De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

Functional and Molecular Characterization of Voltage-Gated Sodium Channels in Uteri from Nonpregnant Rats

9 páginas, 8 figuras, 2 tablasWe investigated the function and expression of voltage-gated Naþ channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca2þ] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50–60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca2þ]i, which were abolished by treatment with 0.1 lM TTX. Using end-point and real-time quantitative RTPCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.Supported by a grant from Laboratorios del Dr. Esteve (Barcelona, Spain), and fellowships from Laboratorios del Dr. Esteve (to M.S.) and the MRC (to S.W).Peer reviewe

A role for tachykinins in the regulation of human sperm motility

9 páginas, 6 figuras, 2 tablas.BACKGROUND Tachykinins and tachykinin receptors are widely distributed in the male reproductive tract and appear to be involved in reproduction. However, the function and expression of tachykinins and their receptors in human spermatozoa remain poorly studied. We analysed the effects of tachykinins on sperm motility and characterized the population of tachykinin receptors in human spermatozoa. METHODS AND RESULTS Motility analysis was performed following World Health Organization guidelines and we found that substance P (SP), human hemokinin-1 (hHK-1), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-dependent increases in sperm progressive motility. The effects of tachykinins were antagonized by the NK1 receptor-selective antagonist SR 140333, the NK2 receptor-selective antagonist, SR 48968 and, to a lesser extent, also by the NK3 receptor-selective antagonist SR 142801. Immunocytochemistry studies showed expression of the NK1, NK2 and NK3 tachykinin receptor proteins in spermatozoa with different major sites of localization for each receptor. Western blot analysis confirmed the presence of tachykinin receptors in sperm cell homogenates. RT–PCR demonstrated expression of the genes that encode SP/NKA (TAC1), NKB (TAC3) and hHK-1 (TAC4) but not the genes TACR1, TACR2 and TACR3 encoding NK1, NK2 and NK3 receptors, respectively. CONCLUSIONS These results show for the first time that the NK1, NK2 and NK3 tachykinin receptor proteins are present in human spermatozoa. Our findings suggest that tachykinins, probably acting through these three tachykinin receptors, play a role in the regulation of human sperm motility.We are very grateful to Dr Emonds-Alt for generous gifts of SR 140333, SR 48968 and SR 142801. This work was supported by a grant from Ministerio de Educación y Ciencia (BFU2005-04495-C02-01/BFI), Spain. M.S. is the recipient of a fellowship from Laboratorios del Dr Esteve (Barcelona, Spain).Peer reviewe

Molecular diversity of voltage-gated sodium channel α and β subunit mRNAs in human tissues

8 páginas, 4 figuras, 2 tablas.Voltage-gated Na+ channels are composed of one α subunit and one or more auxiliary β subunits. A reverse transcription–polymerase chain reaction assay was used to analyse the expression of the nine known α subunits (Nav1.1–Nav1.9) in 20 different human tissues. The mRNA expression of the currently known β subunits (β1, β2, β3 and β4) was also assessed. The mRNAs of voltage-gated Na+ channel α and β subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na+ channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases.This work was supported by Laboratorios del Dr. Esteve (Barcelona, Spain). M. Seda is the recipient of a fellowship from Laboratorios del Dr. Esteve.Peer reviewe

A role for tachykinins in the regulation of human sperm motility

9 páginas, 6 figuras, 2 tablas.BACKGROUND Tachykinins and tachykinin receptors are widely distributed in the male reproductive tract and appear to be involved in reproduction. However, the function and expression of tachykinins and their receptors in human spermatozoa remain poorly studied. We analysed the effects of tachykinins on sperm motility and characterized the population of tachykinin receptors in human spermatozoa. METHODS AND RESULTS Motility analysis was performed following World Health Organization guidelines and we found that substance P (SP), human hemokinin-1 (hHK-1), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-dependent increases in sperm progressive motility. The effects of tachykinins were antagonized by the NK1 receptor-selective antagonist SR 140333, the NK2 receptor-selective antagonist, SR 48968 and, to a lesser extent, also by the NK3 receptor-selective antagonist SR 142801. Immunocytochemistry studies showed expression of the NK1, NK2 and NK3 tachykinin receptor proteins in spermatozoa with different major sites of localization for each receptor. Western blot analysis confirmed the presence of tachykinin receptors in sperm cell homogenates. RT–PCR demonstrated expression of the genes that encode SP/NKA (TAC1), NKB (TAC3) and hHK-1 (TAC4) but not the genes TACR1, TACR2 and TACR3 encoding NK1, NK2 and NK3 receptors, respectively. CONCLUSIONS These results show for the first time that the NK1, NK2 and NK3 tachykinin receptor proteins are present in human spermatozoa. Our findings suggest that tachykinins, probably acting through these three tachykinin receptors, play a role in the regulation of human sperm motility.We are very grateful to Dr Emonds-Alt for generous gifts of SR 140333, SR 48968 and SR 142801. This work was supported by a grant from Ministerio de Educación y Ciencia (BFU2005-04495-C02-01/BFI), Spain. M.S. is the recipient of a fellowship from Laboratorios del Dr Esteve (Barcelona, Spain).Peer reviewe

Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.</p

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of <i>Bbs1</i>

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate