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Rachel is a generous and loyal pal to her engaged best friend Darcy. But after celebrating her 30th birthday, perpetual good girl Rachel unexpectedly ends up in the arms of Dex, the guy she's had a crush on since law school, and who happens to be Darcy's fiancé. In the frantic weeks leading up to Darcy's wedding, Rachel finds herself caught between her longtime friendship with Darcy and the prospect of losing the love of her life.
DVD
Mutations in TJP2 cause progressive cholestatic liver disease
Richard Thompson, Melissa Sambrotta and colleagues show that biallelic mutations in
TJP2
cause severe cholestatic liver disease. Their findings suggest that loss of TJP2 protein disrupts the structural integrity of tight junctions in liver tissue, resulting in progressive liver damage.
Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that protein-truncating mutations in the tight junction protein 2 gene (
TJP2
) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.
Journal Article
Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please
Primary cilia are microtubule-based organelles that extend from the apical surface of most mammalian cells, forming when the basal body (derived from the mother centriole) docks at the apical cell membrane. They act as universal cellular “antennae” in vertebrates that receive and integrate mechanical and chemical signals from the extracellular environment, serving diverse roles in chemo-, mechano- and photo-sensation that control developmental signaling, cell polarity and cell proliferation. Mutations in ciliary genes cause a major group of inherited developmental disorders called ciliopathies. There are very few preventative treatments or new therapeutic interventions that modify disease progression or the long-term outlook of patients with these conditions. Recent work has identified at least four distinct but interrelated cellular processes that regulate cilia formation and maintenance, comprising the cell cycle, cellular proteostasis, signaling pathways and structural influences of the actin cytoskeleton. The actin cytoskeleton is composed of microfilaments that are formed from filamentous (F) polymers of globular G-actin subunits. Actin filaments are organized into bundles and networks, and are attached to the cell membrane, by diverse cross-linking proteins. During cell migration, actin filament bundles form either radially at the leading edge or as axial stress fibers. Early studies demonstrated that loss-of-function mutations in ciliopathy genes increased stress fiber formation and impaired ciliogenesis whereas pharmacological inhibition of actin polymerization promoted ciliogenesis. These studies suggest that polymerization of the actin cytoskeleton, F-actin branching and the formation of stress fibers all inhibit primary cilium formation, whereas depolymerization or depletion of actin enhance ciliogenesis. Here, we review the mechanistic basis for these effects on ciliogenesis, which comprise several cellular processes acting in concert at different timescales. Actin polymerization is both a physical barrier to both cilia-targeted vesicle transport and to the membrane remodeling required for ciliogenesis. In contrast, actin may cause cilia loss by localizing disassembly factors at the ciliary base, and F-actin branching may itself activate the YAP/TAZ pathway to promote cilia disassembly. The fundamental role of actin polymerization in the control of ciliogenesis may present potential new targets for disease-modifying therapeutic approaches in treating ciliopathies.
Journal Article
Planar Cell Polarity Acts Through Septins to Control Collective Cell Movement and Ciliogenesis
The planar cell polarity (PCP) signaling pathway governs collective cell movements during vertebrate embryogenesis, and certain PCP proteins are also implicated in the assembly of cilia. The septins are cytoskeletal proteins controlling behaviors such as cell division and migration. Here, we identified control of septin localization by the PCP protein Fritz as a crucial control point for both collective cell movement and ciliogenesis in Xenopus embryos. We also linked mutations in human Fritz to Bardet-Biedl and Meckel-Gruber syndromes, a notable link given that other genes mutated in these syndromes also influence collective cell movement and ciliogenesis. These findings shed light on the mechanisms by which fundamental cellular machinery, such as the cytoskeleton, is regulated during embryonic development and human disease.
Journal Article
RNA-Seq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation
Skeletal muscle satellite cells cultured on soft surfaces (12 kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100 kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2k
b
-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of ɣ-interferon, 37 °C). The largest change in overall gene expression occurred at day 1, as cells switched from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of mRNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNA-Seq dataset will be a useful resource for understanding Pax3 expressing cells.
Journal Article
Cleavage of the Meckel-Gruber syndrome protein TMEM67 by ADAMTS9 uncouples Wnt signaling and ciliogenesis
TMEM67
mutations cause Meckel-Gruber syndrome and other related ciliopathies. 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 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 non-cleavable TMEM67 mouse model which develop severe ciliopathies phenocopying
Tmem67
-/-
mice, but in contrast, transduces normal Wnt signaling, substantiating the existence of two functional forms of TMEM67.
How Meckel-Gruber syndrome protein TMEM67 functions in ciliogenesis and Wnt signaling simultaneously is unknown. Here the authors show that TMEM67 is proteolytically cleaved by the matrix metalloproteinase ADAMTS9 generating two functional isoforms.
Journal Article
TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome
The ciliary transition zone (TZ) at the base of cilia is thought to gate entry of proteins into the cilium. The authors characterize a role for TMEM107, a protein mutated in the ciliopathy Joubert syndrome, in organizing a submodule of the TZ.
The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel–Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome
1
(JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral–facial–digital syndrome and JBTS patients. Mechanistic studies in
Caenorhabditis elegans
showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.
Journal Article
Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration
Rui Chen and colleagues identify mutations in
NMNAT1
as a new cause of Leber congenital amaurosis. They further show that all examined individuals with
NMNAT1
mutations have macular colobomas, a condition marked by severe degeneration of the central retina.
Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene
NMNAT1
encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with
NMNAT1
mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (
Wld
s
) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.
Journal Article
Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1
Primary ciliary defects cause a group of developmental conditions known as ciliopathies. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitises cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. We identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalised, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. We demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1).
Journal Article
The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes
Background Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.
Journal Article