An insoluble protein matrix is required for centrosome function

The centrosome (CE) is the major microtubule (MT) organizing center in animal cells. It is composed of two centrioles surrounded by an undefined pericentriolar material. CEs isolated from Spisula solidissima oocytes retain MT nucleation potential (MNP) and nucleate and organize MTs in defined media. Treatment of isolated CEs with 1.0 M KI removes MNP and 90% of CE protein but leaves a sedimentable nonfunctional KI‐insoluble CE remnant(KICR) that recovers MNP when incubated in cytoplamic extracts of animal cells. Treatment of KICRs with 6.0 M guanidine‐HCl further dissociates proteins yielding a guanidine‐HCl insoluble CE remnant (GUCR) that also recovers MNP when incubated in cytoplasmic extracts. Treatment of KICRs and GUCRs with trypsin destroys the ability to recover MNP indicating that protein(s) are required for recovery of MNP. The SDS‐PAGE gel profiles of CEs, KICRs and GUCRs shows decreasing protein complexity, respectively. We have generated an antibody to a putative GUCR protein. This antibody stains CEs, KICRs and GUCRs by immunofluorescence and immunoblot reveals that the protein is enriched in CE fractions. Electron microscopy reveals that KICRs are composed of a complex lattice, built from 12–15 nm thick filaments. Preliminary GUCR EM data indicates that the lattice structure remains after guanidine treatment. The results indicate that CEs contain a highly insoluble protein matrix that is capable of docking proteins required for MT nucleation. Research funded by NIH R01GM043264