A Cdc42-regulated actin cytoskeleton mediates Drosophila oocyte polarization.

Polarity of the Drosophila oocyte is essential for correct development of the egg and future embryo. The Par proteins Par-6, aPKC and Bazooka are needed to maintain oocyte polarity and localize to specific domains early in oocyte development. To date, no upstream regulator or mechanism for localization of the Par proteins in the oocyte has been identified. We have analyzed the role of the small GTPase Cdc42 in oocyte polarity. We show that Cdc42 is required to maintain oocyte fate, which it achieves by mediating localization of Par proteins at distinct sites within this cell. We establish that Cdc42 localization itself is polarized to the anterolateral cortex of the oocyte and that Cdc42 is needed for maintenance of oocyte polarity throughout oogenesis. Our data show that Cdc42 ensures the integrity of the oocyte actin network and that disruption of this network with Latrunculin A phenocopies loss of Cdc42 or Par protein function in early stages of oogenesis. Finally, we show that Cdc42 and Par proteins, as well as Cdc42/Par and Arp3, interact in the context of oocyte polarity, and that loss of Par proteins reciprocally affects Cdc42 localization and the actin network. These results reveal a mutual dependence between Par proteins and Cdc42 for their localization, regulation of the actin cytoskeleton and, consequently, for the establishment of oocyte polarity. This most likely allows for the robustness in symmetry breaking in the cell.

Functions of endosomal trafficking in Drosophila epithelial cells.

The mechanisms underlying endosomal trafficking have been mostly dissected in yeast and mammalian tissue culture cells. Here, we review recent advances in the understanding of the role of endosomal trafficking in Drosophila epithelial cells. We focus on endosomal pathways that control cell polarization, cell growth, cell fate and epithelial cell rearrangement. We expect that mechanistic studies in mammalian cells and functional studies in invertebrates will continue to synergize to provide a comprehensive view of the role of endosomal trafficking in epithelial tissue organization and functions.

Drosophila Cip4 and WASp define a branch of the Cdc42-Par6-aPKC pathway regulating E-cadherin endocytosis.

BACKGROUND: Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. RESULTS: Here we show that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. We demonstrate that the Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. CONCLUSION: Altogether our results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue.

WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators.

Plants continuously maintain pools of totipotent stem cells in their apical meristems from which elaborate root and shoot systems are produced. In Arabidopsis thaliana, stem cell fate in the shoot apical meristem is controlled by a regulatory network that includes the CLAVATA (CLV) ligand-receptor system and the homeodomain protein WUSCHEL (WUS). Phytohormones such as auxin and cytokinin are also important for meristem regulation. Here we show a mechanistic link between the CLV/WUS network and hormonal control. WUS, a positive regulator of stem cells, directly represses the transcription of several two-component ARABIDOPSIS RESPONSE REGULATOR genes (ARR5, ARR6, ARR7 and ARR15), which act in the negative-feedback loop of cytokinin signalling. These data indicate that ARR genes might negatively influence meristem size and that their repression by WUS might be necessary for proper meristem function. Consistent with this hypothesis is our observation that a mutant ARR7 allele, which mimics the active, phosphorylated form, causes the formation of aberrant shoot apical meristems. Conversely, a loss-of-function mutation in a maize ARR homologue was recently shown to cause enlarged meristems.

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Interplay between the dividing cell and its neighbors regulates adherens junction formation during cytokinesis in epithelial tissue.

How adherens junctions (AJs) are formed upon cell division is largely unexplored. Here, we found that AJ formation is coordinated with cytokinesis and relies on an interplay between the dividing cell and its neighbors. During contraction of the cytokinetic ring, the neighboring cells locally accumulate Myosin II and produce the cortical tension necessary to set the initial geometry of the daughter cell interface. However, the neighboring cell membranes impede AJ formation. Upon midbody formation and concomitantly to neighboring cell withdrawal, Arp2/3-dependent actin polymerization oriented by the midbody maintains AJ geometry and regulates AJ final length and the epithelial cell arrangement upon division. We propose that cytokinesis in epithelia is a multicellular process, whereby the cooperative actions of the dividing cell and its neighbors define a two-tiered mechanism that spatially and temporally controls AJ formation while maintaining tissue cohesiveness.

Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.

In Arabidopsis, the network responsible for the maintenance of the shoot apical meristem (SAM) is built on a negative feedback loop involving the peptide ligand CLAVATA3 (CLV3) and the homeodomain transcription factor WUSCHEL (WUS). The local WUS/CLV3 regulatory module is linked to the organism-wide cytokinin signalling system by direct transcriptional control of A-type ARABIDOPSIS RESPONSE REGULATOR genes (ARRs) by WUS. Here we investigate two A-type ARR genes, ARR7 and ARR15, which are negative regulators of cytokinin signalling. We show that the expression of ARR7, WUS and CLV3 is dependent on cytokinin signalling. While ARR7 expression strongly responds to variations in cytokinin activity, WUS and CLV3 appeared to be much more buffered against this type of variation. As earlier studies had shown that pertubation of A-type ARR activity only causes mild effects on SAM function, we employed tissue regeneration assays as a sensitised background. Root explants pre-treated on auxin and cytokinin-rich callus-inducing medium showed severely suppressed shoot regeneration when ARR7 and ARR15 were overexpressed, whereas loss of function of these genes had a strongly promoting effect. This phenotype was even aggravated in the arr3,4,5,6,7,8,9 septuple mutant. Futhermore, loss-of A-type ARR function in arr7 and arr3,4,5,6,7,8,9 mutants strongly stimulated callus development, indicating that cell proliferation is repressed by A-type ARRs. To elucidate the mechanisms underlying the enhanced capacity of the arr3,4,5,6,7,8,9 septuple mutant to develop shoot tissue in culture, we used whole-genome expression profiling. Among the transcripts with increased abundance in arr3,4,5,6,7,8,9 inflorescence apices a strong enrichment for functions in pollen development was apparent, while the reduced transcripts showed a more heterogeneous distribution of functional categories, ranging from development to pathogen defence.

Transcriptional control of a plant stem cell niche.

Despite the independent evolution of multicellularity in plants and animals, the basic organization of their stem cell niches is remarkably similar. Here, we report the genome-wide regulatory potential of WUSCHEL, the key transcription factor for stem cell maintenance in the shoot apical meristem of the reference plant Arabidopsis thaliana. WUSCHEL acts by directly binding to at least two distinct DNA motifs in more than 100 target promoters and preferentially affects the expression of genes with roles in hormone signaling, metabolism, and development. Striking examples are the direct transcriptional repression of CLAVATA1, which is part of a negative feedback regulation of WUSCHEL, and the immediate regulation of transcriptional repressors of the TOPLESS family, which are involved in auxin signaling. Our results shed light on the complex transcriptional programs required for the maintenance of a dynamic and essential stem cell niche.