Tissue-Scale Mechanical Coupling Reduces Morphogenetic Noise to Ensure Precision during Epithelial Folding.

Morphological constancy is universal in developing systems. It is unclear whether precise morphogenesis stems from faithful mechanical interpretation of gene expression patterns. We investigate the formation of the cephalic furrow, an epithelial fold that is precisely positioned with a linear morphology. Fold initiation is specified by a precise genetic code with single-cell row resolution. This positional code activates and spatially confines lateral myosin contractility to induce folding. However, 20% of initiating cells are mis-specified because of fluctuating myosin intensities at the cellular level. Nevertheless, the furrow remains linearly aligned. We find that lateral myosin is planar polarized, integrating contractile membrane interfaces into supracellular "ribbons." Local reduction of mechanical coupling at the "ribbons" using optogenetics decreases furrow linearity. Furthermore, 3D vertex modeling indicates that polarized, interconnected contractility confers morphological robustness against noise. Thus, tissue-scale mechanical coupling functions as a denoising mechanism to ensure morphogenetic precision despite noisy decoding of positional information.

A homeostatic apical microtubule network shortens cells for epithelial folding via a basal polarity shift.

Epithelial folding is typically driven by localized actomyosin contractility. However, it remains unclear how epithelia deform when myosin levels are low and uniform. In the Drosophila gastrula, dorsal fold formation occurs despite a lack of localized myosin changes, while the fold-initiating cells reduce cell height following basal shifts of polarity via an unknown mechanism. We show that cell shortening depends on an apical microtubule network organized by the CAMSAP protein Patronin. Prior to gastrulation, microtubule forces generated by the minus-end motor dynein scaffold the apical cell cortex into a dome-like shape, while the severing enzyme Katanin facilitates network remodelling to ensure tissue-wide cell size homeostasis. During fold initiation, Patronin redistributes following basal polarity shifts in the initiating cells, apparently weakening the scaffolding forces to allow dome descent. The homeostatic network that ensures size/shape homogeneity is thus repurposed for cell shortening, linking epithelial polarity to folding via a microtubule-based mechanical mechanism.

Nedd4 Deficiency in Vascular Smooth Muscle Promotes Vascular Calcification by Stabilizing pSmad1.

The nonosseous calcification process such as atherosclerosis is one of the major complications in several types of metabolic diseases. In a previous study, we uncovered that aberrant activity of transforming growth factor ß (TGF-ß) signaling pathway could contribute to the vascular smooth muscle cells' (VSMCs) calcification process. Also, we identified NEDD4 E3 ligase as a key suppressor of bone morphogenetic protein (BMP)/Smad pathway via a polyubiquitination-dependent selective degradation of C-terminal phosphorylated Smad1 (pSmad1) activated by TGF-ß. Here, we further validated and confirmed the role of Nedd4 in in vivo vascular calcification progression. First, Nedd4 deletion in SM22α-positive mouse tissues (Nedd4fl/fl ;SM22α-Cre) showed deformed aortic structures with disarranged elastin fibers at 24 weeks after birth. Second, vitamin D-induced aorta vascular calcification rate in Nedd4fl/fl ;SM22α-Cre mice was significantly higher than their wild-type littermates. Nedd4fl/fl ;SM22α-Cre mice showed a development of vascular calcification even at very low-level injection of vitamin D, but this was not exhibited in wild-type littermates. Third, we confirmed that TGF-ß1-induced pSmad1 levels were elevated in Nedd4-deficient primary VSMCs isolated from Nedd4fl/fl ;SM22α-Cre mice. Fourth, we further found that Nedd4fl/fl ;SM22α-Cre mVSMCs gained mesenchymal cell properties toward osteoblast-like differentiation by a stable isotope labeling in cell culture (SILAC)-based proteomics analysis. Finally, epigenetic analysis revealed that methylation levels of human NEDD4 gene promoter were significantly increased in atherosclerosis patients. Collectively, abnormal expression or dysfunction of Nedd4 E3 ligase could be involved in vascular calcification of VSMCs by activating bone-forming signals during atherosclerosis progression. © 2016 American Society for Bone and Mineral Research.

A transport and retention mechanism for the sustained distal localization of Spn-F-IKKε during Drosophila bristle elongation.

There was an error published in Development 142, 2338-2351. Otani et al. reported the genetic interactions between ikkε and spn-F, using the allele ikkε66. This allele was referred to in the Materials and Methods on p. 2349, Fig. 3 on p. 2343 and Table S1. However, they subsequently found that the allele used in the experiments was ikkε1 (also known as ikkε36). This was as a result of misannotation in their laboratory stock list. Both alleles are strong loss-of-function alleles with a missense mutation in the kinase domain and show similar phenotypes (Oshima et al., 2006; Shapiro and Anderson, 2006). Therefore, this error does not affect the conclusions of the paper. The authors apologise to readers for this mistake.

A transport and retention mechanism for the sustained distal localization of Spn-F-IKKε during Drosophila bristle elongation.

Stable localization of the signaling complex is essential for the robust morphogenesis of polarized cells. Cell elongation involves molecular signaling centers that coordinately regulate intracellular transport and cytoskeletal structures. In Drosophila bristle elongation, the protein kinase IKKε is activated at the distal tip of the growing bristle and regulates the shuttling movement of recycling endosomes and cytoskeletal organization. However, how the distal tip localization of IKKε is established and maintained during bristle elongation is unknown. Here, we demonstrate that IKKε distal tip localization is regulated by Spindle-F (Spn-F), which is stably retained at the distal tip and functions as an adaptor linking IKKε to cytoplasmic dynein. We found that Javelin-like (Jvl) is a key regulator of Spn-F retention. In jvl mutant bristles, IKKε and Spn-F initially localize to the distal tip but fail to be retained there. In S2 cells, particles that stain positively for Jvl or Spn-F move in a microtubule-dependent manner, whereas Jvl and Spn-F double-positive particles are immobile, indicating that Jvl and Spn-F are transported separately and, upon forming a complex, immobilize each other. These results suggest that polarized transport and selective retention regulate the distal tip localization of the Spn-F-IKKε complex during bristle cell elongation.

Ubiquitin E3 ligase Nedd4-1 acts as a downstream target of PI3K/PTEN-mTORC1 signaling to promote neurite growth.

Protein ubiquitination is a core regulatory determinant of neural development. Previous studies have indicated that the Nedd4-family E3 ubiquitin ligases Nedd4-1 and Nedd4-2 may ubiquitinate phosphatase and tensin homolog (PTEN) and thereby regulate axonal growth in neurons. Using conditional knockout mice, we show here that Nedd4-1 and Nedd4-2 are indeed required for axonal growth in murine central nervous system neurons. However, in contrast to previously published data, we demonstrate that PTEN is not a substrate of Nedd4-1 and Nedd4-2, and that aberrant PTEN ubiquitination is not involved in the impaired axon growth upon deletion of Nedd4-1 and Nedd4-2. Rather, PTEN limits Nedd4-1 protein levels by modulating the activity of mTORC1, a protein complex that controls protein synthesis and cell growth. Our data demonstrate that Nedd4-family E3 ligases promote axonal growth and branching in the developing mammalian brain, where PTEN is not a relevant substrate. Instead, PTEN controls neurite growth by regulating Nedd4-1 expression.

Development of a mutual-assistance capability training program to safeguard the health of local residents in evacuation shelters after a disaster.

The purpose of this study is to create and evaluate a program to enhance the mutual-assistance capability of community members to safeguard their health during time spent in evacuation shelters after a disaster. In previous research, "Supporting people in need of assistance after a disaster, " the participants' awareness of the need for mutual assistance was low and their relevant knowledge and skills were insufficient. Accordingly, this became a priority in the developed program. Twenty-eight people at six different facilities participated in the program. We collected data using a questionnaire survey and group interview with the participants. After conducting the program, the participants' mean scores of mutual-assistance capability were higher than the mean pre-study scores for 25 out of 26 items. The results of group interview implied that the participants acquired [Realization of issues], and not only shared a [Sense of crisis among participants] but also felt a [Sense of responsibility for mutual assistance in the community]. We considered that our mutual-assistance training program at the time of a disaster is effective for developing mutual assistance for safeguarding health where local residents are unprepared to support those in evacuation shelters requiring assistance after a disaster.

The ubiquitin ligase Nedd4-1 participates in denervation-induced skeletal muscle atrophy in mice.

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein:protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myo(Cre);Nedd4-1(flox/flox)) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.

IKKε regulates cell elongation through recycling endosome shuttling.

IKK-related kinases are key regulators of innate immunity and oncogenesis. While their effects on transcription are well characterized, their cytoplasmic functions remain poorly understood. Drosophila IKK-related kinase, IKKɛ, regulates cytoskeletal organization and cell elongation. Here, we demonstrate that IKKɛ is activated locally at the tip of growing mechanosensory bristles and regulates the rapid shuttling of recycling endosomes, independent of its roles in F-actin organization and caspase signaling. IKKɛ regulates the localization of recycling endosome regulators Rab11 and Dynein and phosphorylates their adaptor molecule, Nuclear fallout (Nuf). Nuf's negative regulation by IKKɛ suggests that local activation of IKKɛ inhibits Dynein on incoming recycling endosomes, converting them for outward transport. Mammalian IKK-related kinases also regulate the recycling endosomes' distribution by phosphorylating the Nuf homolog Rab11-FIP3. Our results establish an evolutionarily conserved function of IKK-related kinases in regulating recycling endosome dynamics and point to a key role of endosome dynamics in cell morphogenesis.

Regulation of Rap2A by the ubiquitin ligase Nedd4-1 controls neurite development.

Nedd4-1 is a "neuronal precursor cell expressed and developmentally downregulated protein" and among the most abundant E3 ubiquitin ligases in mammalian neurons. In analyses of conventional and conditional Nedd4-1-deficient mice, we found that Nedd4-1 plays a critical role in dendrite formation. Nedd4-1, the serine/threonine kinase TNIK, and Rap2A form a complex that controls Nedd4-1-mediated ubiquitination of Rap2A. Ubiquitination by Nedd4-1 inhibits Rap2A function, which reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth. We conclude that a Nedd4-1/Rap2A/TNIK signaling pathway regulates neurite growth and arborization in mammalian neurons.

IKK epsilon regulates F actin assembly and interacts with Drosophila IAP1 in cellular morphogenesis.

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKK epsilon is a member of the IkappaB kinase family, and its developmental role has not been clear. Drosophila IKK epsilon was localized to the ruffling membrane of cultured cells and was required for F actin turnover at the cell margin. In IKK epsilon mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibited aberrantly branched morphology. These phenotypes were sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKK epsilon destabilized F actin-based structures. Expression of a dominant-negative form of IKK epsilon increased the amount of DIAP1. The results suggest that at the physiological level, IKK epsilon acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKK epsilon function involves the negative regulation of the nonapoptotic activity of DIAP1.