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.

The germline of Caenorhabditis elegans derives from a single founder cell, the germline blastomere P4. P4 is the product of four asymmetric cleavages that divide the zygote into distinct somatic and germline (P) lineages. P4 inherits a specialized cytoplasm (“germ plasm”) containing maternally encoded proteins and RNAs. The germ plasm has been hypothesized to specify germ cell fate, but the mechanisms involved remain unclear. Three processes stand out: (1) inhibition of mRNA transcription to prevent activation of somatic development, (2) translational regulation of the nanos homolog nos-2 and of other germ plasm mRNAs, and (3) establishment of a unique, partially repressive chromatin. Together, these processes ensure that the daughters of P4, the primordial germ cells Z2 and Z3, gastrulate inside the embryo, associate with the somatic gonad, initiate the germline transcriptional program, and proliferate during larval development to generate ~2,000 germ cells by adulthood.

Highlights d Global calcineurin signaling in humans revealed through systematic substrate mapping d Discovery of calcineurin-binding sequences enables robust in silico SLiM predictions d BioID uncovers SLiM-dependent calcineurin proximity to nuclear pores and centrosomes d Calcineurin dephosphorylates nuclear pore proteins and regulates transport in vivo

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5 end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.The 7 m GpppN cap structure found at the 5Ј terminus of eukaryotic mRNAs is required for efficient translation (27) and mRNA stability (13). The cap structure is formed through a series of enzymatic reactions. An RNA triphosphatase removes a single phosphate from the 5Ј end of the RNA to yield 5Ј ppN. The diphosphate is then capped by an RNA guanylyltransferase which transfers Gp from a Gppp donor to form the cap structure, GpppN. The cap is further modified by a guanine-N-7 (G-N-7) methylase which transfers a methyl group from S-adenosyl-L-methionine (SAM) to yield 7 m GpppN and S-adenosyl homocysteine (14). This methylated cap can then be subsequently methylated by a ribose-2Ј-O (2Ј-O) methylase to yield 7 m GpppN m . Many viruses of eukaryotes have evolved unusual strategies to generate capped mRNA, including the nonsegmented negative-sense (NNS) RNA viruses. Studies with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, demonstrated that the capping enzyme transfers RNA onto Gpp, rather than Gp onto the 5Ј end of the RNA (1, 28). The mRNA cap methylase activities of VSV are also unusual in that the G-N-7 and 2Ј-O methylase activities use a single SAM binding site (24). Thus, for these viruses, each step of mRNA cap formation is distinct from that of the host.Much understanding of gene expression strategies of NNS RNA viruses comes from studies of VSV. The VSV genome comprises 11,161 nucleotides (nt) of RNA. This is delivered into the cytoplasm of the host cell as a transcription-competent core in which the RNA is completely encapsidated by the nucleocapsid (N) protein and associated with the RNA-dependent RNA polymerase (RdRP). The viral components of the RdRP are a 29-kDa phosphoprotein (P) and a 241-kDa large (L) polymerase subunit (10, 11). In response to a promoter, the VSV RdRP initiates synthesis at the first gene-start (GS) sequence (3Ј UUGUCNNUAC 5Ј) (25,40,41). The nascent mRNA chain is capped and methylated (1, 26), and in response to a specific gene-end (GE) sequence (3Ј-AUACUUUUUUU GA-5Ј), the RdRP polyadenyla...