mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

The RNA-binding protein Igf2bp3 is critical for embryonic and germline development in zebrafish.

The ability to reproduce is essential in all branches of life. In metazoans, this process is initiated by formation of the germline, a group of cells that are destined to form the future gonads, the tissue that will produce the gametes. The molecular mechanisms underlying germline formation differs between species. In zebrafish, development of the germline is dependent on the specification, migration and proliferation of progenitors called the primordial germ cells (PGCs). PGC specification is dependent on a maternally provided cytoplasmic complex of ribonucleoproteins (RNPs), the germplasm. Here, we show that the conserved RNA-binding protein (RBP), Igf2bp3, has an essential role during early embryonic development and germline development. Loss of Igf2bp3 leads to an expanded yolk syncytial layer (YSL) in early embryos, reduced germline RNA expression, and mis-regulated germline development. We show that loss of maternal Igf2bp3 function results in translational de-regulation of a Nodal reporter during the mid-blastula transition. Furthermore, maternal igf2bp3 mutants exhibit reduced expression of germplasm transcripts, defects in chemokine guidance, abnormal PGC behavior and germ cell death. Consistently, adult igf2bp3 mutants show a strong male bias. Our findings suggest that Igf2bp3 is essential for normal embryonic and germline development, and acts as a key regulator of sexual development.

N-terminal acetylation and arginylation of actin determines the architecture and assembly rate of linear and branched actin networks.

The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis, and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and cofilin, recent work also implicates posttranslational acetylation or arginylation of the actin N terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing and modification of the N terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in cofilin-mediated severing. Taken together, these results suggest that cells can employ these differently modified actins to regulate actin dynamics. In addition, unprocessed actin with an N-terminal methionine residue shows very different effects on formin-mediated elongation, Arp2/3-mediated nucleation, and severing by cofilin. Altogether, this study shows that the nature of the N terminus of actin can promote distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge.

Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.

Actin is one of the most abundant eukaryotic cytoskeletal polymer-forming proteins, which, in the filamentous form, regulates a number of physiological processes, ranging from cell division and migration to development and tissue function. Actins have different post-translational modifications (PTMs) in different organisms, including methionine, alanine, aspartate and glutamate N-acetylation, N-arginylation and the methylation of the histidine at residue 73 (His-73), with different organisms displaying a distinct signature of PTMs. Currently, methods are not available to produce actin isoforms with an organism-specific PTM profile. Here, we report the Pick-ya actin method, a method to express actin isoforms from any eukaryote with its own key characteristic PTM pattern. We achieve this using a synthetic biology strategy in a yeast strain that expresses, one, actin isoforms with the desired N-end via ubiquitin fusion and, two, mammalian enzymes that promote acetylation and methylation. Pick-ya actin should greatly facilitate biochemical, structural and physiological studies of the actin cytoskeleton and its PTMs.

Rapid production of pure recombinant actin isoforms in Pichia pastoris.

Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.

Translational co-regulation of a ligand and inhibitor by a conserved RNA element.

In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3'UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3'UTR, the lft1 and lft2 3'UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a 'translational regulon'.

Molecular mechanisms that restrict yeast centrosome duplication to one event per cell cycle.

BACKGROUND: The spindle pole body (SPB) of budding yeast is the functional equivalent of the mammalian centrosome. Like the centrosome, the SPB duplicates once per cell cycle. The new SPB assembles adjacent to the mother SPB at a substructure called the bridge. The half-bridge, the bridge precursor, is a one-sided extension of the SPB central plaque layered on both sides of the nuclear envelope. Parallel Sfi1 molecules longitudinally span the half-bridge with their N termini embedded in the SPB central plaque, whereas their C termini mark the half-bridge distal end. In early G1, half-bridge elongation by antiparallel C-to-C dimerization of Sfi1 exposes free N-Sfi1 where the new SPB assembles. After SPB duplication, the dimerized Sfi1 is severed to allow spindle formation and SPB reduplication. RESULTS: We show that Sfi1 C-terminal domain harbors phosphorylation sites for Cdk1 and the polo-like kinase Cdc5. Cdk1 and, to a lesser extent, Cdc5 inhibit SPB duplication as phosphomimetic sfi1 mutations lead to metaphase cells with a single SPB. In contrast, phosphoinhibitory sfi1 mutations in Cdk1 sites are lethal because cells fail to sever the bridge after SPB duplication. Moreover, Cdc14 dephosphorylates C-Sfi1 to prepare it for a new round of duplication, and the kinase Mps1 promotes Sfi1 extension in G1. CONCLUSIONS: Positive (Cdc14) and negative (Cdk1 and Cdc5) SPB duplication signals are integrated at the level of the half-bridge component Sfi1. In addition, Mps1 activates Sfi1 duplication. Fluctuating activities of these regulators ensure one SPB duplication event per cell cycle.

N-terminal regions of Mps1 kinase determine functional bifurcation.

Mps1 is a conserved kinase that in budding yeast functions in duplication of the spindle pole body (SPB), spindle checkpoint activation, and kinetochore biorientation. The identity of Mps1 targets and the subdomains that convey specificity remain largely unexplored. Using a novel combination of systematic deletion analysis and chemical biology, we identified two regions within the N terminus of Mps1 that are essential for either SPB duplication or kinetochore biorientation. Suppression analysis of the MPS1 mutants defective in SPB duplication and biochemical enrichment of Mps1 identified the essential SPB components Spc29 and the yeast centrin Cdc31 as Mps1 targets in SPB duplication. Our data suggest that phosphorylation of Spc29 by Mps1 in G1/S recruits the Mps2-Bbp1 complex to the newly formed SPB to facilitate its insertion into the nuclear envelope. Mps1 phosphorylation of Cdc31 at the conserved T110 residue controls substrate binding to Kar1 protein. These findings explain the multiple SPB duplication defects of mps1 mutants on a molecular level.