Spindle assembly on immobilized chromatin micropatterns.

We describe a method to assemble meiotic spindles on immobilized micropatterns of chromatin built on a first layer of biotinylated BSA deposited by microcontact printing. Such chromatin patterns routinely produce bipolar spindles with a yield of 60%, and offer the possibility to follow spindle assembly dynamics, from the onset of nucleation to the establishment of a quasi steady state. Hundreds of spindles can be recorded in parallel for different experimental conditions.

Mitotic spindle assembly on chromatin patterns made with deep UV photochemistry.

We provide a detailed method to generate arrays of mitotic spindles in vitro. Spindles are formed in extract prepared from unfertilized Xenopus laevis eggs, which contain all the molecular ingredients of mitotic spindles. The method is based on using deep UV photochemistry to attach chromatin-coated beads on a glass surface according to a pattern of interest.

Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts.

Female meiotic spindles in many organisms form in the absence of centrosomes, the organelle typically associated with microtubule (MT) nucleation. Previous studies have proposed that these meiotic spindles arise from RanGTP-mediated MT nucleation in the vicinity of chromatin; however, whether this process is sufficient for spindle formation is unknown. Here, we investigated whether a recently proposed spindle-based MT nucleation pathway that involves augmin, an 8-subunit protein complex, also contributes to spindle morphogenesis.

Chromatin shapes the mitotic spindle.

In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles.

Regulation of microtubule dynamics by reaction cascades around chromosomes.

During spindle assembly, chromosomes generate gradients of microtubule stabilization through a reaction-diffusion process, but how this is achieved is not well understood. We measured the spatial distribution of microtubule aster asymmetry around chromosomes by incubating centrosomes and micropatterned chromatin patches in frog egg extracts. We then screened for microtubule stabilization gradient shapes that would generate such spatial distributions with the use of computer simulations.

A sensitized PiggyBac-based screen for regulators of border cell migration in Drosophila.

Migration of border cells during Drosophila melanogaster oogenesis is a good model system for investigating the genetic requirements for cell migration in vivo. We present a sensitized loss-of-function screen used to identify new genes required in border cells for their migration. Chromosomes bearing FRTs on all four major autosomal arms were mutagenized by insertions of the transposable element PiggyBac, allowing multiple parallel clonal screens and easy identification of the mutated gene.

Splicing regulates NAD metabolite binding to histone macroH2A.

Histone macroH2A is a hallmark of mammalian heterochromatin. Here we show that human macroH2A1.1 binds the SirT1-metabolite O-acetyl-ADP-ribose (OAADPR) through its macro domain.

The macro domain is an ADP-ribose binding module.

The ADP-ribosylation of proteins is an important post-translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules.

Reef-coral proteins as visual, non-destructive reporters for plant transformation.

Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study.