Search Results (21)

Serrated adenocarcinoma (SAC) represents a molecularly heterogeneous subtype of colorectal carcinoma (CRC) linked to the serrated pathway. It is aimed to clarify the molecular mechanisms underlying SAC development. Digital slides from The Cancer Genome Atlas (TCGA) colorectal adenocarcinoma Firehose Legacy dataset (632 cases) were reviewed, and cases were classified as SAC, partial-SAC, or classical CRC. Genomic alterations, mRNA expression, and DNA hypermethylation were compared using cBioPortal. Enrichment analyses were performed via WebGestalt, and protein–protein interaction (PPI) networks with hub genes were identified using STRING and Cytoscape. Statistical significance was defined as p < 0.05 and q < 0.05. The results revealed that the groups showed significant differences in the expression of 327 genomic alterations, 20 mRNAs, and 21 methylated genes (p < 0.0001, q < 0.0001). Hub genes were PSMC1, FLT3LG, SNW1, H3C2, H1-2, H2BC14, H1-5, RPS16, SUPT5H, and MYOD1. The pathways associated with differently expressed genes were the following: cell structure and morphology (phagocytic vesicle, microvillus, endocytosis, and immobile cilium), protein kinase activity (particularly MAPK), and immunological mechanisms. The hub genes act as molecular bridges connecting the observed genomic and epigenetic variations, particularly driving chromatin-related regulation and MAPK signaling pathways. In particular, PSMC1, SNW1, H3C2, H1-2, and H2BC14 genes offer promising molecular targets for future therapeutic approaches in SACs. Full article

Collagen VI-related myopathies (COL6-RM) encompass a spectrum of disorders characterized by muscle weakness, joint contractures, and connective tissue abnormalities resulting from mutations in the collagen VI genes. While muscle pathology has been extensively studied, tendon dysfunction has emerged as a critical yet underexplored contributor to disease severity, particularly in the development of joint contractures. Tendons from patients and animal models show disrupted collagen fibrillogenesis, altered extracellular matrix (ECM) composition, and impaired cellular mechanotransduction. Various defects in ECM remodeling pathways further exacerbate tendon pathology. Importantly, current clinical management remains limited to orthopedic interventions with modest outcomes, and targeted pharmacological strategies or gene-editing therapies are not yet available for clinical application. Therefore, understanding the basic pathogenic mechanisms underlying tendon dysfunction is essential for identifying novel therapeutic targets. This review provides a comprehensive synthesis of current understanding and recent advances concerning the role of mutated collagen VI in cellular and molecular mechanisms underlying tendon dysfunction. Emphasis is placed on the role of mutated collagen VI in the modulation of key signaling pathways related to mechanotransduction and primary cilium function in COL6-RM. By discussing these multifaceted contributions to disease pathogenesis, this review outlines future research directions in the field and highlights potential pathways for targeted therapeutic interventions. Full article

Background: Reproductive efficiency is a significant hurdle to the sustainability of the beef cattle industry. Method: This study employed transcriptomic profiling to investigate endometrial gene expression differences in heifers with divergent fertility outcomes. Caruncular endometrial samples from fertile (n = 7) and subfertile (n = 5) heifers were subjected to RNA-Seq analysis, yielding 894 differentially expressed genes (DEGs) (p ≤ 0.05 and |log2FC| ≥ 0.5). Results: The MAPK (Mitogen-activated protein kinase) and Rap1 (Ras-associated protein 1) signaling pathways and immune response regulation were identified among the over-represented pathways underlying the DEGs. Transcriptional regulators, such as DUSP2, DUSP10, and MAPK13, were downregulated in subfertile heifers, suggesting disrupted signal transduction and immune function. Gene co-expression network analysis showed network rewiring and increased connectivity of genes related to cilium organization, motility, and microtubule-based processes in the subfertile group. Over-represented hub genes were enriched in the subfertile endometrium, including DNAH2, DNAI2, DNAAF4, CCDC65, and the transcription factor FOXJ1. Our results suggest that impaired ciliary function and disrupted MAPK and immune signaling could potentially contribute to subfertility. Conclusions: This study highlights novel molecular signatures in the uterine endometrium that may serve as predictive markers of fertility potential in beef heifers, providing a foundation for targeted strategies to improve reproductive performance in cattle. Full article

The primary cilium is generally viewed as a sensory organelle that transduces chemical and mechanical stimuli from the environment. In the adult hippocampus, primary cilia mediate the effects of sonic hedgehog (Shh) and other signals on neurogenesis and hippocampal function, and loss of cilia leads to cognitive and behavioral deficits. The secreted peptide noggin is a bone morphogenetic protein (BMP) antagonist and plays a critical role in regulating adult hippocampal neurogenesis (AHN) and hippocampus-dependent behavior. Here, we show that noggin is expressed by mature granule cell neurons, that it is apically targeted and localized intracellularly near the pocket region of primary cilia, and that cilia regulate noggin release through Shh and somatostatin (SST) pathways. Further, granule cell activation modulates noggin dynamics both in vitro and in vivo. Together, these findings demonstrate synergy between Shh and noggin and the positive regulatory action of neuronal activity on regulating BMP antagonism within the neurogenic niche. Thus, the primary cilium is not only an organelle that transduces signals to neurons but also one that mediates extracellular signaling. Significance statement: Primary cilia are organelles that protrude from the surface of most vertebrate cell types. Defects in primary ciliary structure and function are associated with human disease. Primary cilia are generally viewed as exclusively sensory organelles that respond to environmental signals to regulate both cell development and adult cell function. This study demonstrates that the primary cilia in hippocampal granule cell neurons mediate the release of the BMP antagonist, noggin. These observations expand the current understanding of ciliary signaling and may inform future studies exploring the connection between hippocampal activity and cognition in ciliopathies. Full article

A class of retinal dystrophies known as retinitis pigmentosa (RP) is caused by the loss of photoreceptor cells. RP can be genetically transmitted as an autosomal dominant, autosomal recessive, or X-linked trait. About one-third of genes implicated in retinal degeneration encode for proteins whose functional dysregulation affects the “connecting cilium” in photoreceptors, altering its structure and function. Here we report on a 33-year-old woman who was referred for clinical genetic testing following a previous diagnosis of degenerative retinopathy, which was not informative. She was enrolled in a research program dedicated to undiagnosed retinal disorders, where a whole genome sequencing approach was employed to understand the underlying genetic basis. The genomic analysis documented the occurrence of compound heterozygosity for two functionally relevant missense variants in BAIAP3, which encodes a protein with a well-documented role in SNARE-mediated trafficking and ciliogenesis. Confocal microscopy analysis showed elongated cilia in patient-derived and BAIAP3-depleted fibroblasts compared to control cells. Real-time PCR analyses showed a consistent significant reduction of GLI1 mRNA levels in patient-derived and BAIAP3-depleted cells, both in basal conditions and after treatment with Smoothened agonist, SAG, indicating Sonic hedgehog signaling dysregulation. Collectively, these data suggest that biallelic loss-of-function variants of BAIAP3 may cause photoreceptor degeneration and underlie isolated RP. Full article

Multiciliated cells generate fluid flow along epithelial surfaces, and defects in their development or function cause primary ciliary dyskinesia. The fluid flow is generated by the coordinated beating of motile cilia, which are microtubule-based organelles. The base of each cilium, the basal body, is anchored to the apical cell membrane and surrounded by a dense apical cytoskeleton of actin, microtubules, and intermediate filaments. Several cell adhesion proteins play a role in the connection of the basal body to the apical cytoskeleton. Here, we show that the desmosomal protein plakophilin3, a member of the armadillo family of proteins, localizes to the striated rootlet in Xenopus laevis multiciliated cells. Knockdown of plakophilin 3 leads to significant defects in cilia-generated fluid flow and basal body docking. These defects are cell-autonomous and independent of cell intercalation and gross changes in the actin cytoskeleton. These findings suggest a crucial role for PKP3 in basal body apical migration and docking in multiciliated cells, highlighting a novel connection between desmosomal proteins and ciliary function. Full article

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGR^ORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGR^ORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial. Full article

Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1^rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1^rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1^rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1^rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP. Full article

The primary cilium plays critical roles in the homeostasis and development of neurons. Recent studies demonstrate that cilium length is regulated by the metabolic state of cells, as dictated by processes such as glucose flux and O-GlcNAcylation (OGN). The study of cilium length regulation during neuron development, however, has been an area left largely unexplored. This project aims to elucidate the roles of O-GlcNAc in neuronal development through its regulation of the primary cilium. Here, we present findings suggesting that OGN levels negatively regulate cilium length on differentiated cortical neurons derived from human-induced pluripotent stem cells. In neurons, cilium length increased significantly during maturation (after day 35), while OGN levels began to drop. Long-term perturbation of OGN via drugs, which inhibit or promote its cycling, during neuron development also have varying effects. Diminishing OGN levels increases cilium length until day 25, when neural stem cells expand and undergo early neurogenesis, before causing cell cycle exit defects and multinucleation. Elevating OGN levels induces greater primary cilia assembly but ultimately results in the development of premature neurons, which have higher insulin sensitivity. These results indicate that OGN levels and primary cilium length are jointly critical in proper neuron development and function. Understanding the interplays between these two nutrient sensors, O-GlcNAc and the primary cilium, during neuron development is important in paving connections between dysfunctional nutrient-sensing and early neurological disorders. Full article

An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin in a physiological context remains unclear. Here, we take advantage of the observation that ΔC2-α-tubulin accumulates to high levels in cells lacking tubulin tyrosine ligase (TTL) to screen for cytosolic carboxypeptidases (CCPs) that generate ΔC2-α-tubulin. We identify CCP1 as the sole peptidase that produces ΔC2-α-tubulin in TTLΔ HeLa cells. Interestingly, we find that the levels of ΔC2-α-tubulin are only modestly reduced in photoreceptors of ccp1^−/− mice, indicating that other peptidases act synergistically with CCP1 to produce ΔC2-α-tubulin in post-mitotic cells. Moreover, the production of ΔC2-α-tubulin appears to be under tight spatial control in the photoreceptor cilium: ΔC2-α-tubulin persists in the connecting cilium of ccp1^−/− but is depleted in the distal portion of the photoreceptor. This work establishes the groundwork to pinpoint the function of ΔC2-α-tubulin in proliferating and post-mitotic mammalian cells. Full article

Primary cilia are microtube-based organelles that extend from the cell surface and function as biochemical and mechanical extracellular signal sensors. Primary cilia coordinate a series of signaling pathways during development. Cilia dysfunction leads to a pleiotropic group of developmental disorders, termed ciliopathy. Phosphoinositides (PIs), a group of signaling phospholipids, play a crucial role in development and tissue homeostasis by regulating membrane trafficking, cytoskeleton reorganization, and organelle identity. Accumulating evidence implicates the involvement of PI species in ciliary defects and ciliopathies. The abundance and localization of PIs in the cell are tightly regulated by the opposing actions of kinases and phosphatases, some of which are recently discovered in the context of primary cilia. Here, we review several cilium-associated PI kinases and phosphatases, including their localization along cilia, function in regulating the ciliary biology under normal conditions, as well as the connection of their disease-associated mutations with ciliopathies. Full article

Mutations in the extracellular matrix protein eyes shut homolog (EYS) are a common cause of retinitis pigmentosa, a blinding disease characterized by photoreceptor degeneration. EYS binds to matriglycan, a carbohydrate modification on O-mannosyl glycan substitutions of the cell-surface glycoprotein α-dystroglycan. Patients with mutations in enzymes required for the biosynthesis of matriglycan exhibit syndromic retinal atrophy, along with brain malformations and congenital muscular dystrophy. Protein O-mannosyltransferase 2 (POMT2) is an enzyme required for the synthesis of O-mannosyl glycans. To evaluate the roles of O-mannosyl glycans in photoreceptor health, we generated protein O-mannosyltransferase 2 (pomt2) mutant zebrafish by CRISPR. pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to α-dystroglycan. Mutant zebrafish presented with hydrocephalus and hypoplasia of the cerebellum, as well as muscular dystrophy. EYS protein was enriched near photoreceptor connecting cilia in the wild-type, but its presence and proper localization was significantly reduced in mutant animals. The mutant retina exhibited mis-localization of opsins and increased apoptosis in both rod and cone photoreceptors. Immunofluorescence intensity of G protein subunit alpha transducin 2 (GNAT2) antibody (a general cone marker) and 1D4 antibody (a long double cone marker) in mutant retinas did not differ from wild-type retinas at 1-month post fertilization, but was reduced at 6 months post fertilization, indicating significant cone degeneration. These data suggest that POMT2-mediated O-mannosyl glycosylation is required for EYS protein localization to the connecting cilium region and photoreceptor survival. Full article

The primary cilium is a solitary, sensory organelle that extends from the surface of nearly every vertebrate cell, including craniofacial cells. This organelle converts chemical and physical external stimuli into intracellular signaling cascades and mediates several well-known signaling pathways simultaneously. Thus, the primary cilium is considered a cellular signaling nexus and amplifier. Primary cilia dysfunction directly results in a collection of diseases and syndromes that typically affect multiple organ systems, including the face and teeth. Despite this direct connection, primary cilia are largely unexplored in craniofacial research. In this review, I briefly summarize craniofacial abnormalities tied to the primary cilium and examine the existing information on primary cilia in craniofacial development and repair. I close with a discussion on preliminary studies that motivate future areas of exploration that are further supported by studies performed in long bone and kidney cells. Full article

The embryonic mouse fibroblast cell line NIH3T3 is widely used in life science research, including the study of cell cycle control and primary cilia. Fibroblasts are the most important cell type in connective tissue, as they produce components of the extracellular matrix and determine tissue architecture. However, they are very heterogeneous and consist of subtypes specific to their organ of residence, among others. The NIH3T3 cell line was derived from whole mouse embryos that developed to pre-birth and is therefore most likely composed of different fibroblast subtypes. Furthermore, prolonged proliferation may have influenced their cellular composition. A heterogeneous cell population is unsuitable for any sophisticated research project. We found that the proportion of ciliated cells in the total NIH3T3 cell population was highly variable and asked whether this was a consequence of cellular heterogeneity and what molecular signatures were associated with it. We have established sub-cell lines by clonal expansion of single cells and characterized them morphologically and molecularly. Eventually, a myofibroblast-like and a fibroblast-like cell line were generated that differ in ciliation and proliferation. These homogeneous cell lines are valuable for a more detailed study of their molecular signatures, not least to uncover further the molecular pathways that contribute to the formation of the primary cilium. Full article

The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell’s environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders. Full article