The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.
Bottom Line: We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1.SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes.These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.
Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.Show MeSH
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Mentions: To confirm that the protein interactions we observed in cytoplasmic extracts also occur in living embryos, we employed fluorescence correlation spectroscopy (FCS) to assess the diffusion of fluorescently tagged proteins in vivo. Protein–protein interactions involving fluorescently tagged proteins can be deduced by combining FCS with RNA interference (RNAi)–mediated depletion of candidate binding partners because depletion of a binding partner is expected to decrease the hydrodynamic radius of complexes containing the tagged protein, thereby increasing its diffusion (Figure 3A). Such differences in diffusion can be observed as time shifts of the autocorrelation curve of the fluorescent protein or by changes in the diffusion coefficient obtained from fitting the autocorrelation curve with a diffusion model. Changes in diffusion strongly depend on the relative change in hydrodynamic radius of the particle and thus are best tested by observing a small molecule under depletion of its larger interaction partner.
Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.