Oscillatory movement of a dynein-microtubule complex crosslinked with DNA origami.

Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein's oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

Novel Caffeic Acid Phenethyl Ester-Mortalin Antibody Nanoparticles Offer Enhanced Selective Cytotoxicity to Cancer Cells.

Caffeic acid phenethyl ester (CAPE) is a key bioactive ingredient of honeybee propolis and is claimed to have anticancer activity. Since mortalin, a hsp70 chaperone, is enriched in a cancerous cell surface, we recruited a unique cell internalizing anti-mortalin antibody (MotAb) to generate mortalin-targeting CAPE nanoparticles (CAPE-MotAb). Biophysical and biomolecular analyses revealed enhanced anticancer activity of CAPE-MotAb both in in vitro and in vivo assays. We demonstrate that CAPE-MotAb cause a stronger dose-dependent growth arrest/apoptosis of cancer cells through the downregulation of Cyclin D1-CDK4, phospho-Rb, PARP-1, and anti-apoptotic protein Bcl2. Concomitantly, a significant increase in the expression of p53, p21WAF1, and caspase cleavage was obtained only in CAPE-MotAb treated cells. We also demonstrate that CAPE-MotAb caused a remarkably enhanced downregulation of proteins critically involved in cell migration. In vivo tumor growth assays for subcutaneous xenografts in nude mice also revealed a significantly enhanced suppression of tumor growth in the treated group suggesting that these novel CAPE-MotAb nanoparticles may serve as a potent anticancer nanomedicine.

Direct optical microscopic observation of the microtubule polymerization intermediate sheet structure in the presence of gas7.

The process of microtubule elongation is thought to consist of two stages-formation of a tubulin sheet structure and its closure into a tube. However, real-time observation of this process has been difficult. Here, by utilizing phospho-tau binding protein Gas7 (growth-arrest-specific protein 7), we visualized the polymer transformation process by dark-field microscopy. Upon elongation, thin and flexible structures, often similar to a curved hook, appeared at the end of microtubules. Electron microscopic observations supported the idea that these flexible structures are tubulin sheets. They maintained their length until they gradually became thick and rigid beginning in the central portion, resulting in straight microtubules. In the absence of Gas7, the sheet-like structure was rarely observed; moreover, when observed, it was fragile and engaged in typical dynamic instability. With Gas7, no catastrophe was observed. These results suggest that Gas7 enhances microtubule polymerization by stabilizing sheet intermediates and is a useful tool for analyzing microtubule transformation.