Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems.

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2APPTR−½. Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.DOI: http://dx.doi.org/10.7554/eLife.04591.001

Nonsegmented negative-sense (nsNS) RNA viruses typically replicate within the host cell cytoplasm and do not have access to the host mRNA capping machinery. These viruses have evolved a unique mechanism for mRNA cap formation in that the guanylyltransferase transfers GDP rather than GMP onto the 5 end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype nsNS RNA virus, we now provide genetic and biochemical evidence that its mRNA cap methylase activities are also unique. Using recombinant VSV, we determined the function in mRNA cap methylation of a predicted binding site in the polymerase for the methyl donor, S-adenosyl-L-methionine. We found that amino acid substitutions to this site disrupted methylation at the guanine-N-7 (G-N-7) position or at both the G-N-7 and ribose-2-O (2-O) positions of the mRNA cap. These studies provide genetic evidence that the two methylase activities share an S-adenosyl-L-methioninebinding site and show that, in contrast to other cap methylation reactions, methylation of the G-N-7 position is not required for 2-O methylation. These findings suggest that VSV evolved an unusual strategy of mRNA cap methylation that may be shared by other nsNS RNA viruses.capping ͉ evolution ͉ methyltransferase ͉ S-adenosyl-L-methionine

SUMMARY Asymmetric segregation of P granules during the first four divisions of the C. elegans embryo is a classic example of cytoplasmic partitioning of germline determinants. It is thought that asymmetric partitioning of P granule components during mitosis is essential to distinguish germline from soma. We have identified a mutant (pptr-1) where P granules become unstable during mitosis and P granule proteins and RNAs are distributed equally to somatic and germline blastomeres. Despite symmetric partitioning of P granule components, pptr-1 mutants segregate a germline that uniquely expresses P granules during post-embryonic development. pptr-1 mutants are fertile, except at high temperatures. Hence, asymmetric inheritance of maternal P granules is not essential to specify germ cell fate. Instead, it may serve to protect the nascent germline from stress.