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Exported mRNAs are targeted for translation or can undergo degradation by several decay mechanisms. The 5'3' degradation machinery localizes to cytoplasmic P bodies (PBs). We followed the dynamic properties of PBs in vivo and investigated the mechanism by which PBs scan the cytoplasm. Using proteins of the decapping machinery we asked whether PBs actively scan the cytoplasm of if a diffusion-based mechanism is sufficient. Live-cell imaging showed that PBs were anchored mainly to microtubules. Quantitative single-particle tracking demonstrated that most PBs exhibited spatially confined motion dependent on microtubule motion, while stationary PB pairs were identified at the centrosome. Some PBs translocated in long-range movements on microtubules. PB mobility was compared with mitochondria, ER, peroxisomes, SMN bodies and stress granules, and diffusion coefficients were calculated. Disruption of the microtubule network caused a significant reduction in PB mobility together with an induction of PB assembly. However, FRAP measurements showed that the dynamic flux of assembled PB components was not affected by such treatments. FRAP analysis showed that the decapping enzyme Dcp2 is a nondynamic PB core protein, while Dcp1 proteins continuously exchanged with the cytoplasm. This study reveals the mechanism of PB transport, and demonstrates how PB assembly and disassembly integrate with the presence of an intact cytoskeleton. |
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