ArfGAP1 regulates the endosomal sorting of guidance receptors to promote directed collective cell migration in vivo.

Chemotaxis drives diverse migrations important for development and involved in diseases, including cancer progression. Using border cells in the Drosophila egg chamber as a model for collective cell migration, we characterized the role of ArfGAP1 in regulating chemotaxis during this process. We found that ArfGAP1 is required for the maintenance of receptor tyrosine kinases, the guidance receptors, at the plasma membrane. In the absence of ArfGAP1, the level of active receptors is reduced at the plasma membrane and increased in late endosomes. Consequently, clusters with impaired ArfGAP1 activity lose directionality. Furthermore, we found that the number and size of late endosomes and lysosomes are increased in the absence of ArfGAP1. Finally, genetic interactions suggest that ArfGAP1 acts on the kinase and GTPase Lrrk to regulate receptor sorting. Overall, our data indicate that ArfGAP1 is required to maintain guidance receptors at the plasma membrane and promote chemotaxis.

Distinctive Participation of Transcription-Coupled and Global Genome Nucleotide Excision Repair of Pyrimidine Dimers in the Transcribed Strand of Yeast rRNA Genes.

UV light causes the formation of pyrimidine dimers (PDs). Transcription-coupled (TC) nucleotide excision repair (NER) and global genome (GG) NER remove PDs from the transcribed strand (TS) of active genes and the inactive genome, respectively. TC-NER is triggered by elongating RNA polymerases that are blocked at PDs. The yeast rRNA genes are densely loaded with RNA polymerase-I. After UV irradiation, their density increases at the 5'-end of the gene, which results from continuous transcription initiation, followed by elongation and pausing/release at the first encountered PD, from the transcription start site. RNA polymerase-I posed at downstream PDs are released from the TS and are replaced by nucleosomes. Consequently, discrete chromatin structures are formed in the damaged transcribed rRNA genes. Singular assignation of the two NER sub-pathways could therefore be required to eliminate PDs from the TS. To advance our understanding of NER in the dynamic structure of transcribed chromatin, we investigated the repair of PDs at nucleotide resolution in separate rRNA gene coding regions. In the TS, the TC-NER efficiency reflected the density of RNA polymerase-I, and PDs were removed faster in the 5'-end than in the 3'-end of the gene. GG-NER removed PDs from the TS where RNA polymerase-I was transiently replaced by a nucleosome. The two NER sub-pathways inversely participated to remove PDs from the TS. In the non-TS of both nucleosome and non-nucleosome rRNA gene coding regions, GG-NER was solely responsible to remove UV-induced DNA lesions.

Misshapen coordinates protrusion restriction and actomyosin contractility during collective cell migration.

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.

The ArfGAP Drongo Promotes Actomyosin Contractility during Collective Cell Migration by Releasing Myosin Phosphatase from the Trailing Edge.

Collective cell migration is involved in various developmental and pathological processes, including the dissemination of various cancer cells. During Drosophila melanogaster oogenesis, a group of cells called border cells migrate collectively toward the oocyte. Herein, we show that members of the Arf family of small GTPases and some of their regulators are required for normal border cell migration. Notably, we found that the ArfGAP Drongo and its GTPase-activating function are essential for the initial detachment of the border cell cluster from the basal lamina. We demonstrate through protein localization and genetic interactions that Drongo controls the localization of the myosin phosphatase in order to regulate myosin II activity at the back of the cluster. Moreover, we show that toward the class III Arf, Drongo acts antagonistically to the guanine exchange factor Steppke. Overall, our work describes a mechanistic pathway that promotes the local actomyosin contractility necessary for border cell detachment.

Proteomics Screen Identifies Class I Rab11 Family Interacting Proteins as Key Regulators of Cytokinesis.

The 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human and Drosophila 14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 in Drosophila We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.

RNA Polymerase-I-Dependent Transcription-coupled Nucleotide Excision Repair of UV-Induced DNA Lesions at Transcription Termination Sites, in Saccharomyces cerevisiae.

If not repaired, ultraviolet light-induced DNA damage can lead to genome instability. Nucleotide excision repair (NER) of UV photoproducts is generally fast in the coding region of genes, where RNA polymerase-II (RNAP2) arrest at damage sites and trigger transcription-coupled NER (TC-NER). In Saccharomyces cerevisiae, there is RNA polymerase-I (RNAP1)-dependent TC-NER, but this process remains elusive. Therefore, we wished to characterize TC-NER efficiency in different regions of the rDNA locus: where RNAP1 are present at high density and start transcription elongation, where the elongation rate is slow, and in the transcription terminator where RNAP1 pause, accumulate and then are released. The Rpa12 subunit of RNAP1 and the Nsi1 protein participate in transcription termination, and NER efficiency was compared between wild type and cells lacking Rpa12 or Nsi1. The presence of RNAP1 was determined by chromatin endogenous cleavage and chromatin immunoprecipitation, and repair was followed at nucleotide precision with an assay that is based on the blockage of Taq polymerase by UV photoproducts. We describe that TC-NER, which is modulated by the RNAP1 level and elongation rate, ends at the 35S rRNA gene transcription termination site.