Giant cadherins Fat and Dachsous self-bend to organize properly spaced intercellular junctions.

The cadherins Fat and Dachsous regulate cell polarity and proliferation via their heterophilic interactions at intercellular junctions. Their ectodomains are unusually large because of repetitive extracellular cadherin (EC) domains, which raises the question of how they fit in regular intercellular spaces. Cadherins typically exhibit a linear topology through the binding of Ca(2+) to the linker between the EC domains. Our electron-microscopic observations of mammalian Fat4 and Dachsous1 ectodomains, however, revealed that, although their N-terminal regions exhibit a linear configuration, the C-terminal regions are kinked with multiple hairpin-like bends. Notably, certain EC-EC linkers in Fat4 and Dachsous1 lost Ca(2+)-binding amino acids. When such non-Ca(2+)-binding linkers were substituted for a normal linker in E-cadherin, the mutant E-cadherins deformed more extensively than the wild-type molecule. To simulate cadherin structures with non-Ca(2+)-binding linkers, we used an elastic network model and confirmed that bent configurations can be generated by deformation of non-Ca(2+)-binding linkers. These findings suggest that Fat and Dachsous self-bend due to the loss of Ca(2+)-binding amino acids from specific EC-EC linkers, and can therefore adapt to confined spaces.

Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of noncentrosomal microtubules.

Major microtubules in epithelial cells are not anchored to the centrosome, in contrast to the centrosomal radiation of microtubules in other cell types. It remains to be discovered how these epithelial microtubules are generated and stabilized at noncentrosomal sites. Here, we found that Nezha [also known as calmodulin-regulated spectrin-associated protein 3 (CAMSAP3)] and its related protein, CAMSAP2, cooperate in organization of noncentrosomal microtubules. These two CAMSAP molecules coclustered at the minus ends of noncentrosomal microtubules and thereby stabilized them. Depletion of CAMSAPs caused a marked reduction of microtubules with polymerizing plus ends, concomitantly inducing the growth of microtubules from the centrosome. In CAMSAP-depleted cells, early endosomes and the Golgi apparatus exhibited irregular distributions. These effects of CAMSAP depletion were maximized when both CAMSAPs were removed. These findings suggest that CAMSAP2 and -3 work together to maintain noncentrosomal microtubules, suppressing the microtubule-organizing ability of the centrosome, and that the network of CAMSAP-anchored microtubules is important for proper organelle assembly.

Non-centrosomal microtubules regulate F-actin organization through the suppression of GEF-H1 activity.

Animal cells contain two populations of microtubules: one radiating from the centrosome and the other growing from non-centrosomal sites. Whether or not they have differing roles in cellular architecture and function remains not fully understood. The cytoplasmic protein Nezha (also known as CAMSAP3) stabilizes non-centrosomal microtubules by attaching to their minus ends. Here, we found that depletion of CAMSAP3 in HeLa cells resulted in a relative increase in centrosomal microtubules, and this change was accompanied by accelerated actin stress fiber formation. In these cells, RhoA activity was upregulated, and the soluble fraction of GEF-H1, a RhoGEF whose activity is inhibited by binding to microtubules, increased, explaining why stress fiber formation was promoted. We further found that CAMSAP3 depletion led to an increase in detyrosinated microtubules, and these microtubules did not interact with GEF-H1. These findings suggest that CAMSAP3-anchored non-centrosomal microtubules capture GEF-H1 more efficiently than other microtubules do and that a balance between these microtubules is important to maintain proper actin organization.

Driver Drowsiness Estimation by Parallel Linked Time-Domain CNN with Novel Temporal Measures on Eye States.

This paper presents a vision-based driver drowsiness estimation system from sequences of driver images. We propose a stage-by-stage system instead of an end-to-end system for driver drowsiness estimation. The stage-by-stage system (1) calculates features related to eyes on a frame-by-frame basis, (2) calculates temporal measures on eye states, and (3) estimates drowsiness levels by time-domain convolution with a parallel linked structure. Furthermore, we propose average eye closed time (AECT) and soft percentage of eyelid closure (Soft PERCLOS) as novel temporal measures on eye states to extract information related to driver drowsiness. Extensive experiments have been conducted on a driving movie dataset recorded in a real car. Our system achieves a high accuracy of 95.86% and mean absolute error (MAE) of 0.4007 on the dataset.

Minus end-directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions.

The adherens junction (AJ) plays a crucial role in maintaining cell-cell adhesion in epithelial tissues. Previous studies show that KIFC3, a minus end-directed kinesin motor, moves into AJs via microtubules that grow from clusters of CAMSAP3 (also known as Nezha), a protein that binds microtubule minus ends. The function of junction-associated KIFC3, however, remains to be elucidated. Here we find that KIFC3 binds the ubiquitin-specific protease USP47, a protease that removes ubiquitin chains from substrates and hence inhibits proteasome-mediated proteolysis, and recruits it to AJs. Depletion of KIFC3 or USP47 promotes cleavage of E-cadherin at a juxtamembrane region of the cytoplasmic domain, resulting in the production of a 90-kDa fragment and the internalization of E-cadherin. This cleavage depends on the E3 ubiquitin protein ligase Hakai and is inhibited by proteasome inhibitors. E-cadherin ubiquitination consistently increases after depletion of KIFC3 or USP47. These findings suggest that KIFC3 suppresses the ubiquitination and resultant degradation of E-cadherin by recruiting USP47 to AJs, a process that may be involved in maintaining stable cell-cell adhesion in epithelial sheets.

Temporal and spatial expression profiles of the Fat3 protein, a giant cadherin molecule, during mouse development.

Cadherins constitute a superfamily of cell-cell interaction molecules that participate in morphogenetic processes of animal development. Fat cadherins are the largest members of this superfamily, with 34 extracellular cadherin repeats. Classic Fat, identified in Drosophila, is known to regulate cell proliferation and planar cell polarity. Although 4 subtypes of Fat cadherin, Fat1, Fat2, Fat3, and Fat4/Fat-J, have been identified in vertebrates, their protein localization remains largely unknown. Here we describe the mRNA and protein distributions of Fat3 during mouse development. We found that Fat3 expression was restricted to the nervous system. In the brain, Fat3 was expressed in a variety of regions and axon fascicles. However, its strongest expression was observed in the olfactory bulb and retina. Detailed analysis of Fat3 in the developing olfactory bulb revealed that Fat3 mRNA was mainly expressed by mitral cells and that its proteins were densely localized along the dendrites of these cells as well as in their axons to some extent. Fat3 transcripts in the retina were expressed by amacrine and ganglion cells, and its proteins were concentrated in the inner plexiform layer throughout development. Based on these observations, we suggest that Fat3 plays a role in the interactions between neurites derived from specific subsets of neurons during development.