The Importance of the Position of the Nucleus in Oocyte Development.

Oogenesis is a developmental process leading to the formation of an oocyte, a haploid gamete, which upon fertilisation and sperm entry allows the male and the female pronuclei to fuse and give rise to a zygote. In addition to forming a haploid gamete, oogenesis builds up a store of proteins, mRNAs, and organelles in the oocyte needed for the development of the future embryo. In several species, such as , the polarity axes determinants of the future embryo must be asymmetrically distributed prior to fertilisation.

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts.

Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte.

Microtubules and their associated motors are important players in nucleus positioning. Although nuclear migration in Drosophila oocytes is controlled by microtubules, a precise role for microtubule-associated molecular motors in nuclear migration has yet to be reported. We characterize novel landmarks that allow a precise description of the pre-migratory stages.

GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in .

In cell biology, detection of protein subcellular localizations is often achieved by optical microscopy techniques and more rarely by electron microscopy (EM) despite the greater resolution offered by EM. One of the possible reasons was that protein detection by EM required specific antibodies whereas this need could be circumvented by using fluorescently-tagged proteins in optical microscopy approaches. Recently, the description of a genetically encodable EM tag, the engineered ascorbate peroxidase (APEX), whose activity can be monitored by electron-dense DAB precipitates, has widened the possibilities of specific protein detection in EM.

Nuclear Migration in the Drosophila Oocyte.

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools.

Autoinhibition of Cnn binding to γ-TuRCs prevents ectopic microtubule nucleation and cell division defects.

γ-Tubulin ring complexes (γ-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within γ-TuRC-tethering proteins, including Drosophila Centrosomin (Cnn). Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown.

Microtubules originate asymmetrically at the somatic golgi and are guided via Kinesin2 to maintain polarity within neurons.

Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. We show that γ-tubulin localises asymmetrically to the somatic Golgi within neurons.

Regulation of cortical stability by RhoGEF3 in mitotic Sensory Organ Precursor cells in .

In epithelia, mitotic cells round up and push against their neighbors to divide. Mitotic rounding results from increased assembly of F-actin and cortical recruitment of Myosin II, leading to increased cortical stability. Whether this process is developmentally regulated is not well known.

Nucleus positioning within Drosophila egg chamber.

Both types of Drosophila egg chamber germ cells, i.e. oocyte and nurse cells, have to control their nucleus positions in order to produce a viable gamete.

Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte.

Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-mediated force analysis reveal that nuclear positioning is ensured with an unexpected level of robustness.

Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC).

Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme.

The diverse functions of Notch signalling imply that it must elicit context-specific programmes of gene expression. With the aim of investigating how Notch drives cells to differentiate, we have used a genome-wide approach to identify direct Notch targets in Drosophila haemocytes (blood cells), where Notch promotes crystal cell differentiation. Many of the identified Notch-regulated enhancers contain Runx and GATA motifs, and we demonstrate that binding of the Runx protein Lozenge (Lz) is required for enhancers to be competent to respond to Notch.

Notch targets and their regulation.

The proteolytic cleavages elicited by activation of the Notch receptor release an intracellular fragment, Notch intracellular domain, which enters the nucleus to activate the transcription of targets. Changes in transcription are therefore a major output of this pathway. However, the Notch outputs clearly differ from cell type to cell type.

Specificity of Notch pathway activation: twist controls the transcriptional output in adult muscle progenitors.

Cell-cell signalling mediated by Notch regulates many different developmental and physiological processes and is involved in a variety of human diseases. Activation of Notch impinges directly on gene expression through the Suppressor of Hairless [Su(H)] DNA-binding protein. A major question that remains to be elucidated is how the same Notch signalling pathway can result in different transcriptional responses depending on the cellular context and environment.

The cytolinker Pigs is a direct target and a negative regulator of Notch signalling.

Gas2-like proteins harbour putative binding sites for both the actin and the microtubule cytoskeleton and could thus mediate crosstalk between these cytoskeletal systems. Family members are highly conserved in all metazoans but their in vivo role is not clear. The sole Drosophila Gas2-like gene, CG3973 (pigs), was recently identified as a transcriptional target of Notch signalling and might therefore link cell fate decisions through Notch activation directly to morphogenetic changes.

Direct response to Notch activation: signaling crosstalk and incoherent logic.

Notch is the receptor in one of a small group of conserved signaling pathways that are essential at multiple stages in development. Although the mechanism of transduction impinges directly on the nucleus to regulate transcription through the CSL [CBF-1/Su(H)/LAG-1] [corrected] DNA binding protein, there are few known direct target genes. Thus, relatively little is known about the immediate cellular consequences of Notch activation.