Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging

Abstract

Structure of the meiotic spindle T he meiotic spindle is made up of shorter microtubules than previously believed, suggest results (Rockefeller University, New York, NY), and colleagues. Current models of the spindle, as a bipolar array of overlapping fi laments extending from opposite spindle poles, will require revision. To get a closer look at the architecture of the meiotic spindle, Yang et al. incorporated labeled tubulin subunits into the spindle in a cell-free system. By refi ning their fl uorescent speckle microscopy techniques, the authors were able for the fi rst time to track individual tubulin subunits (seen as speckles) in a single tubulin polymer. The authors identifi ed pairs of speckles representing subunits on the same fi lament. Speckle separation supplied them with the minimum length of that fi lament. They then fi tted a mathematical model to these observed lengths to predict overall fi lament lengths: most fi laments were only 40% of the total spindle length. The short fi laments were also scattered throughout the spindle. The researchers now propose that the spindle is a tiled array of overlapping short fi laments. The group next examined how spindle-associated proteins might control fi lament and spindle size. By inhibiting microtubule motor proteins, they found that dynein-dynactin limited individual fi ber lengths and thus overall spindle length. Kinesin 5 activity limited the overlap between fi bers by sliding them apart. "Our work suggests the spindle is a self-organizing system, whose stability and functional characteristics are built on these kind of local interactions," says Kapoor. Localized mRNA is the norm L ocation, location, location. It's critical for real estate, proteins, and-according to work by Eric Lécuyer, Henry Krause, and colleagues (University of Toronto, Canada)-mRNAs, too. Several localized mRNAs have been previously studied, but just how many transcripts are localized in the cell, and in what patterns, is unknown. Lécuyer et al. approached this problem by optimizing fl uorescence in situ hybridization (FISH) in a global analysis of developmentally expressed mRNAs. They found that 71% of the mRNAs in early fl y embryos showed specifi c patterns of subcellular localization. In several cases, they found new examples of mRNAs that colocalized with their protein products. Less energy is probably required to transport a few copies of an mRNA than to move around many more copies of the protein. And the proteins will be created where they are needed and possibly prevented from straying where they are not wanted. "We need to revise the textbook image of proteins being made in a centralized location near the nucleus, then traffi cking to their ultimate locations," says Krause. "Our work shows that the mRNAs are an intelligent actor, not just a dumb vehicle for creating proteins." With their new database, the group can now further investigate how and why mRNAs are localized.

The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells

“…The reason for the reduced tension in augmin-depleted cells may be that chromosomes are never stably attached to MTs, possibly because of fewer kinetochore MTs. Alternatively, augmin may impact chromosome tension by producing cross-linked, short antiparallel MTs that act as substrates for motor-dependent sliding forces, as suggested for meiotic metaphase spindles reconstituted in Xenopus extracts (23). A reduction in sister kinetochore tension might be responsible for checkpoint activation after augmin depletion.…”

Microtubule assembly during mitosis – from distinct origins to distinct functions?

“…In spindles assembled in Xenopus egg extracts, interpolar MTs are particularly abundant and comprise 95% of the spindle MTs (Ohi et al, 2007). They form a 'polar' array of parallel MTs near the spindle poles and a 'barrel' array of antiparallel, overlapping MTs close to the chromosomes (Yang et al, 2007). During anaphase, interpolar MTs are rearranged and become the major component of the central spindle, where they form an array of antiparallel MTs with bundled overlapping plus-ends (Glotzer, 2009).…”

Op18 reveals the contribution of nonkinetochore microtubules to the dynamic organization of the vertebrate meiotic spindle

“…Two velocity modes for poleward flux were revealed by statistical analysis, and led to the proposal that non-kMTs in the spindle can be subdivided into a ''barrel'' array of antiparallel overlapping microtubules centered on chromosomes, and 2 ''polar'' arrays of microtubules at each end of the spindle. Single-fluorophore qFSM has allowed the flux of individual microtubules in meiotic spindles to be examined, and the length and spatial distribution of microtubules to be estimated (14). Together, these recent FSM studies suggest that the vertebrate meiotic spindle is comprised mostly of microtubules that are shorter than half the length of the spindle, and are organized into a barrel array that is dynamically coupled to the 2 polar arrays.…”