Biology subjects

Christensen, S. T.

Publications and source records attributed to Christensen, S. T..

14 recordsLinked to original sources

bioRxiv2026

Primary cilia coordinate c-KIT signaling induced proliferation in alpha cells

Circulating glucagon levels are elevated in patients with diabetes and obesity and contribute to hyperglycemia. The mechanisms underlying hyperglucagonemia remain poorly understood, but expansion of pancreatic -cell mass is thought to play an important role. Primary cilia are sensory organelles that act as signaling hubs, for pathways controlling cell differentiation, proliferation, and function, yet their contribution to -cell biology remains poorly defined. Here we investigated the role of primary cilia in the regulation of -cell proliferation. To this end we generated the first -cell primary cilia proteome identifying 167 cilia-enriched proteins. Among these, we identified and validated the proto-oncogene receptor tyrosine kinase, c-KIT, as a novel ciliary receptor in -cells. We further show that c-KIT signaling depends on intact primary cilia and that stimulation of isolated mouse islets with its endogenous ligand, stem cell factor (SCF), promotes -cell proliferation. In human pancreatic islets KITLG mRNA expression, but not KIT mRNA expression, correlated positively with donor BMI, suggesting that increased ligand availability drives c-KIT signaling in obesity. Together, our findings identify the primary cilium as a signaling platform c-KIT in cells and reveal a ciliary axis that may drive -cell expansion and hyperglucagonemia in obesity and diabetes.

physiology↗

bioRxiv2026

Distribution of the glucagon receptor in periventricular brain barrier interfaces including motile and primary cilia in rat brain

Glucagon is a key metabolic hormone regulating blood glucose and appetite, yet little is known about its actions within the brain. Here, we investigated its receptor (GCGR) localization in periventricular brain barrier interfaces in young rats using immunohistochemical and immunofluorescence approaches. GCGR was enriched in the proximal region of motile ependymal cilia lining the ventricles, as well as in tanycytic primary cilia and cytoplasmic extensions within the hypothalamus. Additional immunostaining was observed in ciliated cells of the subcommissural organ and, more heterogeneously, in choroid plexus epithelium and associated primary cilia, while other circumventricular organs lacked detectable GCGR. These findings identify brain cilia and tanycytes as previously unrecognized sites of glucagon receptor localization and suggest that glucagon signaling at brain barrier interfaces may contribute to integrating peripheral metabolic cues with central homeostatic circuits.

neuroscience↗

bioRxiv2026

Targeted mRNA restoration of ciliary function in DNAI1-related primary ciliary dyskinesia: ex vivo rescue in patient-derived nasal spheroids, A pilot study

RationalePrimary Ciliary Dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, defective mucociliary clearance, and progressive lung disease. Pathogenic variants in the DNAI1 gene are a well-known cause of PCD. Currently, no approved therapies address the underlying genetic defect. RCT1100 is an inhaled mRNA therapy encoding DNAI1 currently under clinical development. This study evaluates the functional effects of RCT1100 using a fast three-dimensional explant spheroid (3DE-S) model consisting of apical-out undifferentiated nasal epithelial cells derived from patients with DNAI1 PCD. Methods3DE-S were generated from nasal brushings of five patients with confirmed biallelic DNAI1 variants. RCT1100 was administered from day 5 directly to culture wells three times weekly for two weeks. Spheroid motility was assessed throughout treatment by quantifying the proportion of moving spheroid rolling and their movement velocity. Following six doses, spheroids were harvested for high-speed video microscopy for assessment of ciliary beat frequency. ResultsEvaluable data were obtained from three of five patient samples; two samples were excluded due to contamination. After six doses of RCT1100, ciliary beat frequency increased from a baseline range of 2.8-3.5 Hz to 6.7-6.8 Hz post-harvesting. Mean spheroid movement velocity increased from 0.11 {micro}m/sec to 3.87 {micro}m/sec following dosing with 10 {micro}g/mL RCT1100, with more than 80% of spheroids exhibiting coordinated rolling motion pattern. ConclusionThe 3DE-S is a robust platform for evaluating targeted therapies. RCT1100 significantly restored ciliary function, supporting its therapeutic potential and highlighting the utility of spheroid-based systems for precision medicine approaches in DNAI1 PCD.

physiology↗

bioRxiv2026

Cell Type Architecture and Positional Gene Gradients in an Adult Animal at Subcellular Resolution

The spatial organization of cell types and gene expression underlies tissue architecture and organismal physiology. However, resolving complete cellular and molecular landscapes within a three-dimensional context remains challenging, particularly in organisms with high cellular heterogeneity and complex geometry. Here, we developed a semi-supervised workflow to reconstruct high-resolution three-dimensional spatial transcriptomic models of the planarian Schmidtea mediterranea at single-cell resolution. Planarians are basal bilaterians capable of regenerating body structures after injury. Our reconstruction maps the distribution of cell types and spatially patterned gene expression across the intact organism and identifies 119 candidate positional control genes (PCGs). These genes are expressed across muscle, neural, and epidermal populations. Functional perturbation of selected candidates, including pitx3, ptpn11, pi4ka, upf3b, and cul1, revealed roles in regeneration following injury. In addition, we identify intestinal cells as prominent components of the microenvironment surrounding adult tgs1 pluripotent stem cells (neoblasts). Together, these results demonstrate the utility of three-dimensional spatial transcriptomic reconstruction for mapping cellular architecture and positional gene regulation in complex adult organisms.

developmental biology↗

bioRxiv2026

4D Single-Cell Spatial Transcriptomics Reveals Dynamic Morphogenetic Gradients and Regenerative Domains in Planarians

Regeneration relies on precise spatiotemporal gene expression and cellular responses to establish tissue identity and body patterning. Using high-resolution Stereo-seq (715 nm) on 353 sections from 16 whole animals at 8 regeneration timepoints, we constructed a 4D spatiotemporal transcriptomic map of planarian regeneration. Our analysis captured 36 refined cell types from 3,508,004 segmented cells, enabling genome-wide transcriptional imputation of gene expression dynamics across body axes at cellular, tissue, and organismal scales. We identified dynamic positional gradients and distinct spatially distributed cell types during regeneration, including an injury-induced Anterior Regenerative Zone (ARZ). The ARZ exhibited enriched positional signals in epidermal, muscle, and neural cells and was regulated by Mediator 8, which is crucial for polarity remodeling and blastema formation. This study provides a comprehensive spatial molecular and cellular map of regenerative processes, highlighting injury-induced spatial domains and key regulatory factors in planarian regeneration. We also provide an interactive web portal, offering a valuable resource for exploring and analyzing regeneration mechanisms in a spatiotemporal context.

developmental biology↗

bioRxiv2026

INPP5E interactome reveals novel connections to growth factor signaling

Primary cilia are sensory cell membrane protrusions whose malfunction causes diseases known as ciliopathies. Joubert syndrome (JBTS) is a rare recessive ciliopathy causing brain malformations and kidney cysts, among other manifestations. A key player in JBTS is INPP5E, a ciliary phosphoinositide lipid phosphatase. Although INPP5E regulates ciliary growth factor signaling, the molecular mechanisms remain poorly understood. Herein, we show that a constitutively active growth factor receptor (PDGFR-D842V) stimulates INPP5E tyrosine phosphorylation, as does the SRC tyrosine kinase. INPP5E tyrosine phosphorylation did not affect its enzyme activity but was associated with stronger binding to SH3GL1, an endocytic regulator, and SNX9, a phosphoinositide-binding protein involved, like INPP5E, in growth factor-induced ciliary ectovesicle release. Our INPP5E interactomic studies identified other growth factor signaling regulators, including among others: SIN1 (the phosphoinositide-binding subunit of the mTORC2 complex), STRAP (a TGF{beta} and PI3K/AKT signaling regulator), GRB2 (a growth factor receptor adaptor) and the JBTS-associated proteins AHI1 and NPHP1. Moreover, INPP5E strongly interacted with the phosphopeptide-binding 14-3-3 proteins, and did so only in presence of serine-85, a phosphorylated INPP5E residue. Lastly, we found that INPP5E differentially regulates two cilium-dependent growth factor signaling pathways in fibroblasts. Thus, while INPP5E downregulated PDGF-induced AKT phosphorylations, it upregulated TGF{beta}-induced SMAD2 and ERK phosphorylations. Altogether, our work provides important clues on the cilia-dependent actions of growth factors, with implications for ciliopathies like JBTS.

biochemistry↗

bioRxiv2024

Intraflagellar transport protein IFT172 contains a C-terminal ubiquitin-binding U-box-like domain involved in ciliary signaling

Intraflagellar transport (IFT) is a fundamental process driving ciliogenesis in most eukaryotic organisms. IFT172, the largest protein of the IFT complex, plays a crucial role in cilium formation and several disease-causing IFT172 variants have been identified in ciliopathy patients. While IFT172 is tethered to the IFT-B complex via its N-terminal domains, the function of its C-terminal domains has remained elusive. Here, we reveal that the C-terminal part of IFT172 interacts with IFT-A complex subunits, providing a molecular basis for the role of IFT172 in bridging IFT-A and IFT-B complexes. We determine the crystal structure of the C-terminal part of IFT172, uncovering a conserved U-box-like domain often found in E3 ubiquitin ligases. This domain exhibits ubiquitin-binding properties and IFT172 undergoes ubiquitin conjugation in vitro, an activity which is reduced in the C1727R patient ciliopathy variant. We use CRISPR-engineered RPE-1 cells to demonstrate that the U-box-like domain is essential for IFT172 protein stability and proper cilium formation. Notably, RPE-1 cells with heterozygous deletion of the U-box domain show altered TGF-{beta} signaling responses, particularly in SMAD2 phosphorylation levels and AKT activation. Our findings suggest that IFT172, beyond its structural role in bridging IFT-A and IFT-B complexes within IFT trains, harbors a conserved U-box-like domain with potential involvement in ciliary ubiquitination processes and signaling, providing new insights into the molecular mechanisms underlying IFT172-related ciliopathies.

biochemistry↗

bioRxiv2024

Depletion of BBSome Subunits Alters Receptor Endocytosis and Promotes EMT via TGF-β Signaling

Background: Ciliopathies are genetic disorders caused by defects in the structure or function of the cilia and their related structures. Bardet-Biedl syndrome (BBS) is a complex ciliopathy with varied symptoms, probably due to altered membrane receptor signalling pathways. Methods: We investigated the role of BBS1 and BBS4 gene deficiencies in retinal epithelial cells using surface membrane proteomics, co-immunoprecipitation, AlphaFold structural modelling, endocytic trafficking colocalization assays, autophagy flux analysis, and epithelial-to-mesenchymal transition (EMT) functional assays including migration and wound healing. Results: Deficiencies in these BBSome components led to shorter cilia and disrupted receptor endocytosis, with accumulation of TGF-{beta}-pathway and EMT-associated proteins on the plasma membrane of BBS1 KO cells. Co-immunoprecipitation and AlphaFold structural modelling indicated that neither BBS1 nor BBS4 forms stable physical complexes with core endocytic components (EEA1, RAB11, RAB7, LAMP2), suggesting an indirect mechanism. BBS1 KO cells showed sustained TGFBR1 recycling, low basal autophagic flux that increases upon TGF-{beta} stimulation, and the most pronounced mesenchymal phenotype. BBS4 KO cells showed reduced receptor recycling, elevated basal autophagic flux that declines upon stimulation, and the strongest canonical TGF-{beta}/SMAD activation with reduced non-canonical ERK1/2 signalling. Conclusions: Increased EMT markers and enhanced migration in BBS1 KO cells highlight the role of BBSome-dependent receptor trafficking as a potential therapeutic target for retinal degeneration in BBS. The divergent autophagic and signalling phenotypes between BBS1 and BBS4 KO reveal subunit-specific contributions to TGF-{beta} pathway dysregulation.

molecular biology↗

bioRxiv2024

TAK1 operates at the primary cilium in non-canonical TGFB/BMP signaling to control heart development

Transforming Growth Factor-Beta-Activated Kinase 1 (TAK1/MAP3K7), along with its upstream regulators TAK1-Binding Protein 2 (TAB2) and the catalytic alpha-subunit of Protein Kinase A (PKA-C/PRKACA), has been identified as a pivotal player in regulation of developmental processes. Haploinsufficiency of TAB2 causes Congenital Heart Disease (CHD) and rare variants in PKA-C and TAK1 cause cardioacrofacial dysplasia (CAFD), and Frontometaphyseal Dysplasia (FMD) and cardiospondylocarpofacial syndrome (CSCFS), respectively, rare multisystem syndromes, where CHD may appear in the clinical spectrum. We hypothesized that TAK1 plays a significant role in heart development and CHD and addressed this by genetic analysis in CHD patient cohorts and experiments in cell and animal models. Exome sequencing data from 1,471 CHD patients with extracardiac anomalies (syndromic CHD, sCHD), 2,405 patients with nonsyndromic CHD (nsCHD) and 45,082 controls showed increased burden of rare TAB2 and TAK1 variants in sCHD, but not in nsCHD. Detailed characterization of tak1-/-and tab2-/- zebrafish mutants revealed cardiac defects (dilated atrium, trabeculation defects, tachycardia and reduced contractility) as well as extracardiac developmental anomalies. RNA sequencing of tak1-/- mutant hearts showed downregulation of genes encoding core cardiac transcription factors, sarcomeric proteins and extracellular matrix proteins. Experiments with cell cultures and analysis of zebrafish larvae and gastruloids indicated that TAK1 via TAB2 and PKA-C is activated at the primary cilium during cardiomyogenesis and that TAK1 activation at this site is enhanced by cardiomyogenic signaling molecules, including ligands of the TGFB/BMP superfamily. Consistent with these findings, CRISPR/Cas9-mediated editing of TAK1 or administration of small molecule inhibitors targeting TAK1 inhibited ciliary signaling and cardiomyocyte differentiation in vitro, while FMD-causing mutations in TAK1 reduced its ciliary localization. In conclusion, our data establishes a central role for TAK1 and its upstream regulators in cardiac development and syndromic CHD, coordinated via the primary cilium.

developmental biology↗

bioRxiv2024

Loss of KIF13B causes time-dependent changes in ciliary polycystin-2 levels and extracellular vesicle release

Dynamic control of ciliary membrane protein content is crucial for the organelles homeostasis and signaling function and involves removal of ciliary components by BBSome-mediated export, endocytic retrieval and/or extracellular vesicle (EV) shedding. We report that KIF13B regulates ciliary protein composition and EV shedding in cultured kidney epithelial cells, with effects that vary over time. In early stages of ciliation Kif13b-/- cells aberrantly accumulate PC2, FLOT1, and HGS within cilia. These cells also produce fewer small EVs through the GW4869-sensitive, nSMase2 pathway, and release large EVs enriched with CCDC198 and the centriole distal appendage protein CCDC92, which also localizes to the ciliary tip. Upon cilia maturation, Kif13b-/- cells accelerate large EV release of numerous ciliary proteins, including PC2, BBSome components, and IFT proteins, which correlates with gradual depletion of CCDC92 and PC2 from the ciliary tip and shaft, respectively. Furthermore, over time, Kif13b-/- cells show an upregulation in the release of small EVs, which differ in composition from wild-type small EVs. Specifically, the mutant small EVs lack several proteins that are enriched in small EVs from BBSome-deficient cells, such as the palmitoyl transferase ZDHHC5, which localizes to cilia, accumulates within cilia of BBSome-deficient cells, and regulates ciliary length and PC2 levels. Collectively, our work suggests that KIF13B acts at the level of centriole distal appendages to limit ciliary protein entrance and promote endocytic retrieval downstream of the BBSome. Furthermore, this study shows for the first time that CCDC198 and ZDHHC5 localize to primary cilia, suggesting they are potential novel ciliopathy candidates.

cell 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↗

bioRxiv2021

Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at the base of primary cilia and suppresses ciliary elongation

The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport, and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to KIF13Bs MAGUK-binding stalk (MBS) domain relieves motor autoinhibition and promotes microtubule plus end-directed cargo transport. Here we characterize Angiomotin isoform 2 (Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live cell imaging analysis indicated that Ap80 is concentrated at the base of primary cilia and recruits PALS1 to this site, but is not itself a cargo of KIF13B. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia while its overexpression caused ciliary shortening. Our results suggest that Ap80 may specifically activate KIF13B cargo binding at the base of primary cilia to regulate ciliary length.

cell biology↗

bioRxiv2020

CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

cell biology↗

bioRxiv2019

RRP7A links primary microcephaly to radial glial cells and dysfunction of ribosomal biogenesis, neurogenesis and ciliary resorption

Introductory paragraphPrimary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability1. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role1. Using homozygosity mapping and whole exome sequencing, we identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, which segregated with MCPH in a consanguineous family with 10 affected individuals. RRP7A is expressed in neural stem cells/radial glial cells of the developing human forebrain, and targeted mutation of Rrp7a leads to defects in both neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in processing of ribosomal RNA, resorption of primary cilia and cell cycle progression. Finally, analysis of zebrafish embryos with loss-of-function mutation in rrp7a confirmed that RRP7A depletion causes reduced brain size, impaired neurogenesis and cell proliferation as well as defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

neuroscience↗