In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. Here we describe a transmembrane protein, Anakonda (Aka), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka causes epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organizes cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiates TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain has an unusual tripartite repeat structure that may mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly.

Efficient neuronal conductance requires that axons are insulated by glial cells. For this, glial membranes need to wrap around axons. Invertebrates show a relatively simple extension of glial membranes around the axons, resembling Remak fibers formed by Schwann cells in the mammalian peripheral nervous system. To unravel the molecular pathways underlying differentiation of glial cells that provide axonal wrapping, we are using the genetically amenable Drosophila model. At the end of larval life, the wrapping glia differentiates into very large cells, spanning more than 1 mm of axonal length. The extension around axonal membranes is not influenced by the caliber of the axon or its modality. Using cell typespecific gene knockdown we show that the extension of glial membranes around the axons is regulated by an autocrine activation of the EGF receptor through the neuregulin homolog Vein. This resembles the molecular mechanism employed during cell-autonomous reactivation of glial differentiation after injury in mammals. We further demonstrate that Vein, produced by the wrapping glia, also regulates the formation of septate junctions in the abutting subperineurial glia. Moreover, the wrapping glia indirectly controls the proliferation of the perineurial glia. Thus, the wrapping glia appears center stage to orchestrate the development of the different glial cell layers in a peripheral nerve.

Cell migration is an important feature of glial cells. Here, we used the Drosophila eye disc to decipher the molecular network controlling glial migration. We stimulated glial motility by pan-glial PDGF receptor (PVR) activation and identified several genes acting downstream of PVR. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of Integrin results in ECM softening. Moreover, we show that lox expression is regulated by Integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration.

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.