Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Joubert syndrome (JBTS), related disorders (JSRD) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and JBTS2 loci are allelic and due to mutations in TMEM216, encoding an uncharacterized tetraspan transmembrane protein. JBTS2 patients displayed frequent nephronophthisis and polydactytly, and two cases conformed to the Oro-Facio-Digital type VI phenotype, whereas skeletal dysplasia was common in MKS fetuses. A single p.R73L mutation was identified in all patients of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in patient fibroblasts or following siRNA knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 complexed with Meckelin, encoded by a gene also mutated in JSRD and MKS. Abrogation of tmem216 expression in zebrafish led to gastrulation defects that overlap with other ciliary morphants. The data implicate a new family of proteins in the ciliopathies, and further support allelism between ciliopathy disorders.

Meckel-Gruber syndrome (MKS) is a severe autosomal recessively inherited disorder caused by mutations in genes that encode components of the primary cilium and basal body. Here we show that two MKS proteins, MKS1 and meckelin, that are required for centrosome migration and ciliogenesis interact with actin-binding isoforms of nesprin-2 (nuclear envelope spectrin repeat protein 2, also known as Syne-2 and NUANCE). Nesprins are important scaffold proteins for maintenance of the actin cytoskeleton, nuclear positioning and nuclear-envelope architecture. However, in ciliated-cell models, meckelin and nesprin-2 isoforms colocalized at filopodia prior to the establishment of cell polarity and ciliogenesis. Loss of nesprin-2 and nesprin-1 shows that both mediate centrosome migration and are then essential for ciliogenesis, but do not otherwise affect apical-basal polarity. Loss of meckelin (by siRNA and in a patient cell-line) caused a dramatic remodelling of the actin cytoskeleton, aberrant localization of nesprin-2 isoforms to actin stress-fibres and activation of RhoA signalling. These findings further highlight the important roles of the nesprins during cellular and developmental processes, particularly in general organelle positioning, and suggest that a mechanistic link between centrosome positioning, cell polarity and the actin cytoskeleton is required for centrosomal migration and is essential for early ciliogenesis.

On the basis of marker profile, the majority of breast carcinomas are thought to be derived from luminal epithelial cells; however, a subgroup of tumours with more mesenchymal characteristics are associated with a worse prognosis. The hypothesis of our study is that some breast carcinomas exhibit myoepithelial rather than pure mesenchymal differentiation and that acquisition of myoepithelial characteristics confers an aggressive phenotype. Pure luminal epithelial cells and fibroblasts are readily distinguished by many markers but distinguishing between myoepithelial and fibroblast cell lineages is more problematic. The markers found to be most discriminating in our study were CK14, ␣6␤4 integrin and the myoepithelial-associated desmosomal cadherin DSg3. These markers were applied to a series of breast cell lines and purified normal breast cell populations and the expression profile related to in vitro invasive behaviour. This demonstrated that expression of one or more myoepithelial markers by tumour cells (MDA MB 231, MDA MB 468, MDA MB 436) was associated with a high invasive capacity compared with cells with a pure luminal phenotype (MCF-7, T47D, ZR75). To address why myoepithelial characteristics are associated with higher invasion, the in vitro behaviour of normal myoepithelial cells and two other nontumourigenic breast cell lines (MCF-10A, HBL100) was also analysed. Primary myoepithelial cells from normal human breast exhibit a high invasive capacity when grown at low density, suggesting that invasive capacity is part of the myoepithelial phenotype. In keeping with this, both nontumourigenic cell lines exhibited features of the myoepithelial phenotype and a high invasive capacity. These results suggest that tumours that exhibit a myoepithelial phenotype may be clinically more aggressive because a high invasive capacity is intrinsic to the myoepithelial phenotype. © 2003 Wiley-Liss, Inc. Key words: myoepithelial cell; invasion; breast cancerThe normal human nonpregnant breast comprises a system of branching ducts, terminating in lobules embedded in interstitial stroma. The glandular structures are composed of 2 distinct epithelial cell types: the inner luminal epithelium and the outer attenuated myoepithelium, which forms the predominant interface with the basement membrane. 1,2 The myoepithelial cells are contractile cells, as indicated by their expression of ␣-smooth muscle actin, but they are likely to have a more central role in controlling the breast microenvironment. This is reflected in their high-level expression of the integrin receptors ␣2␤1, ␣3␤1, ␣6␤4, 3,4 growth factors and their receptors including members of the fibroblast growth factor family 5 and epidermal growth factor receptor 6 and other proteins including the common acute lymphoblastic leukaemia antigen (CALLA), which is identical to the neutral endopeptidase Ϫ24.11. This is involved in the hydrolysis of biologically active peptides and has been implicated in the control of growth and differentiation in the mammary gland. 7,8 In contra...

Primary cilia have a broad tissue distribution and are present on most cell types in the human body. Until recently, they were considered to be redundant organelles, but progress over the past 5 years has led to an understanding of their role in normal mammalian development. The class of inherited disorders that involve aberrant ciliary function are known as ciliopathies, and although their range of severity can vary, they share some common and unexpected clinical phenotypes. The aim of this review is to assess recent insights into the structure, function and formation of primary cilia, and relate this to the pathology, molecular genetics and cell biology of the ciliopathies.

MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel-Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin-filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from Flna(Dilp2) null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin-filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.

Seagrass ecosystems are inherently dynamic, responding to environmental change across a range of scales. Habitat requirements of seagrass are well defined, but less is known about their ability to resist disturbance. Specific means of recovery after loss are particularly difficult to quantify. Here we assess the resistance and recovery capacity of 12 seagrass genera. We document four classic trajectories of degradation and recovery for seagrass ecosystems, illustrated with examples from around the world. Recovery can be rapid once conditions improve, but seagrass absence at landscape scales may persist for many decades, perpetuated by feedbacks and/or lack of seed or plant propagules to initiate recovery. It can be difficult to distinguish between slow recovery, recalcitrant degradation, and the need for a window of opportunity to trigger recovery. We propose a framework synthesizing how the spatial and temporal scales of both disturbance and seagrass response affect ecosystem trajectory and hence resilience.

A feedback between seagrass presence, suspended sediment and benthic light can induce bistability between two ecosystem states: one where the presence of seagrass reduces suspended sediment concentrations to increase benthic light availability thereby favoring growth, and another where seagrass absence increases turbidity thereby reducing growth. This literature review identifies (1) how the environmental and seagrass meadow characteristics influence the strength and direction (stabilizing or destabilizing) of the seagrass-sediment-light feedback, and (2) how this feedback has been incorporated in ecosystem models proposed to support environmental decision making. Large, dense seagrass meadows in shallow subtidal, noneutrophic systems, growing in sediments of mixed grain size and subject to higher velocity flows, have the greatest potential to generate bistability via the seagrass-sediment-light feedback. Conversely, seagrass meadows of low density, area and height can enhance turbulent flows that interact with the seabed, causing water clarity to decline. Using a published field experiment as a case study, we show that the seagrass-sedimentlight feedback can induce bistability only if the suspended sediment has sufficient light attenuation properties. The seagrass-sediment-light feedback has been considered in very few ecosystem models. These models have the potential to identify areas where bistability occurs, which is information that can assist in spatial prioritization of conservation and restoration efforts. In areas where seagrass is present and bistability is predicted, recovery may be difficult once this seagrass is lost. Conversely, bare areas where seagrass presence is predicted (without bistability) may be better targets for seagrass restoration than bare areas where bistability is predicted.