Biology subjects

Pereira, G.

Publications and source records attributed to Pereira, G..

17 recordsLinked to original sources

bioRxiv2026

Structural cell biology by mega-expansion microscopy

Structural characterization of macromolecular assemblies within intact cells remains one of the central challenges in cell biology. While cryo-electron tomography provides unparalleled structural information, its applicability is limited by sample thickness, imaging throughput, and accessibility. Fluorescence microscopy offers molecular specificity and compatibility with intact biological specimens but has so far lacked the spatial resolution required to visualize cellular ultrastructure. Here we introduce Mega-expansion microscopy (Mega-ExM), a fluorescence imaging approach that enables structural visualization of whole cells using conventional confocal microscopes. Mega-ExM combines iterative hydrogel expansion with whole-proteome NHS-dye labeling and post-expansion immunostaining to achieve tunable expansion factors of up to [~]1,500-fold while preserving ultrastructure. At expansion factors of 40-260x, Mega-ExM resolves centrioles, mitochondrial cristae, protein-dense domains within mitochondrial cristae consistent with respiratory-chain supercomplexes, the synaptonemal complex, and nuclear pore complexes (NPCs) with high fidelity. Particle averaging of [~]200x expanded NPCs yields reconstructions with a structural resolution of [~]35 [A], approaching what cryo-electron tomography has achieved for selected protein assemblies. By combining molecular specificity, large imaging volumes, and nanoscale structural resolution on conventional fluorescence microscopes, Mega-ExM establishes a broadly accessible platform for in situ structural biology.

biophysics↗

bioRxiv2026

Taxonomic filtering accompanies functional expansion during long-term soil restoration

The restoration of species-rich calcareous grasslands is a critical conservation objective, yet the recovery of the invisible below-ground microbiome remains poorly quantified compared to above-ground vegetation. Using a unique 143-year land-use chronosequence on Salisbury Plain, UK, we investigated the trajectory of ecosystem reassembly across arable, regenerating (23 and 67 years), and ancient grasslands. By integrating vegetation surveys with soil physiochemistry, microbial profiling, and shotgun metagenomics, we identified a profound functional decoupling between floral and edaphic recovery. While vegetation diversity recovered relatively rapidly, approaching saturation within 23-67 years, soil properties exhibited persistent legacy effects and slow convergence. Bacterial richness decreased with restoration age, reflecting a transition from disturbance-adapted copiotrophs in arable soils to a specialised, oligotrophic community in ancient sites. This taxonomic contraction was conversely matched by an expansion in functional potential, driven by the emergence of specific taxa (e.g., Microthrixaceae, Aquihabitans sp.) and metabolic pathways associated with complex carbon cycling and stress tolerance. Crucially, the soil ecosystem did not reach equilibrium even after 67 years, characterised by persistent legacy phosphorus and a slow accumulation of soil organic matter. These findings suggest that passive regeneration alone may be insufficient for full soil functional recovery, and that strategies targeting microbial assembly and long-term carbon dynamics warrant further evaluation.

ecology↗

bioRxiv2025

Alternate Splicing Directs PMCA2 to Lysosomes and is Linked to Neurodegeneration

Plasma membrane calcium ATPases (PMCAs) are believed to function exclusively at the plasma membrane where they expel calcium from the cytosol. We have unexpectedly identified a splice variant-dependent localisation of the PMCA isoform PMCA2 to the lysosome, where it forms an evolutionarily conserved complex with NPC1, the lysosomal membrane protein defective in the rare lysosomal storage disease Niemann-Pick disease type C (NPC). This interaction is required for lysosomal Ca2+ homeostasis and implicates PMCA2 as a mediator of Ca2+ uptake into lysosomes. Disruption of the NPC1-PMCA2 complex contributes to the pathophysiology of both Niemann-Pick disease type C and Parkinsons disease, revealing an unrecognised intracellular function for PMCA2 and a shared mechanism linking lysosomal Ca2+ and lipid regulation in neurodegeneration.

cell biology↗

bioRxiv2025

Quantitative high-resolution imaging of mouse nephron formation to study Wnt signaling dynamics

IntroductionNephron formation is initiated when Wnt9b from the ureteric bud acts on nephron progenitor cells in the metanephric mesenchyme. For mouse embryonic kidneys, this process can be studied in real time using ex vivo organ cultures. Previous imaging methods relied on Transwell filters with a long distance between objective and sample as well as low signal-to-noise ratio due to the filter membrane. Moreover, Wnt signaling was previously visualized with the expression reporters TCF/Lef:H2B-GFP. MethodsWe developed an ex vivo culture system based on low-volume media and fibronectin coating for real time imaging. We further made use of endogenously tagged Cherry-Lef1 and Ctnnb1-Venus reporter mouse lines as reporters for Wnt signaling during kidney development. Furthermore, we established an adaptive feedback microscopy pipeline to track the signal with high resolution. ResultsUsing these approaches, we found that Cherry-Lef1 proteins are only expressed in the nascent nephron and derivatives. Expression is graded along distal-proximal nephron axis suggesting that the ureteric bud-derived Wnt9b is controlling its expression. Interestingly, we observed that Cherry-Lef1 gradient shifts from distal to proximal axis during nephron patterning. {beta}-catenin-Venus, on the other hand, first became visible in the ureteric bud, then in the nascent nephron after Lef1. ConclusionsUsing a novel imaging approach we demonstrate a dynamic regulation of Wnt signaling that correlates with nephrogenesis in mouse kidney development. Combined with our two new Wnt signaling reporters, nephron formation can be studied in real-time with high resolution.

developmental biology↗

bioRxiv2025

Commensal to pathogen switch in Streptococcus pneumoniae is governed by a thermosensing master regulator

Opportunistic pathogens switch from a commensal to pathogenic state by sensing and responding to a variety of environmental cues, including temperature fluctuations. Minor temperature oscillations can alert the pathogen to a changing niche ecosystem, necessitating efficient sensing and rapid integration to trigger behavioral change. This is typically achieved through master regulators, which act as umbrella systems dictating pleiotropic phenotypes. Here, we uncover a pivotal role of minor temperature shifts in transition of Streptococcus pneumoniae (SPN) from commensal to virulent lifestyles, mediated via an RNA thermosensing (RNAT) element within the untranslated region of the global regulator CiaR. By positively regulating the expression of the surface adhesin, Phosphorylcholine (PCho), in response to elevated temperature, CiaR potentiates pneumococcal infection. Engineering the RNAT structure to create translation restrictive or permissive versions allowed us to demonstrate how modulation in expression of CiaR could alter pneumococcal invasion capability, influencing infection outcomes. Moreover, intranasal administration of PCho mitigated SPN-induced bacteraemic pneumonia. Since a majority of opportunistic respiratory bacterial pathogens decorate their surface with PCho, this signaling arm could be exploited for anti-infective interventions. Significance statementOur nasopharyngeal area is dominated by opportunistic pathogens, such as Streptococcus pneumoniae (SPN), which typically reside as harmless bystanders. However, in individuals with heightened nasopharyngeal inflammation, resulting from allergy, viral infection or an immature or senescent immune system, these seemingly innocuous microbes, including SPN, switch to virulent lifestyles. We reveal an elegant mechanism for rewiring of SPN virulence genes upon sensing temperature oscillations in the nasopharynx by the master regulator CiaR. Elevated nasopharyngeal temperatures due to pathologic conditions is sensed by thermosensing ciaR mRNA. This primarily promotes SPN surface decoration by PCho which facilitates improved invasion, triggering virulence phenotypes. Our findings point towards adoption of a common mechanism for switch to virulent lifestyles by variety of microbes sharing the specific respiratory niche.

microbiology↗

bioRxiv2025

Separable Dorsal Raphe Dopamine Projections mediate the Facets of Loneliness-like state

Affiliative social connections facilitate well-being and survival in numerous species. Engaging in social interactions requires positive or negative motivational drive, elicited through coordinated activity across neural circuits. However, the identity, interconnectivity, and functional encoding of social information within these circuits remains poorly understood. Here, we focus on downstream projections of dorsal raphe nucleus (DRN) dopamine neurons (DRNDAT), which we previously implicated in social motivation alongside an aversive affective state. We show that three prominent DRNDAT projections - to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP) - play separable roles in behavior, despite substantial collateralization. Photoactivation of the DRNDAT-CeA projection promoted social behavior and photostimulation of the DRNDAT-BNST projection promoted exploratory behavior, while the DRNDAT-BLP projection supported place avoidance, suggesting a negative affective state. Downstream regions showed diverse receptor expression, poising DRNDAT neurons to act through dopamine, neuropeptide, and glutamate transmission. Furthermore, we show ex vivo that the effect of DRNDAT photostimulation on downstream neuron excitability depended on region and baseline cell properties, resulting in excitatory responses in BNST cells and diverse responses in CeA and BLP. Finally, in vivo microendoscopic cellular-resolution recordings in the CeA with DRNDAT photostimulation revealed a correlation between social behavior and neurons excited by social stimuli- suggesting that increased dopamine tone may recruit different CeA neurons to social ensembles. Collectively, these circuit features may facilitate a coordinated, but flexible, response in the presence of social stimuli that can be flexibly guided based on the internal social homeostatic need state of the individual.

neuroscience↗

bioRxiv2025

Wnt/LRP6 signaling imbalance impairs ciliogenesis in human retina epithelial cells

The primary cilium is a microtubule-based organelle essential for various cellular functions, particularly signal transduction. While the role of cilia in regulating signaling pathways has been extensively studied, the impact of signaling pathways on cilia formation remains less well understood. Wnt signals are critical modulators of cell fate. In this study, we investigate how modulating Wnt signaling affects cilia formation in human retinal epithelial cells. Our findings show that enhancement of Wnt/LRP6 signaling before serum starvation impairs ciliogenesis. Cells with high baseline Wnt activity exhibited defective distal appendage formation, failure to remove CP110/CEP97 from mother centrioles, and reduced Rab8-vesicular docking, which are critical events for cilia membrane establishment and axoneme extension. Additionally, these cells displayed reduced autophagic flux, increased mTOR kinase activity, and elevated OFD1 levels at centriolar satellites. Importantly, mTOR inhibition rescued ciliogenesis in cells with elevated Wnt activity, underscoring the interplay between these signaling pathways. Our data also indicate that insufficient Wnt signaling activation disrupts ciliogenesis, emphasizing the need for precisely regulated Wnt levels. SummaryThis study investigates how modulating Wnt signaling affects ciliogenesis in human retinal epithelial cells, revealing that both excessive and insufficient Wnt activity disrupt cilia formation and highlighting the interplay between Wnt and mTOR signaling in this process.

cell biology↗

bioRxiv2024

Spatio-temporal analysis of the innate immune response to cytoplasmic dsDNA using a novel cGAMP biosensor

The cGAS-STING signalling pathway acts as gatekeeper in the innate immune response towards extrinsic and intrinsic sources of cytoplasmic double stranded DNA (dsDNA). At the core of this pathway is the cGAS-dependent production of the intra- and extra-cellular messenger 23-cyclic GMP-AMP (cGAMP), which activates STING and leads to IRF3-dependent expression of cytokines and interferon. Despite its relevance to monitor viral and bacterial infections, cell death, and genome instability, the lack of specific live reporters has precluded spatio-temporal analyses of cGAS-STING signalling. Here, we generate a fluorescent cGAMP biosensor by engineering the functional interaction of activated STING and IRF3 at the Golgi. We show that cells encoding for the cGAMP biosensor react within 45 minutes, and in a time- and concentration-dependent manner to STING agonists and cGAMP. We demonstrate that the cGAMP biosensor is suitable for single cell characterisation of the dynamics of Herpes Simplex Virus 1 infection, mtDNA release upon apoptosis, and other sources of cytoplasmic dsDNA. Intriguingly, we show that STING signalling is not activated by ruptured micronuclei, suggesting that other cytosolic pattern recognition receptors underlie the interferon response upon chromosomal instability. Furthermore, we demonstrate that the biosensor sensitivity is sufficient to report microenvironmental cGAMP, allowing to analyse how STING signalling spreads through neighbouring cells, including upon viral infection. Taken together, we provide a tool to easily monitor the activation cGAS-STING signalling pathway by live cell imaging, plate reader or immunofluorescence analyses; and to capture the spatio-temporal and heterogenous dynamics of the response to cGAMP.

cell biology↗

bioRxiv2023

Elucidating the heterogeneity of immunotherapy response and immune-related toxicities by longitudinal ctDNA and immune cell compartment tracking in lung cancer

PurposeAlthough immunotherapy is the mainstay of therapy for advanced non-small cell lung cancer (NSCLC), robust biomarkers of clinical response are lacking. The heterogeneity of clinical responses together with the limited value of radiographic response assessments to timely and accurately predict therapeutic effect -especially in the setting of stable disease-call for the development of molecularly-informed real-time minimally invasive predictive biomarkers. In addition to capturing tumor regression, liquid biopsies may be informative in evaluating immune-related adverse events (irAEs). Experimental designWe investigated longitudinal changes in circulating tumor DNA (ctDNA) in patients with metastatic NSCLC who received immunotherapy-based regimens. Using ctDNA targeted error-correction sequencing together with matched sequencing of white blood cells and tumor tissue, we tracked serial changes in cell-free tumor load (cfTL) and determined molecular response for each patient. Peripheral T-cell repertoire dynamics were serially assessed and evaluated together with plasma protein expression profiles. ResultsMolecular response, defined as complete clearance of cfTL, was significantly associated with progression-free (log-rank p=0.0003) and overall survival (log-rank p=0.01) and was particularly informative in capturing differential survival outcomes among patients with radiographically stable disease. For patients who developed irAEs, peripheral blood T-cell repertoire reshaping, assessed by significant TCR clonotypic expansions and regressions were noted on-treatment. ConclusionsMolecular responses assist with interpretation of heterogeneous clinical responses especially for patients with stable disease. Our complementary assessment of the tumor and immune compartments by liquid biopsies provides an approach for monitoring of clinical benefit and immune-related toxicities for patients with NSCLC receiving immunotherapy. Statement of translational relevanceLongitudinal dynamic changes in cell-free tumor load and reshaping of the peripheral T-cell repertoire capture clinical outcomes and immune-related toxicities during immunotherapy for patients with non-small cell lung cancer.

genomics↗

bioRxiv2023

Identification of post-transcriptional modifications in nucleic acid sequences using propose-designed molecular beacons

Post-transcriptional RNA modifications (PTxMs) present in small RNA species, specifically circulating extracellular RNAs, were recently identified as clinically relevant readouts, often more indicative of disease severity than the classical "up and down" changes in their copy number alone. While identification of PTxMs requires multiple and complex sample preparation steps, microgram-range amounts of RNA, followed by expensive and protracted bioinformatics analyses, the clinically relevant information is usually a yes/no for a particular genetic variant(s), and an up/down answer for relevant biomarkers. We have previously shown that molecular beacons (MBs) can identify specific nucleic acid sequences with picomolar sensitivity and single nucleotide specificity by exploiting the target-dependent change in their electrophoretic mobility profile. We now present a method for direct identification of miRNAs and isomiRs in cells and extracellular vesicles using gel electrophoresis, without the need for RNA isolation and purification. The detection is based on discreet changes in the hydrodynamic surface profile, the overall size, charge and charge distribution of the MB-target hybrid. Furthermore, using an RNA tertiary structure prediction algorithm (iFoldRNA) and a custom molecular dynamics simulation (DMD), we designed modified MBs specific for m6A-modified nucleotides in target RNA sequences. The sample preparation method coupled to the software package affords the design of specific MBs and sensitive, multiplex-type detection of targets in a wide variety of biofluids and cells, in a simple mix and read approach.

molecular biology↗

bioRxiv2023

Rational Prediction of PROTAC-compatible Protein-Protein Interfaces by Molecular Docking

Proteolysis targeting chimeras (PROTACS) are heterobifunctional ligands that mediate the interaction between a protein target and an E3 ligase, resulting in a ternary complex whose interaction with the ubiquitination machinery leads to target degradation. This technology is emerging as an exciting new avenue for therapeutic development, with several PROTACS currently undergoing clinical trials targeting cancer. Here, we describe a general and computationally efficient methodology combining restraint-based docking, energy-based rescoring, and a filter based on minimal solvent-accessible surface distance to produce PROTAC-compatible PPIs suitable for when there is no a priori known PROTAC ligand. In a benchmark employing a manually curated dataset of 13 ternary complex crystals, we achieved accuracy of 92% when starting from bound structures, and 77% when starting from unbound structures, respectively. Our method only requires that the ligand-bound structures of the monomeric forms of the E3 ligase and target proteins be given to run, making it general, accurate and highly efficient, with the ability to impact early stage PROTAC-based drug design campaigns where no structural information about the ternary complex structure is available.

bioinformatics↗

bioRxiv2021

SIN-like pathway kinases regulate the end of mitosis in the methylotrophic yeast Ogataea polymorpha

The Mitotic exit network (MEN) is a conserved signalling pathway essential for termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Amongst O. polymorpha protein kinases that have some similarity to ScCdc15, only two had no other obvious homologues in S. cerevisiae and these were named OpHCD1 and OpHCD2 for homologue candidate of ScCdc15. A search in other yeast species revealed that OpHcd2 has an armadillo type fold in the C-terminal region as found in SpCdc7 kinases of the fission yeast Schizosaccharomyces pombe, which are homologues of ScCdc15; while OpHcd1 is homologous to SpSid1 kinase, a component of the Septation Initiation Network (SIN) of S. pombe not present in the MEN. Since the deletion of either OpHCD1 or OpHCD2 resulted in lethality under standard growth conditions, conditional mutants were constructed by introducing an ATP analog sensitive mutation. For OpHCD2, we constructed and used new genetic tools for O. polymorpha that combined the Tet promoter and the improved auxin-degron systems. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. These results suggest a SIN-like signalling pathway regulates termination of mitosis in O. polymorpha and that the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

molecular biology↗

bioRxiv2021

Balancing the length of distal tip is key for stability and signalling function of primary cilia

Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia are formed by middle and distal segments, in analogy to sensory cilia of lower eukaryotes. The analysis of middle/distal segmentation indicated that perturbations leading to cilia over-elongation influenced middle or distal segment length with a different impact on cilia behaviour. We identified Septins as novel repressors of distal segment growth. We show that Septins control the localisation of MKS3 and CEP290 required for a functional transition zone, and through this the entrance of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor, into the cilium. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that distal segment over-extension increased cilia excision events and decreased SHH activation. Our data underlies the importance of understanding cilia segmentation for length control and cilia-dependent signalling.

cell biology↗

bioRxiv2021

The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota

The diets of industrialized countries reflect the increasing use of processed foods, often with the introduction of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent1. However, little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other, chemically-distinct dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears to be contingent on the activity of a single bacterium that is a member of an uncultured bacterial genus in the family Ruminococcaceae. We used a combination of enrichment culture, multi-omics, and recombinant enzyme studies to identify and characterize a complete pathway in this uncultured bacterium for the degradation of xanthan gum. Our data reveal that this keystone degrader cleaves the xanthan gum backbone with a novel glycoside hydrolase family 5 (GH5) enzyme before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides species that is capable of consuming oligosaccharide products generated by the keystone Ruminococcaceae or a purified form of the GH5 enzyme. This Bacteroides symbiont is equipped with its own distinct enzymatic pathway to cross-feed on xanthan gum breakdown products, which still harbor the native linkage complexity in xanthan gum, but it cannot directly degrade the high molecular weight polymer. Thus, the introduction of a common food additive into the human diet in the past 50 years has promoted the establishment of a food chain involving at least two members of different phyla of gut bacteria.

microbiology↗

bioRxiv2019

SWR1 Chromatin Remodeling Complex Prevents Mitotic Slippage during Spindle Position Checkpoint Arrest

Faithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well-understood. Here, we performed a genome-wide genetic screen to search for components required for SPOC. We identified the SWR1 chromatin-remodeling complex (SWR1-C) among the several novel factors that are essential for SPOC integrity. Cells lacking SWR1-C were able to activate SPOC upon spindle misorientation but underwent mitotic slippage upon prolonged SPOC arrest. This mitotic slippage required the Cdc14-early anaphase release pathway and other factors including the SAGA histone acetyltransferase complex, proteasome components, the mitotic cyclin-dependent kinase inhibitor Sic1 and the mitogen-activated protein kinase Slt2/Mpk1. Together, our data establish a novel link between chromatin remodeling and robust checkpoint arrest in late anaphase. AUTHORS SUMMARYBefore it physically divides into two, the cell must duplicate its genetic material and separate the duplicated copies to the opposite poles of the cell with the help of the spindle machinery. The direction along which the genetic material is separated has different consequences on cell division, especially when the opposite poles of the cell differ from each other, as is the case of asymmetric cell division. Every cell division in budding yeast is asymmetric. The new (daughter) cell grows on the old (mother) cell and pinches of from this location at the end of the cell division, giving rise to a new and an old cell. The daughter and mother cells differ in size and composition, thus the cell division is asymmetric. In order for the daughter cell to receive one copy of the duplicated genetic material, budding yeast has to separate the copies of its genetic material along the mother to daughter cell direction, which is possible by placing the spindle apparatus along this direction. A surveillance mechanism named the Spindle Position Checkpoint (SPOC) in budding yeast monitors the position of the mitotic spindle and prevents cells from dividing if the spindle fails to align in the mother to daughter direction. The cell can resume cell division only after correcting the position of the spindle followed by inactivation of SPOC. This way SPOC prevents multi-nucleation and enucleation, and hence it is a crucial mechanism to maintain the correct ploidy. It has been known that about five proteins play a role in positively supporting the SPOC. Yet, how SPOC works on a molecular level remains ill understood. In this study, we aimed to find out novel components of SPOC. Through an unbiased genome-wide genetic screen, we successfully identified several new components of the SPOC machinery. Among several other novel proteins identified, we investigated the role of the SWR1 chromatin remodeling complex (SWR1-C) in more detail. We show that the SWR1-C has a function in preventing cells with mis-positioned spindles from resuming cell division when the spindle stays mis-positioned for a prolonged time (mitotic slippage). Our data indicated that SWR1-C is not required to start the immediate SPOC response, rather it is important to keep the prolonged SPOC arrest.

cell biology↗

bioRxiv2018

CRISPR/Cas12a-assisted PCR tagging of mammalian genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

cell biology↗

bioRxiv2017

Analysis of LRRC45 indicates cooperative functions of distal appendages at early steps of ciliogenesis

Cilia perform essential signalling functions during development and tissue homeostasis. Ciliary malfunction causes a variety of diseases, named ciliopathies. The key role that the mother centriole plays in cilia formation can be attributed to appendage proteins that associate exclusively with the mother centriole. The distal appendages form a platform that docks early ciliary vesicles and removes CP110/Cep97 inhibitory complexes from the mother centriole. Here, we analysed the role played by LRRC45 in appendage formation and ciliogenesis. We show that the core appendage proteins Cep83 and SCLT1 recruit LRRC45 to the mother centriole. Once there LRRC45 recruits FBF1. The association of LRRC45 with the basal body of primary and motile cilia in differentiated and stem cells reveals a broad function in ciliogenesis. In contrast to the appendage components Cep164 and Cep123, LRRC45 was neither essential for docking of early ciliary vesicles nor for removal of CP110. Rather, LRRC45 promotes cilia biogenesis in CP110-uncapped centrioles by organising centriolar satellites and promoting the docking of Rab8 GTPase-positive vesicles. We propose that, instead of acting solely as a platform to recruit early vesicles, centriole appendages form discrete scaffolds of cooperating proteins that execute specific functions that promote the initial steps of ciliogenesis.

cell biology↗