The midbody and midbody remnant are assembly sites for RNA and active translation

Abstract

The midbody (MB) is a transient structure at the spindle midzone that is required for cytokinesis, the terminal stage of cell division. Long ignored as a vestigial remnant of cytokinesis, we now know MBs are released post-abscission as extracellular vesicles called MB remnants (MBRs) and can modulate cell proliferation, fate decisions, tissue polarity, neuronal architecture, and tumorigenic behavior. Here, we demonstrate that the MB matrix,the structurally amorphous MB core of unknown composition,is the site of ribonucleoprotein assembly and is enriched in mRNAs that encode proteins involved in cell fate, oncogenesis, and pluripotency, that we are calling the MB granule. Using a quantitative transcriptomic approach, we identified a population of mRNAs enriched in mitotic MBs and confirmed their presence in signaling MBR vesicles released by abscission. The MB granule is unique in that it is translationally active, contains both small and large ribosomal subunits, and has both membrane-less and membrane-bound states. Both MBs and post-abscission MBRs are sites of spatiotemporally regulated translation, which is initiated when nascent daughter cells re-enter G1 and continues after extracellular release. We demonstrate that the MB is the assembly site of an RNP granule. MKLP1 and ARC are necessary for the localization and translation of RNA in the MB dark zone, whereas ESCRT-III was necessary to maintain translation levels in the MB. Our data suggest a model in which the MB functions as a novel RNA-based organelle with a uniquely complex life cycle. We present a model in which the assembly and transfer of RNP complexes are central to post-mitotic MBR function and suggest the MBR serves as a novel mode of RNA-based intercellular communication with a defined biogenesis that is coupled to abscission, and inherently links cell division status with signaling capacity. To our knowledge, this is the first example of an autonomous extracellular vesicle with active translation activity.

The biogenesis of large extracellular vesicles occurs in mitosis

“…5214,6114,38 The implications of this work are promising, because EVs serve as excellent vehicles for therapeutics and as factors for cancer and neurodegenerative disease diagnostics. We also suggest that the knowledge uncovered in this study provides the field a starting point to harness the power of the MBR, which is assembled during mitosis from the spindle midzone and midbody and is comprised of MKLP1, microtubules, RNA, and protein [39][40][41]52 .…”

“…Lastly, dsRNA is a well-known EV marker and the main nucleic acid component transported by extracellular vesicles 18,65 . Our group has recently discovered dsRNA in the spindle midzone, midbody, and MBR 52 . Collectively, these markers that have been published and used to study EVs have now been documented as midbody and MBR markers, further suggesting that the biogenesis of large EVs occurs mainly in mitosis.…”

“…As our lab recently discovered that the midbody and MBR are sites of active translation 52 , we sought to determine if common and commercial methods of isolating EVs also preserve translation capacity. Puromycin antibody labeling was used as a proxy for translation activity because the localization pattern is similar to HPG-Click-IT activity 52,88,89 . Here, cleared culture media was treated with puromycin for 10 minutes at 37°C.…”

“…MBRs arise at the end of cytokinesis during a process called abscission, which separates the newly formed daughter cells 40,41,[51][52][53][54][55] . MBRs have been suggested to play post-mitotic roles in cell-cell communication, cell fate, and signaling, which are similar to the roles of EVs 39,41,[56][57][58] .…”

An oocyte meiotic midbody cap is required for developmental competence in mice

“…Studies on MB functions have extended beyond regulatory functions of cytokinesis, and now indicate their signaling capabilities 9 and ribonucleoprotein (RNP) properties 13,[32][33][34][35] . An array of proteins involved in translation, translational regulation, and RNA molecules are enriched in mitotic MBs 33,34,36 .…”