ABSTRACT
The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.
Subject(s)
Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Protein Structure, Tertiary , Spindle Apparatus/metabolism , Animals , Aurora Kinase B , Aurora Kinases , Cell Cycle/physiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spindle Apparatus/ultrastructure , Tissue Distribution , rhoA GTP-Binding Protein/metabolism , Polo-Like Kinase 1ABSTRACT
GIPC1/synectin, a single PDZ domain-containing protein, binds to numerous proteins and is involved in multiple biological processes, including cell migration. We reported previously that MyoGEF, a guanine nucleotide exchange factor, plays a role in regulating breast cancer cell polarization and invasion. Here, we identify GIPC1 as an interacting partner of MyoGEF. Both in vitro and in vivo binding assays show that the GIPC1 PDZ domain binds to the PDZ-binding motif at the C terminus of MyoGEF. Immunofluorescence analysis shows that GIPC1 and MyoGEF colocalize to the cell leading edge. Depletion of GIPC1 by RNAi in MDA-MB-231 cells causes cells to shift from a polarized to a rounded morphology. Matrigel invasion assays show that RNAi-mediated depletion of GIPC1 dramatically decreases MDA-MB-231 cell invasion. Notably, an anti-MyoGEF peptide antibody, whose epitope is located at the C terminus of MyoGEF, interferes with GIPC1-MyoGEF complex formation. Treatment of MDA-MB-231 cells with the anti-MyoGEF peptide antibody disrupts cell polarization and invasion. Thus, our results suggest that GIPC1-MyoGEF complex formation plays an important role in regulating MDA-MB-231 breast cancer cell polarization and invasion.
Subject(s)
Breast Neoplasms/metabolism , Carrier Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Mammary Neoplasms, Animal/metabolism , Multiprotein Complexes/metabolism , Neoplasm Proteins/metabolism , Neuropeptides/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Motifs , Animals , Antibodies, Neoplasm/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carrier Proteins/genetics , Cell Line, Tumor , Epitopes/genetics , Epitopes/metabolism , Epitopes/pharmacology , Female , Guanine Nucleotide Exchange Factors/genetics , Humans , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Mice , Multiprotein Complexes/genetics , Neoplasm Invasiveness/genetics , Neoplasm Proteins/genetics , Neuropeptides/genetics , Peptides/antagonists & inhibitors , Peptides/genetics , Peptides/metabolism , Peptides/pharmacology , Protein BindingABSTRACT
Prevalence of lung worms belonging to the genus Metastrongylus was surveyed on 42 Japanese wild boars (Sus scrofa leucomystax) captured officially for wildlife damage control in the western parts of Tokyo, Japan from April 2000 to April 2001. The number of parasites was the highest in the caudal lung lobes. Four species, M. elongatus (ME), M. salmi (MS), M. asymmetricus (MA) and M. pudendotectus (MP), were identified. All the boars were infected with 2 or more species, and 64.3% of the boars had all 4 species. The composition of species, ME:MS:MA:MP=1.3:3.4:1.0:1.4, was drastically different from the previous reports. The peak of the average number of the parasites was observed in the period of January to March 2001 because of the increase of MS.
