Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

Spatial Control of Microtubule Length and Lifetime by Opposing Stabilizing and Destabilizing Functions of Kinesin-8

A Developmentally Regulated Two-Step Process Generates a Noncentrosomal Microtubule Network in Drosophila Tracheal Cells

The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys40 residue of α-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2.

Mitosis is a highly regulated process characterized by dramatic and coordinated morphological changes to ensure the fidelity of chromosome segregation. Missegregation of mitotic chromosomes leads to a condition that underlies chromosomal instability(1), which is a hallmark of cancer. In order to assure symmetry and bipolarity of the cell division process, mitotic spindle microtubules properly segregate mitotic chromosomes (2). Among the several isoforms of serine/threonine kinases, PKCε is one of the best understood for its role as a transforming oncogene, and it has been found overexpressed in different types of tumors. In 2008, Saurin and colleagues demonstrated the involvement of PKCε in the regulation of the late stage of mitosis (3). Through its association with 14-3-3 at the midbody, PKCε is essential for the successful completion of cytokinesis, and the inhibition of functional PKCε-14-3- 3 complex leads to abscission failure and multinucleated phenotype in cells. In this study, we found that PKCε is involved in mitotic spindle stability. Using fluorescence microscopy, we found that the active form of PKCε (phosphorylated at Ser-729), colocalizes to the centrosome in cells in metaphase, where the mitotic spindle nucleation occurs. Furthermore, experiments of co-immunoprecipitation revealed that, when cells are synchronized in metaphase, PKCɛ is associated to ɣ-tubulin, a member of the tubulin superfamily localized to the microtubule organizing centers and is essential for microtubule nucleation from centrosomes. Consequently modulation of PKCɛ expression affects spindle stability: PKCɛ downregulation by specific shRNA results in mitotic spindle disorganization with a reduction of the amount of centrosomal and mitotic ɣ-Tubulin and αβ-tubulin fluorescence. Mitotic spindle formation assays using Nocodazole, known to interfere with the polymerization of microtubules, revealed that cells lacking PKCɛ were unable to regrow microtubules after depolymerization. These results reveal a novel role of PKCɛ in mitotic spindle stability, which likely determinant for genome stability.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

To determine whether mutations in the D5 dopamine receptor gene (DRD5) are associated with schizophrenia, the gene was examined in 78 unrelated schizophrenic individuals (156 DRD5 alleles). After amplification by the polymerase chain reaction, products were examined by dideoxy fingerprinting (ddF), a screening method related to single strand conformational polymorphism analysis that detects essentially 100% of mutations. All samples with abnormal ddF patterns were sequenced. Nine different sequence changes were identified. Five of these were sequence changes that would result in protein alterations; of these, one was a nonsense change (C335X), one was a missense change in an amino acid conserved in all dopamine receptors (N351D), two were missense changes in amino acids that are identical in only some dopamine receptors and in only some species (A269V; S453C), and one was a missense change in a non-conserved amino acid (P330Q). To investigate whether the nonsense change (C335X), predicted to prematurely truncate the receptor protein and result in a 50% diminution of functional protein, was associated with schizophrenia, other neuropsychiatric diseases, or specific neuropsychological, psychophysiological, or personality traits, both case-control and family analyses were performed. No statistically-significant associations were detected with schizophrenia or other neuropsychiatric disease. There also were no significant associations between any one measure of neuropsychological function. However, a post-hoc analysis of combined measures of frontal lobe function hinted that heterozygotes for C335X may have a vulnerability to mild impairment, but these findings must be interpreted with caution.(ABSTRACT TRUNCATED AT 250 WORDS)

During exercise, amino acid oxidation and protein breakdown are enhanced while protein synthesis is suppressed, even though protein does not constitute a quantitatively impor- tant energy source. In response to exercise-induced stimulation, various changes in free amino acids occur in skeletal muscle to meet physiological demands. Plasma amino acids are also under the influence of various types of stress, including exercise stress. For example, acute ex- ercise increases alanine and glutamine levels, but decreases glutamate levels in skeletal muscle. At the same time, it increases tryptophan and taurine levels, but decreases glutamine levels in plasma. Prolonged exercise decreases glutamine and glutamate levels, while increasing tyrosine and phenylalanine levels in skeletal muscle. Furthermore, when prolonged exercise-induced changes in amino acid levels are compared between trained and untrained individuals, gluta- mate and taurine levels in skeletal muscle and phenylalanine, leucine, isoleucine, and tyrosine levels in plasma are higher in trained individuals. This review provides an overview of changes in amino acid levels in skeletal muscle and plasma, with a focus on changes induced by exer-

Convergent and parallel evolution occur more frequently than previously thought. Here, we focus on the evolutionary adaptations of angiosperms at sub-zero temperatures. We begin by introducing the history of research on convergent and parallel evolution, defining all independent similarities as convergent evolution. Our analysis reveals that frost zones (periodic or constant), which cover49.1% of Earth's land surface, host 137 angiosperm families, with over 90% of their species thriving in these regions. In this context, we revisit the global biogeography and evolutionary trajectories of plant traits, such as herbaceous form and deciduous leaves, that are thought to be evasion strategies for frost adaptation. At the physiological and molecular levels, many angiosperms have independently evolved cold acclimation mechanisms through multiple pathways in addition to the well-characterized C-repeat binding factor/dehydration-responsive element binding protein 1 (CBF/DREB1) regulatory pathway. These convergent adaptations have occurred across various molecular levels, including amino acid substitutions and changes in gene duplication and expression within the same or similar functional pathways; however, identical amino acid changes are rare. Our results also highlight the prevalence of polyploidy in frost zones and the occurrence of paleopolyploidization events during global cooling. These patterns suggest repeated evolution in cold climates. Finally, we discuss plant domestication and predict climate zone shifts due to global warmingand their effects on plant migration and in situ adaptation. Overall, the integration of ecological and molecular perspectives is essential for understanding and forecasting plant responses to climate change.

Clasps Depolymerize Microtubules in a Nucleotide-Dependent Manner

Studies of the Interaction of a Kinesin-13 Protein with Microtubules

Mitotic spindles are mechanical structures that play a critical role in cell division by generating forces to separate chromosomes. They are ordered assemblages of proteins that make up microtubules (MT) and microtubule connectors whose mechanical properties are responsible for their structural integrity under mitotic forces. We use a continuum mechanics approach to study the stability of equilibrium of a mitotic spindle as a whole. We create and apply a finite element (FE) parameterised model of interpolar MTs, astral MTs and MT connectors varying the number of MT filaments and the arrangement of their interconnections. The model is based on the experimental data on Fission Yeast spindles in late anaphase B and mitotic HeLa cells [1]–[3]. We account for the complex interactions between interpolar MTs, astral MTs, connectors and centrosomes through mechanical coupling. Comparing the results with experiments and Molecular dynamics-based simulations [1], we demonstrate the great potential of Structural mechanics methods to address the stability of spindles. Here we report how buckling states of the spindle get localised towards either of centrosomes due to the irregular placement of microtubules and irregularities in MT coupling. In certain cases, such behaviour may result in nuclear misplacement, asymmetric division or other abnormalities. [1] J. J. Ward, H. Roque, C. Antony, and F. Nedelec, “Mechanical design principles of a mitotic spindle,” eLife, vol. 3, p. e03398, 2014. [2] F. Pampaloni, G. Lattanzi, A. Jonas, T. Surrey, E. Frey, and E.-L. Florin, “Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length,” Proc. Natl. Acad. Sci., vol. 103, no. 27, pp. 10248–10253, 2006. [3] F. M. Nixon, C. Gutierrez-Caballero, F. E. Hood, D. G. Booth, I. A. Prior, and S. J. Royle, “The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle,” eLife, vol. 4, no. JUNE2015, pp. 1–21, 2015.

The incursions of H7 subtype low-pathogenicity avian influenza virus (LPAIV) from wild birds into poultry and its mutations to highly pathogenic avian influenza virus (HPAIV) have been an ongoing concern in North America. Since 2000, 10 phylogenetically distinct H7 virus outbreaks from wild birds have been detected in poultry, six of which mutated to HPAIV. To study the molecular evolution of the H7 viruses that occurs when changing hosts from wild birds to poultry, we performed analyses of the North American H7 hemagglutinin (HA) genes to identify amino acid changes as the virus circulated in wild birds from 2000 to 2019. Then, we analyzed recurring HA amino acid changes and gene constellations of the viruses that spread from wild birds to poultry. We found six HA amino acid changes occurring during wild bird circulation and 10 recurring changes after the spread to poultry. Eight of the changes were in and around the HA antigenic sites, three of which were supported by positive selection. Viruses from each H7 outbreak had a unique genotype, with no specific genetic group associated with poultry outbreaks or mutation to HPAIV. However, the genotypes of the H7 viruses in poultry outbreaks tended to contain minor genetic groups less observed in wild bird H7 viruses, suggesting either a biased sampling of wild bird AIVs or a tendency of having reassortment with minor genetic groups prior to the virus's introduction to poultry. IMPORTANCE Wild bird-origin H7 subtype avian influenza viruses are a constant threat to commercial poultry, both directly by the disease they cause and indirectly through trade restrictions that can be imposed when the virus is detected in poultry. It is important to understand the genetic basis of why the North American lineage H7 viruses have repeatedly crossed the species barrier from wild birds to poultry. We examined the amino acid changes in the H7 viruses associated with poultry outbreaks and tried to determine gene reassortment related to poultry adaptation and mutations to HPAIV. The findings in this study increase the understanding of the evolutionary pathways of wild bird AIV before infecting poultry and the HA changes associated with adaptation of the virus in poultry.

Mitochondrial genes are part of the oxidative phosphorylation pathway and important for energy production. Although evidence for positive selection at the mitochondrial level exists, few studies have investigated the link between amino acid changes and phenotype. Here we test the hypothesis that differences in two life-history related traits, migratory distance between spawning and foraging areas and larval phase duration, are associated with divergent selection within the mitochondrial ATP6 gene in anguillid eels. We compare amino acid changes among 18 species with the sequence of the putative ancestral species, believed to have shown short migratory distance and larval phase duration. We find positive correlations between both life-history related traits and (i) the number of amino acid changes and (ii) the strength of the combined physico-chemical and structural changes at positions previously identified as candidates for positive selection. This supports a link between genotype and phenotype driven by positive selection at ATP6.