High Temperature-induced Spindle Destabilization Results in Aborted Pollen Production in Populus

High temperature can induce the production of 2n gametes and aborted pollen during microsporogenesis in Populus canescens. However, the mechanism by which high temperature induces pollen abortion remains unknown. Here, pollen abortion was induced by exposing male flower buds of P. canescens to 38 and 41 °C; pollen morphology, meiotic abnormalities, defects of the meiotic microtubular cytoskeleton, and tapetum development were characterized, and expression analysis of the Actin gene was conducted. We found that the dominant meiotic stage, temperature, and duration of treatment significantly affected the percentage of high temperature-induced aborted pollen. Damaged spindle microtubules and depolymerized microtubular cytoskeletons were observed, which resulted in many lagging chromosomes at anaphase Ⅰ and Ⅱ, as well as aneuploid male gametes and micronuclei, generating aborted pollen grains. Tapetum disintegration was also delayed. However, the anther dehisced normally, and some viable pollen grains were released, suggesting that the delayed degradation of the tapetum was not responsible for pollen abortion. A significant reduction in PtActin gene expression was detected in treated cells, indicating that spindle actin was disrupted. The spindle actin appeared to protect cells against chromosome segregation errors during meiosis.

Sequential binding of zinc triggers tau aggregation

Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Isothermal Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on hTau40 isoform. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part (H14, H32, H94, and H121), the repeat region (H299, C322, H329 and H330) and the C-terminal part (H362, H374, H388 and H407). We then demonstrated that one zinc ion binds first to the repeat region, thus allowing the binding of a second zinc ion to the C-terminal part, while the N-terminal site is independant. Using molecular simulations, we modeled the structure of each site in complex with zinc. Finally, using turbidity and Dynamic Light Scattering (DLS) assays, we showed that the C-terminal site (in particular H388 and H407) is critical for zinc-induced aggregation of tau. Our study highlights key residues involved in zinc induced aggregation of tau. Given the clinical importance of tau aggregation, our findings pave the way for designing potential therapies for tauopathies. Based on our results, we propose a model of zinc-induced aggregation of tau, allowing a better understanding of both the physiological and pathological processes associated with tau-zinc interaction.

A Hypomorphic Mutant of PHD Domain Protein Male Meiocytes Death 1

Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.

Vulnerability of Subcellular Structures to Pathogenesis Induced by Rotenone in SH-SY5Y Cells

Numerous pathological changes of subcellular structures are characteristic hallmarks of neurodegeneration. The main research has focused to mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomal networks as well as microtubular system of the cell. The sequence of specific organelle damage during pathogenesis has not been answered yet. Exposition to rotenone is used for simulation of neurodegenerative changes in SH-SY5Y cells, which are widely used for in vitro modelling of Parkinson´s disease pathogenesis. Intracellular effects were investigated in time points from 0 to 24 h by confocal microscopy and biochemical analyses. Analysis of fluorescent images identified the sensitivity of organelles towards rotenone in this order: microtubular cytoskeleton, mitochondrial network, endoplasmic reticulum, Golgi apparatus and lysosomal network. All observed morphological changes of intracellular compartments were identified before αS protein accumulation. Therefore, their potential as an early diagnostic marker is of interest. Understanding of subcellular sensitivity in initial stages of neurodegeneration is crucial for designing new approaches and a management of neurodegenerative disorders.

P11.06 Non epigenetic effect of vorinostat in glioblastoma cells

BACKGROUND Glioblastoma multiform (GBM) is the most frequent primitive brain tumor. GBM has a high recurrence and mortality. Histone deacetylase (HDAC) inhibitors have evoked great interest because they are able to change transcriptomic profiles to promote tumor cell death but also show undesirable side effects due to the lack of selectivity.We show new properties of low dose vorinostat, which inhibits cytoplasmic HDAC6 and display interesting non-epigenetics effects, especially on the microtubular cytoskeleton. MATERIAL AND METHODS We used murine (GL261) and human (U87 and GBM6 stem cells) cellular models. The cell proliferation was assessed by MTT tests, the migration by the 24 hours Transwell technic and by wound/healing tests. The expression levels of proteins of interest were assessed by Western Blot. Microtubules dynamics were assessed by time-lapse videomicroscopy. The synergy between drugs was tested by Loewe model. RESULTS Vorinostat inhibited the proliferation and the migration of the three cell lines mentioned above at level below the EC50.Vorinostat induces tubulin acetylation and alpha-tubulin c-terminal detyrosination that signed microtubular stabilization and these effects are independent of histone acetylation (HADC3). Interestingly, vorinostat decreases EB1 expression (a bad prognostic factor in GBM) and decreases microtubule dynamics. Moreover, vorinostat decreases neural markers such as GFAP, beta3-tubulin and CNPase and increases mural markers expression such as SMA/EGFR and PDGFR. Finally, it showed a synergy combined with erlotinib. CONCLUSION Low dose vorinostat, which do not affect histone désacetylase, has antitumor effect on glioblastoma cells by a new mechanism involving microtubule cytoskeleton. Interestingly, combination of low doses vorinostatand erlotinibshowed a strong synergy. Low dose, vorinostat could therefore represent an interesting therapeutic option and fewer side effects and that could be used to increased GBM patient sensitivity to erlotinib.

Stx5-Mediated ER-Golgi Transport in Mammals and Yeast

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 5 (Stx5) in mammals and its ortholog Sed5p in Saccharomyces cerevisiae mediate anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking. Stx5 and Sed5p are structurally highly conserved and are both regulated by interactions with other ER-Golgi SNARE proteins, the Sec1/Munc18-like protein Scfd1/Sly1p and the membrane tethering complexes COG, p115, and GM130. Despite these similarities, yeast Sed5p and mammalian Stx5 are differently recruited to COPII-coated vesicles, and Stx5 interacts with the microtubular cytoskeleton, whereas Sed5p does not. In this review, we argue that these different Stx5 interactions contribute to structural differences in ER-Golgi transport between mammalian and yeast cells. Insight into the function of Stx5 is important given its essential role in the secretory pathway of eukaryotic cells and its involvement in infections and neurodegenerative diseases.

TROAP Promotes Breast Cancer Proliferation and Metastasis

Trophinin-associated protein (TROAP) is a cytoplasmic protein required for microtubular cytoskeleton regulation and spindle assembly, and its expression plays a critical role in the initiation and progression of various types of cancer. However, little is known about the role of TROAP in breast cancer (BC). TROAP mRNA expression levels and clinical data from Gene Expression Omnibus (GEO) datasets (GSE42568, 104 BC patients; GSE1456, 159 BC patients; and GSE21653, 266 BC patients) were analyzed by the R2: Genomics Analysis and Visualization Platform to estimate overall survival (OS). We also analyzed the genes correlated with TROAP by gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to predict potential relationships between TROAP and other genes in BC. Our study verified that both TROAP mRNA and protein expression levels were upregulated in human BC samples and cell lines. In vitro experiments demonstrated that TROAP knockdown significantly inhibited cell proliferation, the G1 to S phase transition, and the migration and invasion abilities of BC cells. The present study suggests that TROAP plays an important role in promoting the proliferation, invasion, and metastasis of BC.