Wide and scalable field-of-view Talbot-grid-based fluorescence microscopy

Here we report a low-cost and simple wide field-of-view (FOV) on-chip fluorescence-imaging platform, termed fluorescence Talbot microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly scalable FOV. The FTM prototype has a resolution of 1.2 μm and an FOV of 3.9  mm×3.5  mm. We demonstrate the imaging capability of FTM on fluorescently labeled breast cancer cells (SK-BR-3) and human embryonic kidney 293 (HEK) cells expressing green fluorescent protein

Extrasynaptic acetylcholine signaling through a muscarinic receptor regulates cell migration

Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans. We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCβ/egl-8, and TRIO/unc-73. This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration

LINKIN, a new transmembrane protein necessary for cell adhesion

In epithelial collective migration, leader and follower cells migrate while maintaining cell-cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG-GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain

Niche-Associated Activation of Rac Promotes the Asymmetric Division of <i>Drosophila</i> Female Germline Stem Cells

Background: Drosophila female germline stem cells (GSCs) reside adjacent to a cellular niche that secretes Bone Morphogenetic Protein (BMP) ligands and anchors the GSCs through adherens junctions. The GSCs divide asymmetrically such that one daughter remains in the niche as a GSC, while the other is born away from the niche and differentiates. However, given that the BMP signal can be diffusible, it remains unclear how a local extracellular asymmetry is sufficient to result in a robust pattern of asymmetric division. Methods and Findings: Here we show that GSCs are polarized with respect to the cellular niche. We first use a modified biosensor to demonstrate that the small GTPase Rac is asymmetrically activated within the GSC at the niche-GSC interface. Experiments using loss-of-function and gain-of-function mutations in Rac indicate that asymmetric Rac activity both localizes the microtubule binding protein Apc2 to orient one GSC centrosome at the niche-GSC interface during interphase and activates the Jun N-terminal kinase pathway to increase the ability of the GSC to respond to BMP ligands. Other processes act in concert with each function of Rac. Specifically, we demonstrate that the GSC cell cycle arrests at prometaphase if centrosomes are misoriented. Conclusions: Thus, the GSCs, an adult stem cell present in a cellular niche, have a niche-associated polarity that couples control of the division plane with increased response to an extracellular maintenance signal. Other processes work in parallel with the Rac-mediated polarity to ensure a robust pattern of asymmetric division. We suggest that all adult stem cells likely employ multiple, independently acting mechanisms to ensure asymmetric division to maintain tissue homeostasis.</p

Functional transcriptomics of a migrating cell in Caenorhabditis elegans

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi–treated L4 larvae. We observed expression of 8,000– 10,000 genes in the linker cell, 22–25% of which were up- or downregulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67–dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans