Real-time study of spatio-temporal dynamics (4D) of physiological activities in alive biological specimens with different FOVs and resolutions simultaneously.

This article reports the development of a microscopy imaging system that gives feasibility for studying spatio-temporal dynamics of physiological activities of alive biological specimens (over entire volume not only for a particular section, i.e., in 4D). The imaging technology facilitates to obtain two image frames of a section of the larger specimen ([Formula: see text]) with different FOVs at different resolutions or magnifications simultaneously in real-time (in addition to recovery of 3D (volume) information). Again, this imaging system addresses the longstanding challenges of housing multiple light sources (6 at the maximum till date) in microscopy (in general) and light sheet fluorescence microscopy (LSFM) (in particular), by using a tuneable pulsed laser source (with an operating wavelength in the range [Formula: see text]-670 nm) in contrast to the conventional CW laser source being adopted for inducing photo-excitation of tagged fluorophores. In the present study, we employ four wavelengths ([Formula: see text] 488 nm, 585 nm, 590 nm, and 594 nm). Our study also demonstrates quantitative characterization of spatio-temporal dynamics (velocity-both amplitude and direction) of organelles (mitochondria) and their mutual correlationships. Mitochondria close to the nucleus (or in clustered cells) are observed to possess a lower degree of freedom in comparison to that at the cellular periphery (or isolated cells). In addition, the study demonstrates real-time observation and recording of the development and growth of all tracheal branches during the entire period ([Formula: see text] min) of embryonic development (Drosophila). The experimental results-with experiments being conducted in various and diversified biological specimens (Drosophila melanogaster, mouse embryo, and HeLa cells)-demonstrate that the study is of great scientific impact both from the aspects of technology and biological sciences.

Live imaging of delamination in Drosophila shows epithelial cell motility and invasiveness are independently regulated.

Delaminating cells undergo complex, precisely regulated changes in cell-cell adhesion, motility, polarity, invasiveness, and other cellular properties. Delamination occurs during development and in pathogenic conditions such as cancer metastasis. We analyzed the requirements for epithelial delamination in Drosophila ovary border cells, which detach from the structured epithelial layer and begin to migrate collectively. We used live imaging to examine cellular dynamics, particularly epithelial cells' acquisition of motility and invasiveness, in delamination-defective mutants during the time period in which delamination occurs in the wild-type ovary. We found that border cells in slow border cells (slbo), a delamination-defective mutant, lacked invasive cellular protrusions but acquired basic cellular motility, while JAK/STAT-inhibited border cells lost both invasiveness and motility. Our results indicate that invasiveness and motility, which are cooperatively required for delamination, are regulated independently. Our reconstruction experiments also showed that motility is not a prerequisite for acquiring invasiveness.

Insulin signalling elicits hunger-induced feeding in Drosophila.

Insulin, a highly conserved peptide hormone, links nutrient availability to metabolism and growth in animals. In fed states insulin levels remain high and in animals that are food deprived insulin signalling drops. Here, we report that in Drosophila, feeding elicited by short periods of starvation is dependent on insulin signalling. The activity of insulin signalling pathway in the abdominal fatbody aids in feeding during short periods of starvation. A feedback regulatory signalling that involves cells that express the Drosophila hunger hormone short-neuropeptide-F (sNPF) and insulin-producing cells sustain the orexigenic function of insulin. Furthermore, the orexigenic phase of insulin activity aids in the efficient management of nutrient stores and survival of flies during starvation.

Effective guidance of collective migration based on differences in cell states.

Directed cell migration is important for normal animal development and physiology. The process can also be subverted by tumor cells to invade other tissues and to metastasize. Some cells, such as leukocytes, migrate individually; other cells migrate together in groups or sheets, called collective cell migration. Guidance of individually migrating cells depends critically on subcellularly localized perception and transduction of signals. For collective cell migration, guidance could result from cells within a group achieving different signaling levels, with directionality then encoded in the collective rather than in individual cells. Here we subject this collective guidance hypothesis to direct tests, using migration of border cells during Drosophila oogenesis as our model system. These cells normally use two receptor tyrosine kinases (RTKs), PDGF/VEGF-related receptor (PVR) and EGFR, to read guidance cues secreted by the oocyte. Elevated but delocalized RTK signaling in one cell of the cluster was achieved by overexpression of PVR in the absence of ligand or by overexpression of fusion receptors unable to detect Drosophila ligands; alternatively, Rac was photoactivated centrally within a single cell. In each case, one cell within the group was in a high signal state, whereas others were in low signal states. The high signal cell directed cluster movement effectively. We conclude that differences in cell signaling states are sufficient to direct collective migration and are likely a substantial contributor to normal guidance. Cell signaling states could manifest as differences in gene expression or metabolite levels and thus differ substantially from factors normally considered when analyzing eukaryotic cell guidance.

Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS).

During myogenesis in Drosophila embryos, a prominent adhesive structure is formed between precursor cells and fusion-competent myoblasts (fcms). Here, we show that Duf/Kirre and its interaction partners Rols7 (found in founder myoblasts and growing myotubes) and Sns (found in fcms) are organized in a ring-structure at the contact points of fcms with precursor cells, while cytoskeletal components like F-actin and Titin are centered in this ring in both cell types. The cytoplasmic protein Blow colocalizes with the actin plugs in fcms after cell adhesion. Furthermore, the requirement of additional as yet unidentified components was demonstrated by using mammalian C2C12 myoblasts. In this study, we propose that the fusion-restricted myogenic-adhesive structure (FuRMAS) is pivotal in linking cell adhesion as well as local F-actin assembly and dynamics to downstream events that ultimately lead to plasma membrane fusion. Moreover, we suggest that the FuRMAS may restrict the area of membrane breakdown.

The adaptor protein X11Lalpha/Dmint1 interacts with the PDZ-binding domain of the cell recognition protein Rst in Drosophila.

The Drosophila cell adhesion molecule Rst plays key roles during the development of the embryonic musculature, spacing of ommatidia in the compound eye and of sensory organs on the antenna, as well as in the neuronal wiring of the optic lobe. In rst(CT) mutants lacking the cytoplasmic domain of the Rst protein, cell sorting and apoptosis in the eye are affected, suggesting a requirement of this domain for Rst function. To identify potential interacting proteins, yeast two-hybrid screens were performed using the cytoplasmic domains of Rst and its paralogue Kirre as baits. Among several putative interactors, two paralogous Drosophila PDZ motif proteins related to X11/Mint were identified. X11/Mint family members in C. elegans (LIN-10) and vertebrates are believed to function as adaptor proteins and to regulate the assembly of multi-subunit complexes at the synapse, thereby linking the vesicle cycle to cell adhesion. Using genetic, cell biological, and biochemical approaches, we show that the interaction of Rst with X11Lalpha is of biological significance. The proteins interact, for example, in the context of cell sorting in the pupal retina.