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Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that can lead to kidney failure. Mutations in the proteins PKD1 and PKD2 are linked to the disease, but the function of these proteins remains unclear, both in physiology and disease. PKD1 has been implicated in the sensing of chemical and mechanical force stimuli, and PKD2 is proposed to be a calcium ion channel. Su et al. show that the transmembrane regions form a PKD1-PKD2 complex assembled in a 1:3 ratio. Their high-resolution cryo–electron microscopy structure confirms that the complex adopts transient receptor potential channel architecture, with some distinctive features. Mapping disease-causing mutations onto the structure suggests that pathogenesis may come from incorrect folding or trafficking of the complex rather than from disruption of channel activity. Science , this issue p. eaat9819 This structure provides a framework for further investigations into a complex involved in polycystic kidney disease. Mutations in two genes, PKD1 and PKD2 , account for most cases of autosomal dominant polycystic kidney disease, one of the most common monogenetic disorders. Here we report the 3.6-angstrom cryo–electron microscopy structure of truncated human PKD1-PKD2 complex assembled in a 1:3 ratio. PKD1 contains a voltage-gated ion channel (VGIC) fold that interacts with PKD2 to form the domain-swapped, yet noncanonical, transient receptor potential (TRP) channel architecture. The S6 helix in PKD1 is broken in the middle, with the extracellular half, S6a, resembling pore helix 1 in a typical TRP channel. Three positively charged, cavity-facing residues on S6b may block cation permeation. In addition to the VGIC, a five–transmembrane helix domain and a cytosolic PLAT domain were resolved in PKD1. The PKD1-PKD2 complex structure establishes a framework for dissecting the function and disease mechanisms of the PKD proteins.

PKD2L1, also termed TRPP3 from the TRPP subfamily (polycystic TRP channels), is involved in the sour sensation and other pH-dependent processes. PKD2L1 is believed to be a nonselective cation channel that can be regulated by voltage, protons, and calcium. Despite its considerable importance, the molecular mechanisms underlying PKD2L1 regulations are largely unknown. Here, we determine the PKD2L1 atomic structure at 3.38 Å resolution by cryo-electron microscopy, whereby side chains of nearly all residues are assigned. Unlike its ortholog PKD2, the pore helix (PH) and transmembrane segment 6 (S6) of PKD2L1, which are involved in upper and lower-gate opening, adopt an open conformation. Structural comparisons of PKD2L1 with a PKD2-based homologous model indicate that the pore domain dilation is coupled to conformational changes of voltage-sensing domains (VSDs) via a series of π–π interactions, suggesting a potential PKD2L1 gating mechanism. Polycystic kidney disease 2-like 1 protein (PKD2L1) is a voltage-dependent calcium-dependent nonselective ion channel involved in sour taste perception and regulation of pH-dependent action potential of spinal cord neurons. Here the authors present the 3.4 Å cryo-EM structure of PKD2L1 in the open state and propose a model for the gating mechanism.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2 , which encode polycystin‐1 (PC1) and polycystin‐2 (PC2), respectively. Earlier work has shown that PC1 and PC2 assemble into a polycystin complex implicated in kidney morphogenesis. PC2 also assembles into homomers of uncertain functional significance. However, little is known about the molecular mechanisms that direct polycystin complex assembly and specify its functions. We have identified a coiled coil in the C‐terminus of PC2 that functions as a homodimerization domain essential for PC1 binding but not for its self‐oligomerization. Dimerization‐defective PC2 mutants were unable to reconstitute PC1/PC2 complexes either at the plasma membrane (PM) or at PM‐endoplasmic reticulum (ER) junctions but could still function as ER Ca 2+ ‐release channels. Expression of dimerization‐defective PC2 mutants in zebrafish resulted in a cystic phenotype but had lesser effects on organ laterality. We conclude that C‐terminal dimerization of PC2 specifies the formation of polycystin complexes but not formation of ER‐localized PC2 channels. Mutations that affect PC2 C‐terminal homo‐ and heteromerization are the likely molecular basis of cyst formation in ADPKD.

Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules. Multiple adeno-associated viruses (AAV) use the same receptor (AAVR), but the binding mode is not clear. Here, the authors determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, identify residues necessary for virus entry and compare the receptor interfaces of different AAV capsids.

Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellings or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliary bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliary bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliary bulb function and structure.

A primary cilium, a sensory organelle present in almost every vertebrate cell, is regularly described in odontoblasts, projecting from the surfaces of the cells. Based on the hypothesis that the primary cilium is crucial both for dentin formation and possibly in tooth pain transmission, we have investigated the expression and localization of the main cilium components and involvement of the OFD1 gene in tooth morphogenesis. Odontoblasts in vitro express tubulin, inversin, rootletin, OFD1, BBS4, BBS6, ALMS1, KIF3A, PC1, and PC2. In vivo, cilia are aligned parallel to the dentin walls, with the top part oriented toward the pulp core. Close relationships between cilium and nerve fibers are evidenced. Calcium channels are concentrated in the vicinity of the basal body. Analysis of these data suggests a putative role of cilia in sensing the microenvironment, probably related to dentin secretion. This hypothesis is enhanced by the huge defects observed on molars from Ofd1 knockout mice, showing undifferentiated dentin-forming cells.

Cilia are traditionally classified as motile or primary. Motile cilia are restricted to specific populations of well-differentiated epithelial cells, including those in the airway, brain ventricles, and oviducts. Primary cilia are nonmotile, solitary structures that are present in many cell types, and often have sensory functions such as in the retina and renal tubules. Primary cilia were also implicated in the regulation of fundamental processes in development. Rare depictions of primary cilia in embryonic airways led us to hypothesize that primary cilia in airway cells are temporally related to motile ciliogenesis. We identified primary cilia in undifferentiated, cultured airway epithelial cells from mice and humans and in developing lungs. The solitary cilia in the airways express proteins considered unique to primary cilia, including polycystin-1 and polycystin-2. A temporal analysis of airway epithelial cell differentiation showed that cells with primary cilia acquire markers of motile ciliogenesis, suggesting that motile ciliated cells originate from primary ciliated cells. Whereas motile ciliogenesis requires Foxj1, primary ciliogenesis does not, and the expression of Foxj1 was associated with a loss of primary cilia, just before the appearance of motile cilia. Primary cilia were not found in well-differentiated airway epithelial cells. However, after injury, they appear in the luminal layer of epithelium and in basal cells. The transient nature of primary cilia, together with the temporal and spatial patterns of expression in the development and repair of airway epithelium, suggests a critical role of primary cilia in determining outcomes during airway epithelial cell differentiation.

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.

The integrin α6β4, a major component of hemidesmosomes (HDs), stabilizes keratinocyte cell adhesion to the epidermal basement membrane through binding to the cytoskeletal linker protein plectin and association with keratin filaments. Disruption of the α6β4-plectin interaction through phosphorylation of the β4 subunit results in a reduction in adhesive strength of keratinocytes to laminin-332 and the dissolution of HDs. Previously, we have demonstrated that phosphorylation of T1736 in the C-terminal end of the β4 cytoplasmic domain disrupts the interaction of β4 with the plakin domain of plectin. Furthermore, we showed that β4-T1736 can be phosphorylated by PKD1 in vitro, and although both PMA and EGF induced T1736 phosphorylation, only PMA was able to activate PKD1. Here, we show that depletion of [Ca2+]i augments PMA- and EGF-induced phosphorylation of β4-T1736 and that this is caused by inhibition of the calcium-sensitive protein phosphatase calcineurin and augmentation of ERK1/2 activation. We also show that in keratinocytes the PMA-stimulated phosphorylation of β4-T1736 primarily is mediated by PKD2 activation downstream of PKCδ. On the other hand, both the EGF-stimulated phosphorylation of T1736 and the EGF-induced dissolution of HDs are dependent on a functional MAPK signaling pathway, and treatment with the RSK inhibitor BI-D1870 prevented EGF-stimulated phosphorylation of β4-T1736. Moreover, phosphorylation of β4-T1736 is enhanced by overexpression of wild-type RSK1, while it is reduced by the expression of kinase-inactive RSK1 or by siRNA-mediated depletion of RSK1. In summary, our data indicate that different stimuli can lead to the phosphorylation of β4-T1736 by either PKD2 or RSK1.

The stereotyped mating behaviour of the Caenorhabditis elegans male is made up of several substeps: response, backing, turning, vulva location, spicule insertion and sperm transfer. The complexity of this behaviour is reflected in the sexually dimorphic anatomy and nervous system. Behavioural functions have been assigned to most of the male-specific sensory neurons by means of cell ablations; for example, the hook sensory neurons HOA and HOB are specifically required for vulva location. We have investigated how sensory perception of the hermaphrodite by the C. elegans male controls mating behaviours. Here we identify a gene, lov-1 (for location of vulva), that is required for two male sensory behaviours: response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine-threonine-rich amino terminus followed by two blocks of homology to human polycystins, products of the autosomal dominant polycystic kidney-disease loci PKD1 and PKD2. LOV-1 is the closest C. elegans homologue of PKD1. lov-1 is expressed in adult males in sensory neurons of the rays, hook and head, which mediate response, vulva location, and potentially chemotaxis to hermaphrodites, respectively. PKD-2, the C. elegans homologue of PKD2, is localized to the same neurons as LOV-1, suggesting that they function in the same pathway.