Biology subjects

Pedersen, S. F.

Publications and source records attributed to Pedersen, S. F..

11 recordsLinked to original sources

bioRxiv2026

SurpHer: a genetically encoded ratiometric sensor for dynamic extracellular pH imaging

Extracellular pH is a key microenvironmental factor shaping cell physiology and disease, creating a need for quantitative biosensors that can capture dynamic changes in pHe at the surface of individual living cells. Here, we develop a genetically encoded, ratiometric extracellular pH biosensor through systematic screening of a modular library of membrane-display designs that combine SEpHluorin with a pH-stable reference fluorophore. Screening identified a cell-surface-localised mKate2-SEpHluorin construct, named SurpHer, that exhibits dynamic ratiometric responses across the pHe range of 6 - 7.8. SurpHer shows robust membrane localisation and extracellular pH responsiveness across diverse human cell types including HEK293T, PANC-1 and MDA-MB231 cells. Following stable integration in MDA-MB-231 cells, SurpHer enabled time-course imaging of pHe gradients in a microfluidic platform for modelling tumour microenvironments. SurpHer enables real-time interrogation of the pericellular pH environment of tumor cells and, more broadly, provides a strategy to probe microenvironmental pH dynamics across diverse biological contexts.

cell biology↗

bioRxiv2026

NBCn1 interacts with DYNLL1 and regulates ciliary length and SUFU localization to control Sonic hedgehog signaling

Primary cilia integrate morphogen signaling with cellular physiology, yet how ion transporters contribute to ciliary organization and function remains poorly understood. Here, we identify the sodium-bicarbonate cotransporter NBCn1 (SLC4A7) as a previously unrecognized ciliary membrane component essential for regulating ciliary length and Sonic hedgehog (Shh) signaling. NBCn1 localizes to primary cilia through distinct Nand Cterminal targeting motifs and inhibition of dynein activity or loss of DLG1, a known NBCn1 interacting protein, augments its ciliary localization. Loss of NBCn1 shortens cilia without altering ciliation frequency or deciliation kinetics, indicating a selective role in ciliary elongation control rather than in ciliogenesis initiation or maintenance. NBCn1 deficiency or -inhibition leads to ciliary enrichment of SUFU even at basal level and markedly attenuates GLI1 transcriptional activation, despite intact SMO ciliary entry. Structural modeling and coimmunoprecipitation reveal that NBCn1 interacts with DYNLL1, VPS45, and the transitionzone protein TMEM216, suggesting that NBCn1 modulates ciliary length and Shh signaling through interactions with these proteins. Together, these findings uncover NBCn1 as a central regulator of ciliary length and Shh signaling, highlighting ion transport as a critical and underappreciated determinant of ciliary signaling competence. Significance statementPrimary cilia are essential cellular signaling hubs, yet the contribution of ion transporters and pH to ciliary architecture and function has remained unclear. This study identifies the bicarbonate transporter NBCn1 as a previously unrecognized regulator of ciliary length and Sonic hedgehog (Shh) signaling. We show that NBCn1 is actively trafficked into cilia through defined Nand Cterminal determinants, is exported from cilia via retrograde intraflagellar transport (IFT), and interacts directly with IFT dynein component DYNLL1. Loss of NBCn1 selectively shortens cilia while leaving ciliogenesis frequency and deciliation dynamics intact. NBCn1 deficiency disrupts the ciliary localization of the key Shh component SUFU, leading to impaired GLI transcriptional responses. By revealing how ion transport intersects with IFT and morphogen signaling, these findings establish NBCn1 as a central integrator of ciliary microenvironmental regulation and developmental signaling output, highlighting pH regulatory ion transport as a critical and underappreciated determinant of ciliary function.

cell biology↗

bioRxiv2025

Integrative structural analysis of the human endosomal Na⁺/H⁺ exchanger NHE6 reveals a lipid-stabilized intracellular gate and a disordered C-terminus

Human NHE6 (HsNHE6) is an endosomal Na+/H+ exchanger essential for maintaining luminal pH and endo-lysosomal trafficking in neurons. HsNHE6 mutations are implicated in devastating neurological syndromes, but mechanistically the transporter remains poorly understood. Here, we present the single particle cryo-electron microscopy structure of HsNHE6 at 3.4 [A], captured in an inward-open conformation. The structure reveals a homodimeric architecture with 13 transmembrane helices per protomer and the conserved ion-binding site. Functional assays demonstrate that HsNHE6 mediates the exchange of both Na+ and K+ for protons. A structured C-terminal helix interacts with the transmembrane core, jointly forming a hydrophobic cavity that accommodates two lipid molecules, potentially modulating cation access to the ion-binding site. The distal C-terminus of HsNHE6 is intrinsically disordered, as revealed by NMR and small-angle X-ray scattering and equips HsNHE6 with a large cytosolic span. Our integrative model of full-length HsNHE6 provides a comprehensive framework for understanding HsNHE6-mediated ion exchange and its disruption in neurological diseases such as Christianson syndrome.

biochemistry↗

bioRxiv2025

A microfluidic model to recapitulate pH and oxygen gradients in solid tumors

The tumor microenvironment (TME) plays critical roles in cancer development, aggressiveness, and treatment resistance. The TME comprises cellular (stromal) components as well as gradients of physicochemical properties, including hypoxia and acidosis. Understanding of how hypoxia and acidosis gradients impact cancer phenotypes is lacking, in large part due to challenges in precisely mimicking and controlling such gradients in a manner compatible with the growth of cancer- and stromal cells. Here, we design and validate a microfluidic device enabling orthogonal gradients of oxygen and pH. Both gradients are established by diffusion from a nearby source and sink in the observation area in the absence of flow. This produces linear gradients at steady state. Our device enables a wide range of spatiotemporally resolved analyses, from omics to live cell imaging, interrogating the impact of the physicochemical TME on disease development. The design is easily adaptable, making it valuable for a wide range of questions involving physicochemical gradients.

cancer biology↗

bioRxiv2024

Tumor microenvironment acidosis favors pancreatic cancer stem cell properties and in vivo metastasis

The acidic tumor microenvironment favors cancer aggressiveness via incompletely understood pathways. Here, we asked whether acidic environments select for cancer stem cell (CSC) properties. Bulk RNA-seq of Panc-1 human pancreatic cancer cells adapted to extracellular pH 6.5 revealed upregulation of CSC markers including CD44, EpCam, Nestin and aldehyde dehydrogenases, and CSC pathway enrichment. We therefore assessed CSC characteristics of acid-adapted (AA) and non-adapted (Ctrl) PaTu8988s and MiaPaca-2 pancreatic cancer cells. Compared to Ctrl, AA cells exhibited increased ALDH- and {beta}-catenin activity and pancreatosphere-forming efficiency, classical CSC characteristics. Panc-1, PaTu8988s and MiaPaCa-2 AA cells differed in CSC marker expression, and AA cells did not exhibit typical flow cytometric CSC populations. However, single-nucleus sequencing identified the acid adaptation-induced emergence of a population with clear CSC characteristics. Finally, in an orthotopic mouse model, AA Panc-1 cells drove strongly increased aggressiveness and liver metastasis compared to Ctrl cells. We conclude that acid-adaptation of pancreatic cancer cells leads to enrichment of a CSC phenotype with unusual traits, providing new insight into how acidic tumor microenvironments favor cancer aggressiveness.

cancer biology↗

bioRxiv2023

DLG1 functions upstream of SDCCAG3 and IFT20 to control targeting of polycystin-2 to the primary cilium

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

cell biology↗

bioRxiv2023

MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

The lactate-proton cotransporter MCT4 and its chaperone CD147 are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were strongly reciprocally interdependent in MDA-MB-231 invasive breast cancer cells. Knockdown (KD) and overexpression (OE) of MCT4 and/or CD174 in- and decreased, respectively, migration, invasion, and fluorescent gelatin degradation. OE of both proteins increased gelatin degradation and appearance of the matrix metalloprotease (MMP)-generated collagen-I cleavage product reC1M more than each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 co-localized strongly with invadopodia markers at the plasma membrane and with MMP14, the lysosomal marker LAMP-1, and in some cases the autophagosome marker LC3, in F-actin-decorated, large intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and support migration and invasiveness of MDA-MB-231 breast cancer cells in an interdependent manner. Mechanistically, this involves the MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

cancer biology↗

bioRxiv2023

Adaptation to an acid microenvironment promotes pancreatic cancer organoid growth and drug resistance in a p53-dependent manner

The harsh environments in poorly perfused tumor regions have been proposed to select for traits that may drive cancer aggressiveness. Here, we tested the hypothesis that tumor acidosis interacts with driver mutations to exacerbate cancer hallmarks, including drug resistance, in pancreatic cancer. We gradually adapted mouse organoids from normal pancreatic duct (mN) and early PDAC (mP, with KRAS G12V mutation and +/- p53 knockout), from pH 7.4 (physiological level) to 6.7, representing acidic tumor niches. Acid adaptation rewired organoid transcriptional activity, increased viability and, strikingly, increased Gemcitabine- and Erlotinib resistance. Importantly, this response only occurred in organoids expressing wild-type p53 and was most pronounced when acid-adapted cells were returned to physiological pH (mimicking increased perfusion or invasion). While the acid adaptation transcriptional change was overall not highly similar to that induced by drug adaptation of the organoids, acid adaptation induced expression of cytidine deaminase (Cda) and ribonucleotide reductase regulatory subunit M2 (Rrm2), both associated with Gemcitabine resistance, and inhibition of these proteins partially restored Gemcitabine sensitivity. Thus, adaptation to the acidic tumor microenvironment increases drug resistance even after cells leave this niche, and this is in part dependent on acid-adaptation-induced transcriptional upregulation of Cda and Rrm2.

cancer biology↗

bioRxiv2022

The prolactin receptor scaffolds Janus kinase 2 via co-structure formation with phosphoinositide-4,5-bisphosphate

Class 1 cytokine receptors transmit signals through the membrane by a single transmembrane helix to an intrinsically disordered cytoplasmic domain that lacks kinase activity. While specific binding to phosphoinositides has been reported for the prolactin receptor (PRLR), the role of lipids in PRLR signalling is unclear. Using an integrative approach combining NMR spectroscopy, cellular signalling experiments, computational modelling and simulation, we demonstrate co-structure formation of the disordered intracellular domain of the human PRLR, the membrane constituent phosphoinositide-4,5-bisphosphate (PI(4,5)P2) and the FERM-SH2 domain of the Janus kinase 2 (JAK2). We find that the complex leads to accumulation of PI(4,5)P2 at the transmembrane helix interface and that mutation of residues identified to interact specifically with PI(4,5)P2 negatively affects PRLR-mediated activation of signal transducer and activator of transcription 5 (STAT5). Facilitated by co-structure formation, the membrane-proximal disordered region arranges into an extended structure. We suggest that the co-structure formed between PRLR, JAK2 and PI(4,5)P2 locks the juxtamembrane disordered domain of the PRLR in an extended structure, enabling signal relay from the extracellular to the intracellular domain upon ligand binding. We find that the co-structure exists in different states which we speculate could be relevant for turning signalling on and off. Similar co-structures may be relevant for other non-receptor tyrosine kinases and their receptors.

biophysics↗

bioRxiv2022

Dynamic subcellular localization of sodium-bicarbonate cotransporter NBCn1/SLC4A7 to plasma membrane, centrosomes, spindle, and primary cilia

Finely tuned regulation of transport protein localization is vital for epithelial function. Sodium-bicarbonate co-transporter NBCn1 (SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal {beta}-sheet and short C-terminal -helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking the transporter to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of localization of native and GFP-tagged NBCn1, subcellular fractionation, co-IP of NBCn1 with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to the centrosome and primary cilium in non-dividing, polarized epithelial cells, and to spindle, centrosome and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, leading edge, and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression. Summary statementWe unravel molecular determinants of plasma membrane localization of the Na+,HCO3- cotransporter NBCn1 and discover that NBCn1 also localizes to centrosomes, spindle, midbody and primary cilia, likely cycling between these compartments.

cell biology↗

bioRxiv2020

Dynamic NHE1-Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts expressing wildtype and mutant NHE1, we discovered multiple accessible states of this important complex existing in different NHE1:CaM stoichiometries and structures. We solved the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities were tunable by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding -helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation or mutating the first CaM-binding helix, whereas Ca2+-induced NHE1 activity was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results reveal the diversity of NHE1:CaM interactions and suggest that CaM may contribute to NHE1 dimerization. We propose that similar structural diversity is relevant to other CaM complexes.

cell biology↗