Emergence of periodic circumferential actin cables from the anisotropic fusion of actin nanoclusters during tubulogenesis.

The periodic circumferential cytoskeleton supports various tubular tissues. Radial expansion of the tube lumen causes anisotropic tensile stress, which can be exploited as a geometric cue. However, the molecular machinery linking anisotropy to robust circumferential patterning is poorly understood. Here, we aim to reveal the emergent process of circumferential actin cable formation in a Drosophila tracheal tube. During luminal expansion, sporadic actin nanoclusters emerge and exhibit circumferentially biased motion and fusion. RNAi screening reveals the formin family protein, DAAM, as an essential component responding to tissue anisotropy, and non-muscle myosin II as a component required for nanocluster fusion. An agent-based model simulation suggests that crosslinkers play a crucial role in nanocluster formation and cluster-to-cable transition occurs in response to mechanical anisotropy. Altogether, we propose that an actin nanocluster is an organizational unit that responds to stress in the cortical membrane and builds a higher-order cable structure.

Inhibition of negative feedback for persistent epithelial cell-cell junction contraction by p21-activated kinase 3.

Actin-mediated mechanical forces are central drivers of cellular dynamics. They generate protrusive and contractile dynamics, the latter of which are induced in concert with myosin II bundled at the site of contraction. These dynamics emerge concomitantly in tissues and even each cell; thus, the tight regulation of such bidirectional forces is important for proper cellular deformation. Here, we show that contractile dynamics can eventually disturb cell-cell junction contraction in the absence of p21-activated kinase 3 (Pak3). Upon Pak3 depletion, contractility induces the formation of abnormal actin protrusions at the shortening junctions, which causes decrease in E-cadherin levels at the adherens junctions and mislocalization of myosin II at the junctions before they enough shorten, compromising completion of junction shortening. Overexpressing E-cadherin restores myosin II distribution closely placed at the junctions and junction contraction. Our results suggest that contractility both induces and perturbs junction contraction and that the attenuation of such perturbations by Pak3 facilitates persistent junction shortening.

Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum.

Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.

Improved split fluorescent proteins for endogenous protein labeling.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila.

Proper function of the neural network results from the precise connections between axons and dendrites of presynaptic and postsynaptic neurons, respectively. In the Drosophila olfactory system, the dendrites of projection neurons (PNs) stereotypically target one of ∼50 glomeruli in the antennal lobe (AL), the primary olfactory center in the brain, and form synapses with the axons of olfactory receptor neurons (ORNs). Here, we show that Eph and Ephrin, the well-known axon guidance molecules, instruct the dendrodendritic segregation during the discrete olfactory map formation. The Eph receptor tyrosine kinase is highly expressed and localized in the glomeruli related to reproductive behavior in the developing AL. In one of the pheromone-sensing glomeruli (DA1), the Eph cell-autonomously regulates its dendrites to reside in a single glomerulus by interacting with Ephrins expressed in adjacent PN dendrites. Our data demonstrate that the trans interaction between dendritic Eph and Ephrin is essential for the PN dendritic boundary formation in the DA1 olfactory circuit, potentially enabling strict segregation of odor detection between pheromones and the other odors.

Live Cell Imaging of Endogenous mRNA Using RNA-Based Fluorescence "Turn-On" Probe.

Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence "turn-on" probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable, and capable of targeting various mRNAs for live E. coli imaging.

A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

Versatile protein tagging in cells with split fluorescent protein.

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh ß-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

Meigo governs dendrite targeting specificity by modulating ephrin level and N-glycosylation.

Neural circuit assembly requires precise dendrite and axon targeting. We identified an evolutionarily conserved endoplasmic reticulum (ER) protein, Meigo, from a mosaic genetic screen in Drosophila melanogaster. Meigo was cell-autonomously required in olfactory receptor neurons and projection neurons to target their axons and dendrites to the lateral antennal lobe and to refine projection neuron dendrites into individual glomeruli. Loss of Meigo induced an unfolded protein response and reduced the amount of neuronal cell surface proteins, including Ephrin. Ephrin overexpression specifically suppressed the projection neuron dendrite refinement defect present in meigo mutant flies, and ephrin knockdown caused a similar projection neuron dendrite refinement defect. Meigo positively regulated the level of Ephrin N-glycosylation, which was required for its optimal function in vivo. Thus, Meigo, an ER-resident protein, governs neuronal targeting specificity by regulating ER folding capacity and protein N-glycosylation. Furthermore, Ephrin appears to be an important substrate that mediates Meigo's function in refinement of glomerular targeting.

Organelles in developing neurons: essential regulators of neuronal morphogenesis and function.

Eukaryotic cells contain multiple intracellular organelles which are structurally and functionally distinct membrane-delimited compartments. Organelles play vital roles in many cellular events in essentially all eukaryotic cells. Although the canonical roles of organelles are well described by classical in vitro studies, little is known about the specific physiological roles of organelles in neurons, which possess extremely polarized cellular structures and have a massive cellular volume compared with most eukaryotic cells. Studies that make use of recently developed genetic and microscopic techniques are currently elucidating the unexpectedly specialized roles of intracellular, membrane-delimited organelles in neuronal morphogenesis and function, and in human disease. Here we review recent advances in understanding the roles of organelles (the ER-Golgi secretory pathway, endosomes and mitochondria) in developing neurons.

Dermatitis due to epiregulin deficiency and a critical role of epiregulin in immune-related responses of keratinocyte and macrophage.

Epidermal growth factor (EGF) family members, including epiregulin (EP), play a fundamental role in epithelial tissues; however, their roles in immune responses and the physiological role of EP remain to be elucidated. The skin has a versatile system of immune surveillance. Biologically active IL-1alpha is released to extracellular space upon damage from keratinocytes and is a major player in skin inflammation. Here, we show that EP is expressed not only in keratinocytes but also in tissue-resident macrophages, and that EP-deficient (EP(-/-)) mice develop chronic dermatitis. Wound healing in the skin in EP(-/-) mice was not impaired in vivo, nor was the growth rate of keratinocytes from EP(-/-) mice different from that of WT mice in vitro. Of interest is that in WT keratinocytes, both IL-1alpha and the secreted form of EP induced down-regulation of IL-18 mRNA expression, which overexpression in the epidermis was reported to induce skin inflammation in mice, whereas the down-regulation of IL-18 induced by IL-1alpha was impaired in EP(-/-) keratinocytes. Although bone marrow transfer experiments indicated that EP deficiency in non-bone-marrow-derived cells is essential for the development of dermatitis, production of proinflammatory cytokines by EP(-/-) macrophages in response to Toll-like receptor agonists was much lower, compared with WT macrophages, whose dysfunction in EP(-/-) macrophages was not compensated by the addition of the secreted form of EP. These findings, taken together, suggested that EP plays a critical role in immune/inflammatory-related responses of keratinocytes and macrophages at the barrier from the outside milieu and that the secreted and membrane-bound forms of EP have distinct functions.

Variation in estrogenic activity among fractions of a commercial nonylphenol by high performance liquid chromatography.

Estrogenic activity by recombinant yeast screen assay of the commercial NP was considerably higher when compared with that of n-nonylphenol (n-NP). Fractionation of the commercial NP by high performance liquid chromatography (HPLC) afforded seven isomers: 4-(1,3-dimethyl-1-propyl-butyl)-phenol, 4-(1,1,3-trimethyl-hexyl)-phenol, 4-(1,1-dimethyl-3-ethyl-pentyl)-phenol, 4-(1,1,4-trimethyl-hexyl)-phenol, 4-(1-methyl-1-propyl-pentyl)-phenol, 4-(1,1,2-trimethyl-hexyl)-phenol and 4-(1-ethyl-1-methyl-hexyl)-phenol. The structures of these isomers were determined by GC-MS and nuclear magnetic resonance spectroscopy (NMR). All of these isomers possessed tertiary alpha-carbon in their chemical structures. Another tertiary NP, 4-(1,1-dimethyl-heptyl)-phenol was synthesized in the present study and this synthetic NP also exhibited the estrogenic activity. One fractionated compound was identified as one of decylphenol, 4-(1-ethyl-1,4,4-trimethyl-pentyl)-phenol. The isomer, 4-(1,1,4-trimethyl-hexyl)-phenol exhibited the highest estrogenic activity corresponding to 1/10000 that of 17beta-estradiol (E2). The activity of n-NP was the least. This suggests that it may be possible to develop a technical NP mixture with relatively low estrogenic activity.