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1.
Development ; 148(20)2021 10 15.
Article in English | MEDLINE | ID: mdl-34532737

ABSTRACT

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.


Subject(s)
Cell Adhesion Molecules/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Epithelium/metabolism , Repressor Proteins/metabolism , Wings, Animal/metabolism , Adherens Junctions/metabolism , Animals , Carrier Proteins , Cytoplasm/metabolism , Morphogenesis/physiology , Pupa/metabolism
2.
Curr Biol ; 19(23): 1969-78, 2009 Dec 15.
Article in English | MEDLINE | ID: mdl-19931458

ABSTRACT

BACKGROUND: Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several "noncore" AJ components have been identified, which modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. RESULTS: We previously identified dASPP as a positive regulator of dCsk (Drosophila C-terminal Src kinase). Here we show that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. Our genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, we observed abnormal E-cadherin localization in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. CONCLUSIONS: Because Src is known to regulate AJs in both Drosophila and mammals, we propose that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. We show that the dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/embryology , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis Regulatory Proteins/genetics , Carrier Proteins/genetics , Cell Adhesion/physiology , Cell Line , Drosophila Proteins/genetics , Gene Expression Regulation, Developmental/physiology , Protein Binding , Proto-Oncogene Mas , Retina/embryology , Tumor Suppressor Proteins/genetics , Wings, Animal
3.
Chromosoma ; 117(5): 457-69, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18521620

ABSTRACT

Polo-like kinase 1 (Plk1) and Aurora A play key roles in centrosome maturation, spindle assembly, and chromosome segregation during cell division. Here we show that the functions of these kinases during early mitosis are coordinated through Bora, a partner of Aurora A first identified in Drosophila. Depletion of human Bora (hBora) results in spindle defects, accompanied by increased spindle recruitment of Aurora A and its partner TPX2. Conversely, hBora overexpression induces mislocalization of Aurora A and monopolar spindle formation, reminiscent of the phenotype seen in Plk1-depleted cells. Indeed, Plk1 regulates hBora. Following Cdk1-dependent recruitment, Plk1 triggers hBora destruction by phosphorylating a recognition site for SCF(Beta-TrCP). Plk1 depletion or inhibition results in a massive accumulation of hBora, concomitant with displacement of Aurora A from spindle poles and impaired centrosome maturation, but remarkably, co-depletion of hBora partially restores Aurora A localization and bipolar spindle formation. This suggests that Plk1 controls Aurora A localization and function by regulating cellular levels of hBora.


Subject(s)
Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , beta-Transducin Repeat-Containing Proteins/metabolism , Amino Acid Sequence , Animals , Aurora Kinase A , Aurora Kinases , Cell Cycle Proteins/drug effects , Centrosome/physiology , Gene Silencing , HeLa Cells , Humans , Mice , Mitosis/physiology , Molecular Sequence Data , RNA, Small Interfering/genetics , SKP Cullin F-Box Protein Ligases/metabolism , Spindle Apparatus/physiology , beta-Transducin Repeat-Containing Proteins/genetics , Polo-Like Kinase 1
4.
J Cell Biol ; 172(3): 363-72, 2006 Jan 30.
Article in English | MEDLINE | ID: mdl-16431929

ABSTRACT

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.


Subject(s)
Cell Cycle Proteins/physiology , Cytokinesis/physiology , Kinesins/physiology , Oncogene Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Binding Sites/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cytoplasm/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Kinesins/genetics , Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Models, Genetic , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , Spindle Apparatus/metabolism , Transfection
5.
Oncogene ; 24(12): 2076-86, 2005 Mar 17.
Article in English | MEDLINE | ID: mdl-15688006

ABSTRACT

Originally identified in Drosophila melanogaster, the Warts(Wts)/Lats protein kinase has been proposed to function with two other Drosophila proteins, Hippo (Hpo) and Salvador (Sav), in the regulation of cell cycle exit and apoptosis. In mammals, two candidate Warts/Lats homologs, termed Lats1 and Lats2, have been described, and the targeted disruption of LATS1 in mice increases tumor formation. Little, however, is known about the function and regulation of human Lats kinases. Here we report that human Mst2, a STE20-family member and purported Hpo ortholog, phosphorylates and activates both Lats1 and Lats2. Deletion analysis revealed that regulation of Lats1 occurs through the C-terminal, catalytic domain. Within this domain, two regulatory phosphorylation sites were identified by mass spectrometry. These sites, S909 in the activation loop and T1079 within a hydrophobic motif, have been highly conserved during evolution. Moreover, a direct interaction was observed between Mst2 and hWW45, a putative ortholog of Drosophila Sav. These results indicate that Mst2-like kinases regulate Lats kinase activities in an evolutionarily conserved regulatory pathway. Although the function of this pathway remains poorly understood in mammals, it is intriguing that, in Drosophila, it has been linked to development and tissue homeostasis.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Cell Line , Drosophila melanogaster , Gene Expression Regulation , Genes, Tumor Suppressor , HeLa Cells , Humans , Kidney , Peptide Fragments/chemistry , Protein Serine-Threonine Kinases/genetics , Serine-Threonine Kinase 3 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tumor Suppressor Proteins/metabolism
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