Dynein-mediated transport and membrane trafficking control PAR3 polarised distribution.

The scaffold protein PAR3 and the kinase PAR1 are essential proteins that control cell polarity. Their precise opposite localisations define plasma membrane domains with specific functions. PAR3 and PAR1 are mutually inhibited by direct or indirect phosphorylations, but their fates once phosphorylated are poorly known. Through precise spatiotemporal quantification of PAR3 localisation in the Drosophila oocyte, we identify several mechanisms responsible for its anterior cortex accumulation and its posterior exclusion. We show that PAR3 posterior plasma membrane exclusion depends on PAR1 and an endocytic mechanism relying on RAB5 and PI(4,5)P2. In a second phase, microtubules and the dynein motor, in connection with vesicular trafficking involving RAB11 and IKK-related kinase, IKKε, are required for PAR3 transport towards the anterior cortex. Altogether, our results point to a connection between membrane trafficking and dynein-mediated transport to sustain PAR3 asymmetry.

Microtubule-dependent apical restriction of recycling endosomes sustains adherens junctions during morphogenesis of the Drosophila tracheal system.

Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive. We carried out a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells shows that MTs are essential during a specific time frame of tracheal cell elongation while the branch extends. In the absence of MTs, one tracheal cell per branch overelongates, ultimately leading to branch break. Three-dimensional quantifications revealed that MTs are crucial to sustain E-Cadherin (Shotgun) and Par-3 (Bazooka) levels at AJs. Maintaining E-Cadherin/Par-3 levels at the apical domain requires de novo synthesis rather than internalisation and recycling from and to the apical plasma membrane. However, apical targeting of E-Cadherin and Par-3 requires functional recycling endosomes, suggesting an intermediate role for this compartment in targeting de novo synthesized E-Cadherin to the plasma membrane. We demonstrate that apical enrichment of recycling endosomes is dependent on the MT motor Dynein and essential for the function of this vesicular compartment. In addition, we establish that E-Cadherin dynamics and MT requirement differ in remodelling tracheal cells versus planar epithelial cells. Altogether, our results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis.

PI(4,5)P2 produced by the PI4P5K SKTL controls apical size by tethering PAR-3 in Drosophila epithelial cells.

BACKGROUND: The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides is controlled by the concerted action of phosphoinositide kinases and phosphatases. RESULTS: Using the Drosophila follicular epithelium as a model system in vivo, we show here that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated by the PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, we observe the disassembly of adherens junctions, actin cytoskeleton reorganization, and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. We provide evidence that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, we show that PI(4,5)P2 is excluded from the cell apex and that PAR-3 diffuses laterally just prior to the apical constriction in a context of endogenous invagination. CONCLUSIONS: All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain.

Nucleoporin98-96 function is required for transit amplification divisions in the germ line of Drosophila melanogaster.

Production of specialized cells from precursors depends on a tightly regulated sequence of proliferation and differentiation steps. In the gonad of Drosophila melanogaster, the daughters of germ line stem cells (GSC) go through precisely four rounds of transit amplification divisions to produce clusters of 16 interconnected germ line cells before entering a stereotypic differentiation cascade. Here we show that animals harbouring a transposon insertion in the center of the complex nucleoporin98-96 (nup98-96) locus had severe defects in the early steps of this developmental program, ultimately leading to germ cell loss and sterility. A phenotypic analysis indicated that flies carrying the transposon insertion, designated nup98-96(2288), had dramatically reduced numbers of germ line cells. In contrast to controls, mutant testes contained many solitary germ line cells that had committed to differentiation as well as abnormally small clusters of two, four or eight differentiating germ line cells. This indicates that mutant GSCs rather differentiated than self-renewed, and that these GSCs and their daughters initiated the differentiation cascade after zero, or less than four rounds of amplification divisions. This phenotype remained unaffected by hyper-activation of signalling pathways that normally result in excessive proliferation of GSCs and their daughters. Expression of wildtype nup98-96 specifically in the germ line cells of mutant animals fully restored development of the GSC lineage, demonstrating that the effect of the mutation is cell-autonomous. Nucleoporins are the structural components of the nucleopore and have also been implicated in transcriptional regulation of specific target genes. The nuclear envelopes of germ cells and general nucleocytoplasmic transport in nup98-96 mutant animals appeared normal, leading us to propose that Drosophila nup98-96 mediates the transport or transcription of targets required for the developmental timing between amplification and differentiation.

A developmentally regulated two-step process generates a noncentrosomal microtubule network in Drosophila tracheal cells.

Microtubules (MTs) are essential for many cell features, such as polarity, motility, shape, and vesicle trafficking. Therefore, in a multicellular organism, their organization differs between cell types and during development; however, the control of this process remains elusive. Here, we show that during Drosophila tracheal morphogenesis, MT reorganization is coupled to relocalization of the microtubule organizing centers (MTOC) components from the centrosome to the apical cell domain from where MTs then grow. We reveal that this process is controlled by the trachealess patterning gene in a two-step mechanism. MTOC components are first released from the centrosome by the activity of the MT-severing protein Spastin, and then anchored apically through the transmembrane protein Piopio. We further show that these changes are essential for tracheal development, thus stressing the functional relevance of MT reorganization for morphogenesis.

Phosphatidylinositol 4,5-bisphosphate directs spermatid cell polarity and exocyst localization in Drosophila.

During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) by low-level expression of the PIP(2) phosphatase SigD or mutation of the PIP(2) biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP(2) localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP(2) levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. Our data are consistent with a mechanism in which localized synthesis of PIP(2) recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts.

Drosophila stathmins bind tubulin heterodimers with high and variable stoichiometries.

In vertebrates, stathmins form a family of proteins possessing two tubulin binding repeats (TBRs), which each binds one soluble tubulin heterodimer. The stathmins thus sequester two tubulins in a phosphorylation-dependent manner, providing a link between signal transduction and microtubule dynamics. In Drosophila, we show here that a single stathmin gene (stai) encodes a family of D-stathmin proteins. Two of the D-stathmins are maternally deposited and then restricted to germ cells, and the other two are detected in the nervous system during embryo development. Like in vertebrates, the nervous system-enriched stathmins contain an N-terminal domain involved in subcellular targeting. All the D-stathmins possess a domain containing three or four predicted TBRs, and we demonstrate here, using complementary biochemical and biophysical methods, that all four predicted TBR domains actually bind tubulin. D-stathmins can indeed bind up to four tubulins, the resulting complex being directly visualized by electron microscopy. Phylogenetic analysis shows that the presence of regulated multiple tubulin sites is a conserved characteristic of stathmins in invertebrates and allows us to predict key residues in stathmin for the binding of tubulin. Altogether, our results reveal that the single Drosophila stathmin gene codes for a stathmin family similar to the multigene vertebrate one, but with particular tubulin binding properties.

A dual role for actin and microtubule cytoskeleton in the transport of Golgi units from the nurse cells to the oocyte across ring canals.

Axis specification during Drosophila embryonic development requires transfer of maternal components during oogenesis from nurse cells (NCs) into the oocyte through cytoplasmic bridges. We found that the asymmetrical distribution of Golgi, between nurse cells and the oocyte, is sustained by an active transport process. We have characterized actin basket structures that asymmetrically cap the NC side of Ring canals (RCs) connecting the oocyte. Our results suggest that these actin baskets structurally support transport mechanisms of RC transit. In addition, our tracking analysis indicates that Golgi are actively transported to the oocyte rather than diffusing. We observed that RC transit is microtubule-based and mediated at least by dynein. Finally, we show that actin networks may be involved in RC crossing through a myosin II step process, as well as in dispatching Golgi units inside the oocyte subcompartments.

Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation.

BACKGROUND: The cytoskeleton and associated motors play an important role in the establishment of intracellular polarity. Microtubule-based transport is required in many cell types for the asymmetric localization of mRNAs and organelles. A striking example is the Drosophila oocyte, where microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. RESULTS: Here, we show that mRNA localization and nuclear positioning at mid-oogenesis depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. CONCLUSION: The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.

A germline-specific gap junction protein required for survival of differentiating early germ cells.

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.

Drosophila stathmin: a microtubule-destabilizing factor involved in nervous system formation.

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins in Drosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin and stathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation of Drosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophila gene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.