MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.

Inherited apicobasal polarity defines the key features of axon-dendrite polarity in a sensory neuron.

Neurons are highly polarized cells with morphologically and functionally distinct dendritic and axonal processes. The molecular mechanisms that establish axon-dendrite polarity in vivo are poorly understood. Here, we describe the initial polarization of posterior deirid (PDE), a ciliated mechanosensory neuron, during development in vivo through 4D live imaging with endogenously tagged proteins. PDE inherits and maintains apicobasal polarity from its epithelial precursor. Its apical domain is directly transformed into the ciliated dendritic tip through apical constriction, which is followed by axonal outgrowth from the opposite basal side of the cell. The apical Par complex and junctional proteins persistently localize at the developing dendritic domain throughout this transition. Consistent with their instructive role in axon-dendrite polarization, conditional depletion of the Par complex and junctional proteins results in robust defects in dendrite and axon formation. During apical constriction, a microtubule-organizing center (MTOC) containing the microtubule nucleator γ-tubulin ring complex (γ-TuRC) forms along the apical junction between PDE and its sister cell in a manner dependent on the Par complex and junctional proteins. This junctional MTOC patterns neuronal microtubule polarity and facilitate the dynein-dependent recruitment of the basal body for ciliogenesis. When non-ciliated neurons are genetically manipulated to obtain ciliated neuronal fate, inherited apicobasal polarity is required for generating ciliated dendritic tips. We propose that inherited apicobasal polarity, together with apical cell-cell interactions drive the morphological and cytoskeletal polarity in early neuronal differentiation.

Centriole-less pericentriolar material serves as a microtubule organizing center at the base of C. elegans sensory cilia.

During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue-specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis, while SPD-2/CEP192 and PCMD-1, which are essential in the one-cell embryo, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans,1-3 we find evidence for "centriole-less PCM" at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules toward the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome.

The centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers, little is known about how the centrosome is inactivated as an MTOC. Analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of partially overlapping territories organized into an inner and outer sphere that are removed from the centrosome at different rates and using different behaviors. We found that phosphatases oppose the addition of PCM by mitotic kinases, ultimately catalyzing the dissolution of inner sphere PCM proteins at the end of mitosis. The nature of the PCM appears to change such that the remaining aging PCM outer sphere is mechanically ruptured by cortical pulling forces, ultimately inactivating MTOC function at the centrosome.

Spindle pole cohesion requires glycosylation-mediated localization of NuMA.

Glycosylation is critical for the regulation of several cellular processes. One glycosylation pathway, the unusual O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) has been shown to be required for proper mitosis, likely through a subset of proteins that are O-GlcNAcylated during metaphase. As lectins bind glycosylated proteins, we asked if specific lectins interact with mitotic O-GlcNAcylated proteins during metaphase to ensure correct cell division. Galectin-3, a small soluble lectin of the Galectin family, is an excellent candidate, as it has been previously described as a transient centrosomal component in interphase and mitotic epithelial cells. In addition, it has recently been shown to associate with basal bodies in motile cilia, where it stabilizes the microtubule-organizing center (MTOC). Using an experimental mouse model of chronic kidney disease and human epithelial cell lines, we investigate the role of Galectin-3 in dividing epithelial cells. Here we find that Galectin-3 is essential for metaphase where it associates with NuMA in an O-GlcNAcylation-dependent manner. We provide evidence that the NuMA-Galectin-3 interaction is important for mitotic spindle cohesion and for stable NuMA localization to the spindle pole, thus revealing that Galectin-3 is a novel contributor to epithelial mitotic progress.

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity.

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.

Basal foot MTOC organizes pillar MTs required for coordination of beating cilia.

Coordination of ciliary beating is essential to ensure mucus clearance in the airway tract. The orientation and synchronization of ciliary motion responds in part to the organization of the underlying cytoskeletal networks. Using electron tomography on mouse trachea, we show that basal bodies are collectively hooked at the cortex by a regular microtubule array composed of 4-5 microtubules. Removal of galectin-3, one of basal-body components, provokes misrecruitment of γ-tubulin, disorganization of this microtubule framework emanating from the basal-foot cap, together with loss of basal-body alignment and cilium orientation, defects in cilium organization and reduced fluid flow in the tracheal lumen. We conclude that galectin-3 plays a crucial role in the maintenance of the microtubule-organizing centre of the cilium and the 'pillar' microtubules, and that this network is instrumental for the coordinated orientation and stabilization of motile cilia.

Mextli is a novel eukaryotic translation initiation factor 4E-binding protein that promotes translation in Drosophila melanogaster.

Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5'-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.