Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.

Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.

Mitosis requires dynamic attachment of chromosomes to spindle microtubules. This interaction is mediated largely by kinetochores. During prometaphase, forces exerted at kinetochores, in combination with polar ejection forces, drive congression of chromosomes to the metaphase plate. A major question has been whether kinetochore-associated microtubule motors play an important role in congression. Using immunodepletion from and antibody addition to Xenopus egg extracts, we show that the kinetochore-associated kinesin-like motor protein CENP-E is essential for positioning chromosomes at the metaphase plate. We further demonstrate that CENP-E powers movement toward microtubule plus ends in vitro. These findings support a model in which CENP-E functions in congression to tether kinetochores to dynamic microtubule plus ends.

Threonine 246 is an active site residue that is conserved in all known L-lactate dehydrogenase (LDH; EC 1.1.1.27) sequences. In order to investigate the role of Thr246 in Bacillus stearothermophilus LDH, this residue was altered by site-directed mutagenesis to valine, alanine, leucine, and serine, respectively. The effects of these mutations, as observed in both steady-state and single-turnover kinetic measurements with different substrates, demonstrated the importance for catalysis of a hydroxyl group in the 246 amino acid residue. In contrast, no significant contribution of the OH group of Thr246 to productive pyruvate binding was observed. Instead, it is proposed that the role of Thr246 may be to facilitate hydride transfer from the nicotinamide ring of the NADH cofactor to the pyruvate carbonyl group.

Members of the kinesin superfamily of motor proteins are essential for mitotic and meiotic spindle organization, chromosome segregation, organelle and vesicle transport, and many other processes that require microtubule-based transport. A compound, adociasulfate-2, was isolated from a marine sponge, Haliclona (also known as Adocia) species, that inhibited kinesin activity by targeting its motor domain and mimicking the activity of the microtubule. Thus, the kinesin-microtubule interaction site could be a useful target for small molecule modulators, and adociasulfate-2 should serve as an archetype for specific inhibitors of kinesin functions.

The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIF1, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus-directed microtubule movement up to 2 microm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.