Emergence of a Bilaterally Symmetric Pattern from Chiral Components in the Planarian Epidermis.

Most animals exhibit mirror-symmetric body plans, yet the molecular constituents from which they are formed are often chiral. In planarian flatworms, centrioles are arranged in a bilaterally symmetric pattern across the ventral epidermis. Here, we found that this pattern is generated by a network of centrioles with prominent chiral asymmetric properties. We identify centriole components required for establishing asymmetric connections between centrioles and balancing their effects to align centrioles along polarity fields. SMED-ODF2, SMED-VFL1, and SMED-VFL3 affect the assembly of centriole appendages that tether cytoskeletal connectors to position the centrioles. We further show that the medio-lateral polarization of centrioles relies on mechanisms that are partly distinct on the left and right sides of the planarian body. Our findings shed light on how bilaterally symmetrical patterns can emerge from chiral cellular organizations.

Dynamic Polarization of the Multiciliated Planarian Epidermis between Body Plan Landmarks.

Polarity is a universal design principle of biological systems that manifests at all organizational scales, yet its coordination across scales remains poorly understood. Here, we make use of the extreme anatomical plasticity of planarian flatworms to probe the interplay between global body plan polarity and local cell polarity. Our quantitative analysis of ciliary rootlet orientation in the epidermis reveals a dynamic polarity field with head and tail as independent determinants of anteroposterior (A/P) polarization and the body margin as determinant of mediolateral (M/L) polarization. Mathematical modeling rationalizes the global polarity field and its response to experimental manipulations as superposition of separate A/P and M/L fields, and we identify the core PCP and Ft/Ds pathways as their molecular mediators. Overall, our study establishes a framework for the alignment of cellular polarity vectors relative to planarian body plan landmarks and establishes the core PCP and Ft/Ds pathways as evolutionarily conserved 2D-polarization module.

Quantitative and Statistical Study of the Dynamics of Clathrin-Dependent and -Independent Endocytosis Reveal a Differential Role of EndophilinA2.

Eukaryotic cells internalize cargos specifically through clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). EndophilinA2 was shown as preferentially implicated in CIE, although initially involved in CME. Here, we investigated the native interplay of endophilinA2 and dynamin2 during CME as compared to CIE. We developed an unbiased integrative approach based on genome engineering, robust tracking methodology, and advanced analytics. We statistically identified CME and CIE subpopulations corresponding to abortive, active, and static endocytic events. Depletion of dynamin2 strongly affected active CME and CIE events, whereas the absence of endophilinA2 impacted only CIE. Accordingly, we demonstrated that endophilinA2 is needed for dynamin2 recruitment during CIE, but not in CME. Despite these differences, endophilinA2 and dynamin2 acted at the latest stage of endocytosis within a similar stoichiometry in both mechanisms. Thus, we propose a conserved function of dynamin2 and endophilinA2 in vesicle scission, but a differential regulation of their recruitment during CME and CIE.

Basal bodies across eukaryotes series: basal bodies in the freshwater planarian Schmidtea mediterranea.

The freshwater planarian Schmidtea mediterranea has recently emerged as a valuable model system to study basal bodies (BBs) and cilia. Planarians are free-living flatworms that use cilia beating at the surface of their ventral epidermis for gliding along substrates. The ventral epidermis is composed of multiciliated cells (MCCs) that are similar to the MCCs in the respiratory airways, the brain ventricles, and the oviducts in vertebrates. In the planarian epidermis, each cell assembles approximately eighty cilia that beat in a coordinate fashion across the tissue. The BBs that nucleate these cilia all assemble de novo during terminal differentiation of MCCs. The genome of the planarian S. mediterranea has been sequenced and efficient methods for targeting gene expression by RNA interference are available. Defects induced by perturbing the expression of BB proteins can be detected simply by analyzing the locomotion of planarians. BBs are present in large numbers and in predictable orientation, which greatly facilitates analyses by immunofluorescence and electron microscopy. The great ease in targeting gene expression and analyzing associated defects allowed to identify a set of proteins required for BB assembly and function in planarian MCCs. Future technological developments, including methods for transgenic expression in planarians and in related species, will achieve turning free-living flatworms into powerful model systems to study MCCs and the associated human pathologies.

Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor.

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.

The planarian Schmidtea mediterranea as a model for studying motile cilia and multiciliated cells.

In the past few years, the freshwater planarian Schmidtea mediterranea has emerged as a powerful model system to study the assembly and function of cilia. S. mediterranea is a free-living flatworm that uses the beating of cilia on its ventral epidermis for locomotion. The ventral epidermis is composed of a single layer of multiciliated cells highly similar to the multiciliated cells that line the airway, the brain ventricles, and the oviducts in humans. The genome of S. mediterranea has been sequenced and efficient methods for targeting gene expression by RNA interference (RNAi) are available. Locomotion defects induced by perturbing the expression of ciliary genes can be often detected by simple visual screening, and more subtle defects can be detected by measuring locomotion speed. Cilia are present in large numbers and are directly accessible, which facilitates analyses by immunofluorescence and electron microscopy. Here we describe a set of methods for maintaining planarians in the lab. These include gene knockout by RNAi, cilia visualization by immunofluorescence, transmission electron microscopy, and live imaging.

Phosphoinositide 3-kinase at the crossroad between endocytosis and signaling of cytokine receptors.

Class I phosphoinositide 3-kinase (PI3K) is a lipid kinase playing key roles in many signaling pathways regulating cell survival and growth. Besides its important role in signal transduction, PI3K is also involved in actin and membrane reorganization such as protrusion, adhesion, phagocytosis and macropinocytosis. Receptor-mediated endocytosis is initiated by plasma membrane reorganization creating buds that then mature to small vesicles. Whereas most of endocytic mechanisms involve actin polymerization, PI3K requirement has not been clearly investigated. Our study identifies class I PI3K as a key player in clathrin-independent endocytosis of the interleukin 2 receptor (IL-2R) in contrast to the clathrin-dependent entry of transferrin (Tf). IL-2R is a cytokine receptor, inducing several signaling cascades such as PI3K, that are essential for the immune response. We have shown previously that IL-2R can be internalized with or without IL-2 and this process requires dynamin, actin and their regulators cortactin, N-WASP, Rac1 and the kinases Pak. Our recent work reveals that PI3K regulates Rac1 during IL-2R uptake in two ways: via its catalytic activity (p110) and via its regulatory factor (p85). Indeed, the catalytic activity of PI3K is required for both constitutive and IL-2 induced uptake of cytokine receptors, in lymphocytes as well as in epithelial cells. Interestingly, Vav2, a Rac1 GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved and recruited during IL-2R uptake. The second action of PI3K is via its regulatory subunit, p85, which binds activated Rac1 and IL-2R; this interaction being enhanced upon IL-2 treatment. Thus, PI3K regulates both the activation of Rac1 and its recruitment during IL-2R endocytosis. Finally, our results identify a link between cytokine receptors signaling and clathrin-independent endocytosis.

The signalling factor PI3K is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors.

Receptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrin-independent endocytic routes exist, but the molecular mechanism of each pathway remains to be uncovered. The present study focuses on a clathrin-independent dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Ras-related C3 botulinum toxin substrate (Rac1) and its targets, the p21-activated kinases (Pak), are specific regulators of this pathway, acting on cortactin and actin polymerization. The present study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI3K) in IL-2R endocytosis. Inhibition of the catalytic activity of PI3K strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved in IL-2R entry. The second action of PI3K is through its regulatory subunit, p85α, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85α mutant missing the Rac1 binding motif leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85α mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85α acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85α, Rac1 and IL-2R that is enhanced by IL-2. In summary, our results indicate a key role of class I PI3K in IL-2R endocytosis that creates a link with IL-2 signalling.