Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135.
A novel 135 kDa centrosomal component (Cep135) was identified by immunoscreening of a mammalian expression library with monoclonal antibodies raised against clam centrosomes. It is predicted to be a highly coiled-coil protein with an extensive alpha-helix, suggesting that Cep135 is a structural component of the centrosome. To evaluate how the protein is arranged in the centrosomal structure, we overexpressed Cep135 polypeptides in CHO cells by transient transfection. HA- or GFP-tagged full (amino acids 1-1144) as well as truncated (#10, 29-1144; Delta3, 29-812) polypeptides become localized at the centrosome and induce cytoplasmic dots of various size and number in CHO cells. Centrosomes are associated with massive approximately 7 nm filaments and dense particles organized in a whorl-like arrangement in which parallel-oriented dense lines appear with a regular approximately 7 nm periodicity. The same filamentous aggregates are also detected in cytoplasmic dots, indicating that overexpressed Cep135 can assemble into elaborate higher-ordered structures in and outside the centrosome. Sf9 cells infected with baculovirus containing Cep135 sequences induce filamentous polymers which are distinctive from the whorl seen in CHO cells; #10 forms highly packed spheroids, but the Delta3-containing structure looks loose. Both structures show an internal repeating unit of dense and less dense stripes. Although the distance between the outer end of two adjacent dense lines is similar between two types of polymers ( approximately 120 nm), the dense stripe of Delta3 polymers ( approximately 40 nm) is wider than #10 ( approximately 30 nm). The light band of Delta3 ( approximately 40 nm) is thus narrower than #10 ( approximately 60 nm). Since thin fibers are frequently seen to extend from one dense line to the next, the coiled-coil rod of Cep135 may span the light band. These results suggest that overexpressed Cep135 assemble into distinctive polymers in a domain-specific manner.
Ryu
J H
JH
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis 55455, USA.
Essner
R
R
Ohta
T
T
Kuriyama
R
R
D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D006224:Cricetinae; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000032:analysis
D008164:Luminescent Proteins; D010455:Peptides; D011506:Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins