The Dynamics of Mammalian P Body Transport, Assembly, and Disassembly In Vivo

Abstract

Exported mRNAs are targeted for translation or can undergo degradation by several decay mechanisms. The 533 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 or whether 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, whereas stationary PB pairs were identified at the centrosome. Some PBs translocated in long-range movements on microtubules. PB mobility was compared with mitochondria, endoplasmic reticulum, 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, whereas Dcp1 proteins continuously exchanged with the cytoplasm. This study reveals the mechanism of PB transport, and it demonstrates how PB assembly and disassembly integrate with the presence of an intact cytoskeleton. INTRODUCTIONGene expression begins with the synthesis of mRNA molecules in the nucleus. After processing events, transcripts are exported to the cytoplasm where they can face several posttranscriptional fates, elicited by a balance between cytoplasmic translation and mRNA degradation pathways. Quality control pathways regulate the degradation of mRNAs and facilitate their sequestration or translational repression (Meyer et al., 2004). In eukaryotes, mRNA degradation typically begins with deadenylation, and then either of two major pathways is used. The exosome protein complex degrades mRNAs in the 3Ј35Ј direction, whereas the 5Ј33Ј direction involves other factors, including a decapping enzyme followed by the Xrn1 exonuclease (Parker and Song, 2004).The removal of the cap structure irreversibly marks the mRNA for degradation. The decapping process is tightly regulated biochemically and spatially. It was first discovered that the Xrn1 nuclease localizes in discrete cytoplasmic foci in eukaryotic cells (Bashkirov et al., 1997). Some years later, a decapping protein termed Dcp2 was found to colocalize in Xrn1-foci (Ingelfinger et al., 2002;Lykke-Andersen, 2002;van Dijk et al., 2002), finally leading to the understanding that both yeast (Sheth and Parker, 2003) and mammalian cells (Cougot et al., 2004) contain discrete areas in which mRNA decapping and 5Ј33Ј degradation can occur (Sheth and Parker, 2006). These cytoplasmic foci are now widely known as P bodies (PBs), and they have been referred to as Dcpbodies, processing bodies, mRNA-decay foci, and GW182 bodies. Since ...

Intracellular trafficking and dynamics of P bodies

“…Interestingly, in about 50% of the cases PBs were found adjacent to mRNPs as if the two entities were docked to each other. In this study 29 and in our study, 18 PBs were not found associated with other organelles such as mitochondria, endoplasmatic reticulum and peroxisomes, but did associate with stress granules that are also RNA-containing structures. Chemical synaptic stimulation of neurons led to a substantial decrease in dendritic PBs, which returned to normal levels after a 30 minute period.…”

“…Fluorescence recovery after photobleaching (FRAP) has shown that most PB components are constantly exchanging between PBs and the cytoplasm, 16,17,35 except for the decapping enzyme Dcp2 that did not exchange. 18 This suggests that Dcp2 is a core component of PBs. However, when there is no microtubule network, this control is overrun and most of the Dcp proteins can now assemble into large PBs.…”

“…In our study, we wished to follow the dynamic aspects of PBs in human cells. 18 We therefore tagged three PB components, Dcp1a, Dcp1b and Dcp2. The Dcp proteins are conserved PB components in yeast, C. elegans, Drosophila and mammalian cells.…”

Viral activation of stress-regulated Rho-GTPase signaling pathway disrupts sites of mRNA degradation to influence cellular gene expression

“…29,30,[32][33][34][35] PBs are extremely dynamic, changing in size and number in response to cell cycle stage, nutrient availability, and stresses such as ultraviolet light (UV), osmotic shock and other inhibitors of global translation. 27,33,35,36 PBs can also transiently associate and exchange cargo with stress granules (SGs), cytoplasmic foci that triage stalled translationally-competent mRNPs. 34 PB assembly and disassembly is influenced by changes in the degradative capacity of the cell; for example, when 5 0 -3 0 exonucleolytic decay is prevented, the resulting accumulation of cytoplasmic mRNA awaiting destruction causes PB size and number to increase, whereas inhibiting de novo transcription or halting translation by trapping mRNA in polysomes has the opposite effect.…”

“…37,38 PBs also maintain a dynamic relationship with the cytoskeleton; stationary PBs associate with actin bundles whereas mobile PBs connect to the microtubule network. 36,39,40 Though PB formation was recently shown to be modified by the cytoskeletal regulator RhoA, 22,27,28 the precise mechanism of action remains to be elucidated.…”