Although mechanisms of embryonic development are similar between mice and humans, the time scale is generally slower in humans. To investigate these interspecies differences in development, we recapitulate murine and human segmentation clocks that display 2- to 3-hour and 5- to 6-hour oscillation periods, respectively. Our interspecies genome-swapping analyses indicate that the period difference is not due to sequence differences in the HES7 locus, the core gene of the segmentation clock. Instead, we demonstrate that multiple biochemical reactions of HES7, including the degradation and expression delays, are slower in human cells than they are in mouse cells. With the measured biochemical parameters, our mathematical model accounts for the two- to threefold period difference between the species. We propose that cell-autonomous differences in biochemical reaction speeds underlie temporal differences in development between species.

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.

During open mitosis in higher eukaryotic cells, the nuclear envelope completely breaks down and then mitotic chromosomes are exposed in the cytoplasm. By contrast, mitosis in lower eukaryotes, including fungi, proceeds with the nucleus enclosed in an intact nuclear envelope. The mechanism of mitosis has been studied extensively in yeast, a closed mitosis organism. Here, we describe a form of mitosis in which the nuclear envelope is torn by elongation of the nucleus in the fission yeast Schizosaccharomyces japonicus. The mitotic nucleus of Sz. japonicus adopted a fusiform shape in anaphase, and its following extension caused separation. Finally, a tear in the nuclear envelope occurred in late anaphase. At the same time, a polarized-biased localization of nuclear pores was seen in the fusiform-shaped nuclear envelope, suggesting a compromise in the mechanical integrity of the lipid membrane. It has been known that nuclear membrane remains intact in some metazoan mitosis. We found that a similar tear of the nuclear envelope was also observed in late mitosis of the Caenorhabditis elegans embryo. These findings provide insight into the diversity of mitosis and the biological significance of breakdown of the nuclear envelope.

The evolutionarily divergent class of kinetoplastid organisms has a set of unconventional kinetochore proteins that drive chromosome segregation, but it is unclear which components interact with spindle microtubules. Llauró et al. now identify KKT4 as the first microtubule-binding kinetochore protein in Trypanosoma brucei, a major human pathogenic parasite.

We report that free tubulin subunits in the Caenorhabditis elegans embryo accumulate in the nascent spindle region, independent of spindle formation. We propose that this newly identified mechanism of accumulation of free tubulin and other molecules at the nascent spindle region contributes to efficient assembly of the mitotic spindle.

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Summary statementTrypanosomes do not have a spindle checkpoint but have an ability to regulate the timing of nuclear division by modulating the cyclin B protein level. AbstractKinetoplastids have a nucleus that contains the nuclear genome and a kinetoplast that contains the mitochondrial genome. These single-copy organelles must be duplicated and segregated faithfully to daughter cells at each cell division. In Trypanosoma brucei, although duplication of both organelles starts around the same time, segregation of the kinetoplast precedes that of the nucleus. Cytokinesis subsequently takes place so that daughter cells inherit a single copy of each organelle. Very little is known about the molecular mechanism that governs the timing of these events.Furthermore, it is thought that T. brucei lacks a spindle checkpoint that delays the onset of nuclear division in response to spindle defects. Here we show that a mitotic cyclin CYC6 has a dynamic localization pattern during the cell cycle, including kinetochore localization. Using CYC6 as a molecular cell cycle marker, we confirmed that T. brucei cannot delay the onset of anaphase in response to a bipolar spindle assembly defect. Interestingly, expression of a stabilized form of CYC6 caused the nucleus to arrest in a metaphase-like state without preventing cytokinesis. We propose that trypanosomes have an ability to regulate the timing of nuclear division by modulating the CYC6 protein level, without a spindle checkpoint.

1While the mechanisms of embryonic development are similar between mouse and human, 2 the tempo is in general slower in human. The cause of interspecies differences in 3 developmental time remains a mystery partly due to lack of an appropriate model system 1 . 4 Since murine and human embryos differ in their sizes, geometries, and nutrients, we use 5 in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells 6 between the species in similar culture conditions. As an example of well-defined 7 developmental time, we focus on the segmentation clock, oscillatory gene expression that 8 regulates the timing of sequential formation of body segments [2][3][4] . In this way we 9 recapitulate the murine and human segmentation clocks in vitro, showing that the species-10 specific oscillation periods are derived from cell autonomous differences in the speeds of 11 biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and 12 human PSCs displayed the oscillatory expression of HES7, the core gene of the 13 segmentation clock 5,6 , with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours 14 (human PSM). Swapping HES7 loci between murine and human genomes did not change 15 the oscillation periods dramatically, denying the possibility that interspecies differences 16 in the sequences of HES7 loci might be the cause of the observed period difference. 17Instead, we found that the biochemical reactions that determine the oscillation period, 18 such as the degradation of HES7 and delays in its expression, are slower in human PSM 19 compared with those in mouse PSM. With the measured biochemical parameters, our 20 mathematical model successfully accounted for the 2-3-fold period difference between 21 mouse and human. We further demonstrate that the concept of slower biochemical 22 reactions in human cells is generalizable to several other genes, as well as to another cell 23 type. These results collectively indicate that differences in the speeds of biochemical 24 reactions between murine and human cells give rise to the interspecies period difference 25 of the segmentation clock and may contribute to other interspecies differences in 26 developmental time.27 28 Main 29 To compare murine and human segmentation clocks in vitro, we induced PSM-like cells 30 from mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells 31 (iPSCs) (Fig. 1a), as other groups have recently reported 7-12 . In essence, our PSM 32 induction protocol is based on activation of WNT and FGF signaling as well as inhibition 33 of TGFβ and BMP signaling 9,12 . Prior to the PSM induction, mouse ESCs, which are in 34 the naïve pluripotent state, were pretreated with ACTIVIN A and bFGF and converted to 35 mouse epiblast-like cells (EpiLCs) that possess primed pluripotency as human iPSCs do.36 3To visualize the segmentation clock in the induced PSM, we introduced a HES7 promoter-1 luciferase reporter 13,14 , detecting clear synchronized oscillations of HES7 expression in 2 both murine a...