the timing of mitotic entry is linked to preparation for mitosis. HighlightsO_LISlow Cdk1 activation through G2 phase coordinates expression of mitotic proteins and activation of mitotic kinases C_LIO_LIThe duration of forced mitosis by WEE1 inhibition depends on when in G2 mitosis is triggered C_LIO_LIForced CDK1 activation decouples CDK1 and PLK1 activation C_LIO_LIThe duration of forced mitosis can be partially rescued by expression of constitutively active PLK1 C_LI

cell biology↗

bioRxiv2020

p53-dependent polyploidisation after DNA damage in G2 phase

Cell cycle progression in the presence of damaged DNA can lead to accumulation of mutations and pose a risk for tumour development. In response to DNA damage in G2 phase, human cells can be forced to exit the cell cycle in a p53-p21- and APC/CCdh1-dependent manner. Cells that exit the cell cycle in G2 phase become senescent, but it is unclear what determines this commitment and whether other cell fates occur. We find that a subset of immortalised RPE-1 cells and primary human fibroblasts spontaneously initiate DNA re-replication several days after forced cell cycle exit in G2 phase. By combining single cell tracking for more than a week with quantitative immunofluorescence, we find that the resulting polyploid cells contain increased levels of damaged DNA and frequently exit the cell cycle again in the next G2 phase. Subsequently, these cells either enter senescence or commit to another round of DNA re-replication, further increasing the ploidy. At least a subset of the polyploid cells show abnormal centrosome numbers or localisation. In conclusion, cells that are forced to exit the cell cycle in G2 phase face multiple choices that lead to various phenotypes, including propagation of cells with different ploidies. Our findings suggest a mechanism by which p53-positive cells can evade senescence that risks genome integrity. Main points-Cell cycle exit from G2 phase does not necessarily lead to senescence -Resumption of proliferation after G2 phase cell cycle exit starts with DNA replication -Successive cell cycle exits lead to propagation of cells with different ploidies -A p53-dependent mechanism allows eventual proliferation after DNA damage

cell biology↗