Multiview tiling light sheet microscopy for 3D high-resolution live imaging.

Light-sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

Kinetochore protein Spindly controls microtubule polarity in Drosophila axons.

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with a minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules.

Microtubule-Associated Serine/Threonine (MAST) Kinases in Development and Disease.

Microtubule-Associated Serine/Threonine (MAST) kinases represent an evolutionary conserved branch of the AGC protein kinase superfamily in the kinome. Since the discovery of the founding member, MAST2, in 1993, three additional family members have been identified in mammals and found to be broadly expressed across various tissues, including the brain, heart, lung, liver, intestine and kidney. The study of MAST kinases is highly relevant for unraveling the molecular basis of a wide range of different human diseases, including breast and liver cancer, myeloma, inflammatory bowel disease, cystic fibrosis and various neuronal disorders. Despite several reports on potential substrates and binding partners of MAST kinases, the molecular mechanisms that would explain their involvement in human diseases remain rather obscure. This review will summarize data on the structure, biochemistry and cell and molecular biology of MAST kinases in the context of biomedical research as well as organismal model systems in order to provide a current profile of this field.

Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila.

Fibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. We show here that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. It is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. We show that both movements are important for layer formation and present evidence suggesting that they are controlled by genetically distinct mechanisms.

Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila.

The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. Here, we found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, our data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.

Functions and Mechanisms of Fibroblast Growth Factor (FGF) Signalling in Drosophila melanogaster.

Intercellular signalling via growth factors plays an important role in controlling cell differentiation and cell movements during the development of multicellular animals. Fibroblast Growth Factor (FGF) signalling induces changes in cellular behaviour allowing cells in the embryo to move, to survive, to divide or to differentiate. Several examples argue that FGF signalling is used in multi-step morphogenetic processes to achieve and maintain a transitional state of the cells required for the control of cell fate. In the genetic model Drosophila melanogaster, FGF signalling via the receptor tyrosine kinases Heartless (Htl) and Breathless (Btl) is particularly well studied. These FGF receptors affect gene expression, cell shape and cell-cell interactions during mesoderm layer formation, caudal visceral muscle (CVM) formation, tracheal morphogenesis and glia differentiation. Here, we will address the current knowledge of the biological functions of FGF signalling in the fly on the tissue, at a cellular and molecular level.

Requirement of the Dynein-Adaptor Spindly for Mitotic and Post-Mitotic Functions in Drosophila.

Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila.