LRRC50, a Conserved Ciliary Protein Implicated in Polycystic Kidney Disease

Abstract

Cilia perform essential motile and sensory functions central to many developmental and physiological processes. Disruption of their structure or function can have profound phenotypic consequences, and has been linked to left-right patterning and polycystic kidney disease. In a forward genetic screen for mutations affecting ciliary motility, we isolated zebrafish mutant hu255H. The mutation was found to disrupt an ortholog of the uncharacterized highly conserved human SDS22-like leucine-rich repeat (LRR)-containing protein LRRC50 (16q24.1) and Chlamydomonas Oda7p. Zebrafish lrrc50 is specifically expressed in all ciliated tissues. lrrc50 hu255H mutants develop pronephric cysts with an increased proliferative index, severely reduced brush border, and disorganized pronephric cilia manifesting impaired localized fluid flow consistent with ciliary dysfunction. Electron microscopy analysis revealed ultrastructural irregularities of the dynein arms and misalignments of the outer-doublet microtubules on the ciliary axonemes, suggesting instability of the ciliary architecture in lrrc50 hu255H mutants. The SDS22-like leucine-rich repeats present in Lrrc50 are necessary for proper protein function, since injection of a deletion construct of the first LRR did not rescue the zebrafish mutant phenotype. Subcellular distribution of human LRRC50-EGFP in MDCK and HEK293T cells is diffusely cytoplasmic and concentrated at the mitotic spindle poles and cilium. LRRC50 RNAi knock-down in human proximal tubule HK-2 cells thoroughly recapitulated the zebrafish brush border and cilia phenotype, suggesting conservation of LRRC50 function between both species. In summary, we present the first genetic vertebrate model for lrrc50 function and propose LRRC50 to be a novel candidate gene for human cystic kidney disease, involved in regulation of microtubule-based cilia and actin-based brush border microvilli.

Mutations in zebrafish leucine-rich repeat-containing six-like affect cilia motility and result in pronephric cysts, but have variable effects on left-right patterning

“…The number of -/-embryos with left expression was extrapolated from the total value on the basis that mutants make up ~25% of the clutch. protein Lrrc50 (Kramer-Zucker et al, 2005;Omori and Malicki, 2006;Sullivan-Brown et al, 2007;Zhao and Malicki, 2007;van Rooijen et al, 2008). We first see dilations in the area we refer to as the medial tubules (this report) .…”

The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle

“…lrrc50 codes for a conserved ciliary protein expressed in the ear, kidney and other ciliated tissues. In the lrrc50 mutant, cilia have dynein arm abnormalities and are immotile; kinocilia length and number are reduced in lateral line organs (Sullivan-Brown et al, 2008;van Rooijen et al, 2008). lrrc50 mutants were indistinguishable from siblings in fixed material (Fig.…”

“…Zebrafish lines used were AB, London wild-type (LWT), igu tm79a , igu ts294e Sekimizu et al, 2004;Wolff et al, 2004), kei m219 (Malicki et al, 1996;Whitfield et al, 1996), lrrc50 hu255H (van Rooijen et al, 2008), mib ta52b Itoh et al, 2003), ovl tz288b (Tsujikawa and Malicki, 2004;Huang and Schier, 2009), slo tu44c , Tg(bactin2:Arl13b-GFP) (Borovina et al, 2010) and Tg(pou4f3:mGFP) (Xiao et al, 2005). All mutant embryos were homozygous for the mutant allele; in MZovl tz288b mutants, both maternal and zygotic gene activity was disrupted (Huang and Schier, 2009).…”

“…Impaired embryo movement might, therefore, contribute to the otolith defects in embryos with defective cilia, as these mutants invariably have a curved body axis and reduced movement Tsujikawa and Malicki, 2004;Kramer-Zucker et al, 2005;van Rooijen et al, 2008;Wilkinson et al, 2009;Clément et al, 2011). Moreover, otolith defects were partially rescued when ciliary mutants were raised under constant agitation.…”

“…These organelles are present on the apical surface of most vertebrate cells and are involved in sensory, signalling and mechanical processes (Bisgrove and Yost, 2006;Ishikawa and Marshall, 2011). Every cell of the zebrafish OV epithelium is monociliated (Riley et al, 1997), and many genes involved in ciliogenesis, intraflagellar transport, ciliary movement, stability and function are expressed in the OV (Tsujikawa and Malicki, 2004;Bisgrove et al, 2005;Omori and Malicki, 2006;Pathak et al, 2007;Omori et al, 2008;SullivanBrown et al, 2008;van Rooijen et al, 2008;Yu et al, 2008;Colantonio et al, 2009;Wilkinson et al, 2009;Gao et al, 2010;Glazer et al, 2010;Kang et al, 2010;May-Simera et al, 2010;Becker-Heck et al, 2011;Clément et al, 2011;Pathak et al, 2011;Ravanelli and Klingensmith, 2011;Rothschild et al, 2011;Yu et al, 2011). Sensory hair cells in the ear each bear a specialised cilium known as the kinocilium.…”