We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the “zero-length cross-linker” 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ~5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ~7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ~20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.
The~1.0 megadalton dynactin complex interacts with cytoplasmic dynein to increase its processivity during minus-end-directed transport of cargo along microtubules (MT). The detailed molecular understanding of how dynactin regulates dynein motility is still elusive due to lack of structural information of the complex. Here we present structure of vertebrate dynactin at 20Å resolution, achieved by negative stain electron microscopy (EM). The reconstruction reveals the overall architecture of dynactin, allowing for delineation of the major known subcomplexes of the molecule. We can clearly discern the individual Arp subunits arranged in actin like helical fashion in the filament, along with the capping density at the barbed-end, and the pointed-end complex. We can also identify the shoulder domain above the filament observing extensive interactions with the Arp subunits. Due to the flexibility of the extended p150Glued coiled-coil arm, whose base interacts with dynein and whose globular tip binds the MT surface, this region was not resolved in the 3D reconstruction. However, focused 2D analysis of the p150Glued arm revealed its attachment point at the shoulder domain, as well as structural details of the globular CAP-Gly domain. This structural study of the dynactin complex establishes a strong foundation for understanding how its architecture is adapted for concerted interaction with dynein, cargo, and MTs during transport processes.
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