Biology subjects

Blacque, O. E.

Publications and source records attributed to Blacque, O. E..

7 recordsLinked to original sources

bioRxiv2025

Glial septins cell non-autonomously regulate a subset of C. elegans sensory cilia via transition zone independent mechanisms

Primary cilia rely on compartmentalisation mechanisms that establish the organelles protein and lipid composition. In mammalian cells, septin GTPases are reported to facilitate cilium formation, function and molecular composition by regulating a membrane diffusion barrier at the ciliary base transition zone (TZ). Here, we examined septins in vivo, within Caenorhabditis elegans sense organs. Unexpectedly, loss of one or both septin genes (unc-59, unc-61) does not cause global defects in cilium structure. Instead, only a subset of ciliated neurons, including the phasmid tail neurons, are affected, with septin mutants displaying short and mispositioned ciliary axonemes due to truncated dendritic processes. Notably, nematode septins do not appear to function at the TZs, with mutants retaining normal gating function and gating complex (MKS & NPHP modules) localisations. Furthermore, double mutant analyses reveal a lack of septin gene interaction with the gating pathways. Strikingly, cell-specific rescue experiments show that UNC-61 regulates phasmid neuronal cilia via a cell non-autonomous mechanism, within supporting glial cells. In addition, UNC-61 localises close to sensory pore adherens cell junctions, and we provide evidence that septin loss disrupts their integrity. Together, our data uncovers an unexpected cell non-autonomous function for glial septins in regulating a subset of sensory neuronal cilia, via a mechanism that may involve cell-cell junctions.

cell biology↗

bioRxiv2024

Two functional forms of the Meckel-Gruber syndrome protein TMEM67 generated by proteolytic cleavage by ADAMTS9 mediate Wnt signaling and ciliogenesis

TMEM67 mutations are the major cause of Meckel-Gruber syndrome. TMEM67 is involved in both ciliary transition zone assembly, and non-canonical Wnt signaling mediated by its extracellular domain. How TMEM67 performs these two separate functions is not known. We identify a novel cleavage motif in the extracellular domain of TMEM67 cleaved by the extracellular matrix metalloproteinase ADAMTS9. This cleavage regulates the abundance of two functional forms: A C-terminal portion which localizes to the ciliary transition zone regulating ciliogenesis, and a non- cleaved form which regulates Wnt signaling. By characterizing three TMEM67 ciliopathy patient variants within the cleavage motif utilizing mammalian cell culture and C. elegans, we show the cleavage motif is essential for cilia structure and function, highlighting its clinical significance. We generated a novel non-cleavable TMEM67 mouse model which develop severe ciliopathies phenocopying Tmem67-/- mice, but in contrast, undergo normal Wnt signaling, substantiating the existence of two functional forms of TMEM67.

cell biology↗

bioRxiv2023

CilioGenics: an integrated method and database for predicting novel ciliary genes

Discovering the entire list of human ciliary genes would help in the diagnosis of cilia-related human disorders known as ciliopathy, but at present the genetic diagnosis of many ciliopathies (over 30%) is far from complete (Bachmann-Gagescu et al., 2015; Knopp et al., 2015; Paff et al., 2018). In a theory, many independent approaches may uncover the whole list of ciliary genes, but 30% of the genes on the ciliary gene list are still ciliary candidate genes (van Dam et al., 2019; Vasquez et al., 2021). All of these cutting-edge techniques, however, have relied on a different single strategy to discover ciliary candidate genes. Because different methodologies demonstrated distinct capabilities with varying quality, categorizing the ciliary candidate genes in the ciliary gene list without further evidence has been difficult. Here, we present a method for predicting ciliary capacity of each human gene that incorporates diverse methodologies (single-cell RNA sequencing, protein-protein interactions (PPIs), comparative genomics, transcription factor (TF)-network analysis, and text mining). By integrating multiple approaches, we reveal previously undiscovered ciliary genes. Our method, CilioGenics, outperforms other approaches that are dependent on a single method. Our top 500 gene list contains 256 new candidate ciliary genes, with 31 experimentally validated. Our work suggests that combining several techniques can give useful evidence for predicting the ciliary capability of all human genes.

bioinformatics↗

bioRxiv2021

Interpreting TMEM67 missense variants of uncertain significance (VUS) in an animal model

PurposeBetter methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of a tractable animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. MethodsCRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function measured if the variants affect mks-3 gene function. Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signaling function. ResultsAssays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile, Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg, Ser961Tyr). ConclusionEfficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

genetics↗

bioRxiv2020

Interpreting the pathogenicity of Joubert Syndrome missense variants in Caenorhabditis elegans

Ciliopathies are inherited disorders caused by cilia defects. Variants in ciliopathy genes are frequently pleiotropic and represent excellent case studies for interrogating genotype-phenotype correlation. We have employed Caenorhabditis elegans and gene editing to characterise two pathogenic biallelic missense variants (P74S, G155S) in B9D2/mksr-2 associated with Joubert Syndrome (JBTS). B9D2 functions within the MKS module at the transition zone (TZ) ciliary subcompartment, and regulates the ciliums molecular composition and signaling function. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm both variant alleles are pathogenic. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that heterozygous worms phenocopy worms homozygous for P74S. This study also reveals a close functional association between the B9 complex and TMEM216/MKS-2. These data establish C. elegans as a paradigm for interpreting JBTS mutations, and provide insight into MKS module organisation.

genetics↗

bioRxiv2019

Rab28 is localised to photoreceptor outer segments, regulates outer segment shedding but is not linked to retinal degeneration in zebrafish

The photoreceptor outer segment is the canonical example of a modified and highly specialised cilium, with an expanded membrane surface area in the form of discs or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localisation of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display a reduction in shed outer segment material in the RPE at 1 month post fertilisation (mpf), but otherwise normal retinal structure and visual function up to 12 mpf. Cone photoreceptor-specific transgenic reporter lines show Rab28 localises almost exclusively to outer segments, independently of nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, Phosphodiesterase 6C and Guanylate Cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy.

cell biology↗

bioRxiv2019

A ciliary BBSome-ARL-6-PDE6D pathway trafficks RAB-28, a negative regulator of extracellular vesicle biogenesis

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that cells shed to influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis and environment release are almost entirely unknown. Here, we show that the ciliary G-protein RAB28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans. We also find that sequential targeting of lipidated RAB28 to periciliary and ciliary membranes is highly dependent on the BBSome and PDE6D, respectively, and that BBSome loss causes excessive and ectopic EV production. Our data indicate that RAB28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane. Our findings also suggest that EVs control sensory organ homeostasis by mediating communication between ciliated neurons and glia, and that defects in ciliary EV biogenesis may contribute to human ciliopathies.

cell biology↗