Anne Royou

Drosophila Rho-Associated Kinase (Drok) Links Frizzled-Mediated Planar Cell Polarity Signaling to the Actin Cytoskeleton

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.

Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: its role in nuclear axial expansion and its regulation by Cdc2 activity

The nuclei of early syncytial Drosophila embryos migrate dramatically toward the poles. The cellular mechanisms driving this process, called axial expansion, are unclear, but myosin II activity is required. By following regulatory myosin light chain (RLC)–green fluorescent protein dynamics in living embryos, we observed cycles of myosin recruitment to the cortex synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the anterocentral part of the embryo at cycle 4. With each succeeding cycle, the patch expands poleward, dispersing at the beginning of each mitosis and reassembling at the end of telophase. Each cycle of actin and myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not require microtubules but correlates inversely with Cdc2/cyclinB (mitosis-promoting factor) activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional with no effect on the cortical myosin cycle, indicating that Cdc2 must be modulating myosin activity by some other mechanism. An inhibitor of Rho kinase blocks the cortical myosin recruitment cycles and provokes a concomitant failure of axial expansion. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to Cdc2 and Rho kinase activity, are directly responsible for the axial expansion of the syncytial nuclei.

ATM is required for telomere maintenance and chromosome stability during Drosophila development.

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.

The Drosophila Grp/Chk1 DNA damage checkpoint controls entry into anaphase

It is well established that DNA damage induces checkpoint-mediated interphase arrest in higher eukaryotes, but recent studies demonstrate that DNA damage delays entry into anaphase as well. Damaged DNA in syncytial and gastrulating Drosophila embryos delays the metaphase/anaphase transition . In human cultured cells, DNA damage also induces a delay in mitosis . However, the mechanism by which DNA damage delays the anaphase onset is controversial. Some studies implicate a DNA damage checkpoint , whereas other studies invoke a spindle checkpoint . To resolve this issue, we compared the effects of random DNA breaks induced by X-irradiation to site-specific I-CreI endonuclease-induced chromosome breaks on cell-cycle progression in wild-type and checkpoint-defective Drosophila neuroblasts. We found that both the BubR1 spindle checkpoint pathway and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation-induced DNA damage, whereas Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-CreI-induced double-strand breaks. On the basis of these results, we propose that DNA damage in nonkinetochore regions produces a Grp/Chk1 DNA-damage-checkpoint-mediated delay in the metaphase/anaphase transition.

BubR1- and Polo-Coated DNA Tethers Facilitate Poleward Segregation of Acentric Chromatids

The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, as CIN contributes to tumorigenesis. To gain insight into these mechanisms, we studied the behavior of cells entering mitosis with damaged chromosomes. We used the endonuclease I-CreI to generate acentric chromosomes in Drosophila larvae. While I-CreI expression produces acentric chromosomes in the majority of neuronal stem cells, remarkably, it has no effect on adult survival. Our live studies reveal that acentric chromatids segregate efficiently to opposite poles. The acentric chromatid poleward movement is mediated through DNA tethers decorated with BubR1, Polo, INCENP, and Aurora-B. Reduced BubR1 or Polo function results in abnormal segregation of acentric chromatids, a decrease in acentric chromosome tethering, and a great reduction in adult survival. We propose that BubR1 and Polo facilitate the accurate segregation of acentric chromatids by maintaining the integrity of the tethers that connect acentric chromosomes to their centric partners.

Grapes(Chk1) prevents nuclear CDK1 activation by delaying cyclin B nuclear accumulation

Entry into mitosis is characterized by a dramatic remodeling of nuclear and cytoplasmic compartments. These changes are driven by cyclin-dependent kinase 1 (CDK1) activity, yet how cytoplasmic and nuclear CDK1 activities are coordinated is unclear. We injected cyclin B (CycB) into Drosophila melanogaster embryos during interphase of syncytial cycles and monitored effects on cytoplasmic and nuclear mitotic events. In untreated embryos or embryos arrested in interphase with a protein synthesis inhibitor, injection of CycB accelerates nuclear envelope breakdown and mitotic remodeling of the cytoskeleton. Upon activation of the Grapes(checkpoint kinase 1) (Grp(Chk1))-dependent S-phase checkpoint, increased levels of CycB drives cytoplasmic but not nuclear mitotic events. Grp(Chk1) prevents nuclear CDK1 activation by delaying CycB nuclear accumulation through Wee1-dependent and independent mechanisms.

Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane

A cortical myosin-dependent mechanism induces cell elongation to ensure clearance of trailing chromatids from the cleavage plane at the correct time during cytokinesis.

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The mechanisms governing the spatial organization of endocytosis and exocytosis are ill defined. A quantitative imaging screen and high-density single-vesicle tracking are used to identify mutants that are defective in endocytic and exocytic vesicle organization. The screen identifies a role for the exocyst complex in connecting the two pathways.

Cdk1-dependent control of membrane-trafficking dynamics

Cyclin-dependent kinase 1 (Cdk1) is required for initiation and maintenance of polarized cell growth in budding yeast. Cdk1 activates Rho-family GTPases, which trigger polarization of the actin cytoskeleton for delivery of membrane to growth sites. It is found that Cdk1s function in polarized growth extends beyond that of actin organization.

The Centrosomal Protein CP190 Regulates Myosin Function during Early Drosophila Development

Centrosomes are the main microtubule (MT)-organizing centers in animal cells, but they also influence the actin/myosin cytoskeleton. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis, and binds to MTs directly in vitro. CP190 has an essential function in the nucleus as a chromatin insulator, but centrosomes and MTs appear unperturbed in Cp190 mutants. Thus, the centrosomal function of CP190, if any, is unclear. Here, we examine the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos.