ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmin(gpg6/gpg6), Atmin(H210Q/H210Q) and Dynll1(GT/GT), revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1(GT/GT) embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathie

Genetic analysis reveals a hierarchy of interactions between polycystin-encoding genes and genes controlling cilia function during left-right determination

During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This ‘nodal flow’ is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo

Microtubule assembly and organisation in normal and cancerous polarised epithelial cells

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Ninein is released from the centrosome and moves bi-directionally along microtubules

Cell-to-cell contact and polarisation of epithelial cells involve a major reorganisation of the microtubules and centrosomal components. The radial microtubule organisation is lost and an apico-basal array develops that is no longer anchored at the centrosome. This involves not only the relocation of microtubules but also of centrosomal anchoring proteins to apical non-centrosomal sites. The relocation of microtubule minus-end-anchoring proteins such as ninein to the apical sites is likely to be essential for the assembly and stabilisation of the apico-basal arrays in polarised epithelial cells. In this study, we establish that ninein is highly dynamic and that, in epithelial cells, it is present not only at the centrosome but also in the cytoplasm as distinct speckles. Live-cell imaging reveals that GFP-ninein speckles are released from the centrosome and move in a microtubule-dependent manner within the cytoplasm and thus establishes that epithelial cells possess the mechanical means for relocation of ninein to non-centrosomal anchoring sites. We also provide evidence for the deployment of ninein speckles to apical anchoring sites during epithelial differentiation in both an in situ tissue and an in vitro culture system. In addition, the findings suggest that the non-centrosomal microtubule anchoring sites associate with adherens junctions in polarised epithelial cells

Cilia and PKD2 Localization and Function.

<p>(A-E) PKD2 localization in nodal cilia of embryos of the indicated genotype. Staining was divided into categories and quantitation is given in (<i>A</i>). In <i>(A)</i>, all genotypes are statistically significantly different from each other (p<0.001) except for <i>Pkd2</i>^{<i>+/lrm4</i>} and <i>Pkd1l1</i>^<i>+/tm1</i> which are statistically not significantly different.</p

The Genetic Relationship between <i>Pkd1l1</i>, <i>Pkd2</i>, and Cilia.

<p>(A-F) <i>Cerl2</i> (<i>A-C</i>) and <i>Nodal</i> (<i>D-F</i>) expression at the node of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and control embryos. Quantitation of <i>in situ</i> signal reveals expression of both genes to be more symmetrical in mutant embryos (<i>C</i>, <i>F</i>). *—p<0.05, unpaired <i>t</i>-test applied. Error bars represent 95% confidence intervals. (G-H) Lung situs <i>(G)</i> (assessed at E13.5) and <i>Pitx2</i> expression <i>(H)</i> (assessed at E8.5) for embryos of the indicated genotypes, with the percentage of embryos exhibiting each phenotype and the total number given.</p

The Relationship Between Nodal Flow and <i>Pkd1l1/Pkd2</i> Function.

<p>(A-C) Nodal flow in embryos of indicated genotypes was examined at the 1–3 somite stages by means of PIV analysis. Flow was normal in <i>Pkd1l1</i>^{<i>tm1/tm1</i>} mutants and wild-type controls but was absent in <i>Dnah11</i>^<i>iv/iv</i> mutants. Black arrowheads denote the direction and speed of flow at that position while the false coloring indicates the direction and magnitude of the flow. Red indicates leftward and blue rightward fluid movements. (D) Lung situs (assessed at E13.5) and <i>Pitx2</i> expression (assessed at E8.5) for embryos of the indicated genotypes, with the percentage of embryos exhibiting each phenotype and the total number given. (E) <i>Pitx2</i> expression for <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and control embryos for each of the 1–7 somite stages. The onset of <i>Pitx2</i> expression is delayed in <i>Dnah11</i>^<i>iv/iv</i> mutants but not in <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryos.</p

Phenotyping of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} Mutants.

<p>(A) Schematic diagram of PKD1L1 and PKD2 showing protein domains and the nature of the <i>Pkd1l1</i>^<i>rks</i> and <i>Pkd2</i>^<i>lrm4</i> point mutations. The double headed red arrow denotes the site of interaction between PKD1L1 and PKD2. PKD—Polycystic Kidney Disease; REJ—Receptor for Egg Jelly; GPS—G-protein Coupled Receptor Proteolytic Site; PLAT—Polycystin-1, Liopoxygenase, Alpha-Toxin. (B) <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and sibling control showing reversed and normal situs, respectively. White arrows indicate stomach position. (C) Heart-stomach discordance (H-S Disc.) in <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and <i>Pkd1l1</i>^{<i>rks/rks</i>} mutants scored at E13.5. Normally, the heart apex and stomach are positioned to the left. H-S Disc. is defined as the heart apex and stomach being on opposite sides. ns—not significant; *—p<0.05; **—p<0.001, Fisher’s Exact Test applied. (D-F) Lung situs assessed at E13.5 for embryos of the indicated genotypes with the ratio of lung lobes between left and right sides given. The percentage and total numbers of embryos showing each phenotype are indicated in <i>(F)</i>. (G-P) Expression patterns of <i>Nodal</i>, <i>Pitx2</i>, and <i>Lefty1/2</i> in embryos at E8.5 of the indicated genotypes, with the percentage number of embryos exhibiting each phenotype and the total number given. Embryos exhibiting bilateral marker expression are further categorized by whether they show equal or biased expression between the left and right sides. The inset in <i>(M)</i> shows a <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryo with bilateral <i>Pitx2</i> expression but with a right-sided bias. Arrowheads in <i>(N)</i> and <i>(O)</i> indicate midline <i>Lefty1</i> expression. <i>t</i> is shorthand for <i>Pkd1l1</i>^<i>tm1</i>. (Q-R) Sonic hedgehog (<i>Shh</i>) expression in the node (n) and notochord (nc) at E8.5.</p

Destabilization of a PKD Domain by the <i>Pkd1l1</i>^<i>rks</i> Mutation.

<p>(A-C) Structure of human PKD1 PKD domain 1 (<i>A</i>) and models of mouse PKD1L1 PKD domain 2; wild-type (<i>B</i>) or <i>rks</i>-mutated (<i>C</i>). Domains are largely composed of β-sheets (block arrows). The aspartic acid mutated in <i>Pkd1l1</i>^<i>rks</i>, or its equivalent in PKD1, is shown in space-fill. The asterisks denote loss of secondary structure in the <i>rks</i>-mutated domain. (D) SRCD spectroscopy of mouse PKD1L1 PKD domain 2 for wild-type and <i>rks</i>-mutated domains. Spectra are consistent with decreased stability (decreased secondary structure) in mutated domains. (E) Thermal denaturation analysis of PKD1L1 PKD domain 2: a reduced melting temperature (Tm) of 56.4°C is evident in the <i>rks</i>-mutated domain; in wild-type controls a Tm of 68.6°C is detected.</p

Multi-repression Model for L-R Asymmetry Determination in Crown Cells.

<p>(A) Schematic of a 3 ss flat-mounted mouse embryo showing somites (yellow), and node (blue). (B) Pictorial representation of the multi-repression model in which flow represses <i>Pkd1l1</i> on the left side, resulting in the derepression of <i>Pkd2</i>, inhibition of <i>Cerl2</i> and, as a result, higher NODAL activity on the left. (C-E) Predictions of the multi-repression model in various genetic mutants including the impact on the crown cell genetic pathway as well as the predicted LPM Nodal cascade activity.</p