Owing to their chemical and magnetic properties, magnetite nanoparticles are an interesting adsorbing material for biomolecules. The understanding of the interactions of simple biomolecules with inorganic nanoparticles is an important approach for research on the bio-nano interface and can constitute the fundamentals to manifold applications in biotechnology, medicine and catalysis. The aim of the work presented here is to compare the interaction of seven different amino acids (L-alanine, L-cysteine, Lglutamic acid, glycine, L-histidine, L-lysine, and L-serine) with magnetite nanoparticles in a colloidal system at pH 6. We investigate the influence of the side chain on the adsorption at a magnetite−water interface with incubation experiments. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and simultaneous thermal analysis (STA) reveal deeper insights into the interactions of amino acids with magnetite nanoparticles. The amino acids that contain polar side chains adsorb on the nanoparticles to a high degree. Cysteine demonstrates the highest adsorption capacity and the formation of cystine is observed. ATR-FTIR spectroscopy results indicate a strong influence of the carboxyl group and side chains on the binding mechanism of amino acids at the iron oxide surface. Our investigation offers novel knowledge into adsorption behavior at the bio-nano interface.

Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long-range transport processes In all organisms studied so far, the kinesin-2 family is essential for long-range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin-2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin-2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin-1, kinesin-2 takes directional, off-axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin-2 to side-track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A- and B-tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co-evolved the heterodimeric kinesin-2 with the ciliary machinery to work on the specialized axonemal surface for two-way traffic.

The fundamental stepping mechanism of kinesins was investigated using the Fluorescence Imaging with One Nanometer Accuracy (FIONA) technique. Kinesin-2 stands out due to it's employment on both axonemes in cilia and microtubules in the cytoplasm. We used FIONA to track head-labeled kinesin-2 motors with high precision on both filaments. Differences in the distribution of step sizes and in the traces show a specialisation of the stepping behavior to the respective track. The motors take off-axis steps to neighbouring protofilaments on microtubules but not on axonemes. In combination, both observations explain the direction-dependent allocation of cargo that is essential for intraflagellar transport. The intrinsic tendency of kinesin-2 to take off-axis steps and structural barriers specific to the axonemal structure restrict the motors to walk on the B-tubules. These results help to explain, why heterodimeric kinesin-2 motors co-evolved with the machinery of cilia to achieve unhindered two-way traffic on axonemes.

No abstract

Specific recognition of cellular cargo and efficient transport to its correct intracellular destination is an infrastructural challenge faced by most eukaryotic cells. This remarkable deed is accomplished by processive motor proteins that are subject to robust regulatory mechanisms. The first level of regulation entails the ability of the motor to suppress its own activity. This autoinhibition is eventually relieved by specific cargo binding. To better understand the role of the cargo during motor activation, we dissected the activation mechanism of the ciliary homodimeric kinesin-2 from Caenorhabditis elegans by its physiological cargo. In functional reconstitution assays, we identified two cargo adaptor proteins that together are necessary and sufficient to allosterically activate the autoinhibited motor. Surprisingly, the orthologous adaptor proteins from the unicellular green algae Chlamydomonas reinhardtii also fully activated the kinesin-2 from worm, even though C. reinhardtii itself lacks a homodimeric kinesin-2 motor. The latter suggested that a motor activation mechanism similar to the C. elegans model existed already well before metazoans evolved, and prompted us to scrutinize predicted homodimeric kinesin-2 orthologs in other evolutionarily distant eukaryotes. We show that the ciliate Tetrahymena thermophila not only possesses a homodimeric kinesin-2 but that it also shares the same allosteric activation mechanism that we delineated in the C. elegans model. Our results point to a much more fundamental role of homodimeric kinesin-2 in intraflagellar transport (IFT) than previously thought and warrant further scrutiny of distantly related organisms toward a comprehensive picture of the IFT process and its evolution.