Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex

Beaver and Aspen: Synergy Among Keystone Species

In the West, climate change is likely to increase the frequency, intensity, and duration of drought. Restoration of soils and water storage capacity can help create resilient uplands and riverscapes (i.e., streams and the valley bottoms). Over the past two centuries, common land uses, the removal of beaver and wood, straightening of streams, and damage to riparian areas have created simplified, structurally starved, riverscapes. Degraded streams are very efficient at transporting water, sediment, and nutrients downstream. Aspen forests are also biological hotspots that have been degraded by past land uses such as overbrowsing ungulates, land clearing, fire suppression, and outright removal in favor of timber species. Loss of riverscape and aspen habitats has a disproportionate impact on biodiversity and landscape resilience. When aspen occur in or near riverscapes they are a preferred food and building material for beavers. Beaver, in-turn, can stimulate aspen regeneration, both through cutting and restoring hydrologic function in riparian areas. Adding beavers can reinstate riparian processes, increase aspen growth and diversity that extends to uplands, and buffer ecosystem sensitivity to extended drought

Autocatalytic plume pinch-off

A localized source of buoyancy flux in a non-reactive fluid medium creates a plume. The flux can be provided by either heat, a compositional difference between the fluid comprising the plume and its surroundings, or a combination of both. For autocatalytic plumes produced by the iodate-arsenous acid reaction, however, buoyancy is produced along the entire reacting interface between the plume and its surroundings. Buoyancy production at the moving interface drives fluid motion, which in turn generates flow that advects the reaction front. As a consequence of this interplay between fluid flow and chemical reaction, autocatalytic plumes exhibit a rich dynamics during their ascent through the reactant medium. One of the more interesting dynamical features is the production of an accelerating vortical plume head that in certain cases pinches-off and detaches from the upwelling conduit. After pinch-off, a new plume head forms in the conduit below, and this can lead to multiple generations of plume heads for a single plume initiation. We investigated the pinch-off process using both experimentation and simulation. Experiments were performed using various concentrations of glycerol, in which it was found that repeated pinch-off occurs exclusively in a specific concentration range. Autocatalytic plume simulations revealed that pinch-off is triggered by the appearance of accelerating flow in the plume conduit.Comment: 10 figures. Accepted for publication in Phys Rev E. See also http://www.physics.utoronto.ca/nonlinear/papers_chemwave.htm

Spindle microtubules in flux

Accurate and timely chromosome segregation is a task performed within meiotic and mitotic cells by a specialized force-generating structure - the spindle. This micromachine is constructed from numerous proteins, most notably the filamentous microtubules that form a structural framework for the spindle and also transmit forces through it. Poleward flux is an evolutionarily conserved mechanism used by spindle microtubules both to move chromosomes and to regulate spindle length. Recent studies have identified a microtubule-depolymerizing kinesin as a key force-generating component required for flux. On the basis of these findings, we propose a new model for flux powered by a microtubule-disassembly mechanism positioned at the spindle pole. In addition, we use the flux model to explain the results of spindle manipulation experiments to illustrate the importance of flux for proper chromosome positioning

Microtubule binding by dynactin is required for microtubule organization but not cargo transport

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150glued, binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150glued, we replaced the wild-type p150glued in Drosophila melanogaster S2 cells with mutant ΔN-p150 lacking residues 1–200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150glued with ΔN-p150glued has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150glued has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo

The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation

ABSTRACTCoordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.Summary: Detailed analysis of the ability of the Drosophila cytolinker protein Pigs to modulate its cytoskeleton-interacting functions (+TIP tracking, microtubule shaft binding, actin binding and actin–microtubule crosslinking)