Biology subjects

Lindqvist, A.

Publications and source records attributed to Lindqvist, A..

8 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

Tracking GAD-specific T-cell expansions in Type 1 diabetes by intradermal GAD-Alum challenge

Identifying and monitoring autoreactive T cells that drive beta cell destruction remains a major obstacle to developing effective immunotherapies for type 1 diabetes (T1D). These cells are extremely rare in peripheral blood and cannot be accessed directly from the pancreas. We used intradermal injection of GAD-Alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Re-expression of selected T cell receptors (TCRs) confirmed antigen specificity. Up to 70% of T cells at the skin injection site were clonally-expanded and 4 of 14 (28%) re-expressed TCRs were GAD-reactive. In draining LNs 1 of 14 (4%) clonally-expanded TCRs was GAD-reactive, representing [~]0.08% of all T-cells. GAD-reactive cells across compartments displayed Th1 and Th17-associated transcription signatures. These results demonstrate the intradermal autoantigen challenge, coupled with scRNAseq, enables direct identification and molecular profiling of autoreactive T cells in vivo. This minimally invasive approach provides a powerful platform for tracking antigen-specific specific T cells to monitor disease activity and evaluate immune interventions in T1D.

immunology↗

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↗

bioRxiv2025

Lysine Potentiates Insulin Secretion via AASS-Dependent Catabolism and Regulation of GABA Content and Signaling

Background and aimLysine is an essential amino acid with insulinotropic effects in humans. In vitro, lysine also potentiates glucose-stimulated insulin secretion (GSIS) in {beta} cell lines and rodent pancreatic islets. For decades it has been assumed that insulinotropic action of lysine is mediated by plasma membrane depolarization similar to arginine. Aminoadipate-Semialdehyde Synthase (AASS) is a mitochondrial-located bifunctional enzyme engaged in the first two steps of the lysine catabolism. Whether AASS-dependent lysine catabolism occurs in {beta} cells and whether it is required for its insulinotropic action has not been investigated. MethodsmRNA expression of lysine catabolism pathway genes was assessed in human islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects. AASS was silenced in human pancreatic islets and in INS1 832/13 {beta} cells. {beta} cell metabolism and function were investigated by ELISA, extracellular flux analysis, live cell calcium imaging, transcriptomics and metabolomics analyses. ResultsExpression of genes involved in lysine catabolism, including AASS, ALDH7A1, DHTKD1 and HADH, was reduced in pancreatic islets from T2D donors. Silencing of AASS resulted in reduced lysine- and glucose-stimulated insulin secretion in human islets and INS1 832/13 {beta} cells. Surprisingly, transcriptomics and metabolomics analysis in Aass-KD {beta} cells with suppressed lysine catabolism identified reduced {gamma}-aminobutyric acid (GABA)/glutamate ratio as well as altered expression of genes implicated in GABA metabolism. This was accompanied by altered mitochondrial TCA cycle and oxidative phosphorylation (OXPHOS) activity, reflected by elevated lactate/pyruvate and reduced whole-cell ATP/ADP content as well as ATP-linked mitochondrial respiration. Glucose-and GABA-stimulated cytosolic calcium was also altered in Aass-KD {beta} cells. Strikingly, addition of GABA recovered impaired insulin secretion in Aass-KD {beta} cells. ConclusionAASS-dependent lysine catabolism is required to maintain adequate GABA shunt metabolism and signaling. In addition, lysine catabolism supports mitochondrial energy production, calcium uptake and insulin secretion. Reduced AASS-dependent lysine catabolism may contribute to {beta} cell GABA depletion and dysfunction in T2D patients.

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↗

bioRxiv2023

Single-cell mRNA-regulation analysis reveals cell type-specific mechanisms of type 2 diabetes

Perturbed secretion of insulin and other pancreatic islet hormones is the main cause of type 2 diabetes (T2D). The islets harbor five cell types that are potentially altered differently by T2D. Whole-islet transcriptomics and single-cell RNA-sequencing (scRNAseq) studies have revealed differentially expressed genes without reaching consensus. Here, we demonstrate that unprecedented insights into disease mechanisms can be obtained by network-based analysis of scRNAseq data. We developed differential gene coordination network analysis (dGCNA) and analyzed islet scRNAseq data from 16 T2D and 16 non-T2D individuals. dGCNA revealed T2D-induced cell type-specific networks of dysregulated genes with remarkable ontological specificity, thus allowing for a comprehensive and unbiased functional classification of genes involved in T2D. In beta cells eleven networks of genes were detected, revealing that mitochondrial electron transport chain, glycolysis, cytoskeleton organization, cell proliferation, unfolded protein response and three networks of beta cell transcription factors are perturbed, whereas exocytosis, lysosomal regulation and insulin translation programs are instead enhanced in T2D. Furthermore, we validated the ability of dGCNA to reveal disease mechanisms and predict the functional context of genes by showing that TMEM176A/B regulates the beta cell cytoskeleton and that CEPBG is a key regulator of the unfolded protein response. In addition, comparing beta- and alpha and cells, we found substantial differences, reproduced across independent datasets, confirming cell type-specific alterations in T2D. We conclude that analysis of networks of differentially coordinated genes provides outstanding insight into cell type-specific gene function and T2D pathophysiology.

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↗

bioRxiv2017

Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate Plk1

Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2-phase, but why active Cyclin A2-CDK2 during S phase does not trigger mitotic kinase activation remains unclear. Here we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation. SynopsisMain mitotic kinases as PLK1 are activated at the S/G2 transition. A change in Cyclin A2 localisation at the S/G2 transition enables activation of PLK1. Main points-Cyclin A2 appears in the cytoplasm at the S/G2 transition -Association with replicating chromatin and p21 restricts Cyclin A2 to the nucleus -DNA damage ensures nuclear Cyclin A2 through p21 -Cytoplasmic Cyclin A2 initiates PLK1 activation Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/191437v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@7e7e82org.highwire.dtl.DTLVardef@a3c9aforg.highwire.dtl.DTLVardef@10e11edorg.highwire.dtl.DTLVardef@baf6bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗