ABSTRACT
Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the separation of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them.
Subject(s)
Cytoplasmic Vesicles/metabolism , Cytoskeleton/metabolism , Epithelial Cells/metabolism , Lysosomal-Associated Membrane Protein 2/metabolism , Microtubules/metabolism , Tubulin/metabolism , Animals , Cell Line , Chlorocebus aethiops , Cytoplasmic Vesicles/drug effects , Cytoplasmic Vesicles/ultrastructure , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Epithelial Cells/drug effects , Epithelial Cells/ultrastructure , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lysosomal-Associated Membrane Protein 2/genetics , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/ultrastructure , Microspheres , Microtubules/drug effects , Microtubules/ultrastructure , Nocodazole/pharmacology , Optical Imaging , Paclitaxel/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tubulin/genetics , Red Fluorescent ProteinABSTRACT
Intracellular transport plays an essential role in maintaining the organization of polarized cells. Motor proteins tether and move cargos along microtubules during long-range transport to deliver them to their proper location of function. To reach their destination, cargo-bound motors must overcome barriers to their forward motion such as intersection points between microtubules. The ability to visualize how motors navigate these barriers can give important information about the mechanisms that lead to efficient transport. Here, we first develop an all-optical correlative imaging method based on single-particle tracking and superresolution microscopy to map the transport trajectories of cargos to individual microtubules with high spatiotemporal resolution. We then use this method to study the behavior of lysosomes at microtubule-microtubule intersections. Our results show that the intersection poses a significant hindrance that leads to long pauses in transport only when the separation distance of the intersecting microtubules is smaller than â¼100 nm. However, the obstructions are typically overcome by the motors with high fidelity by either switching to the intersecting microtubule or eventually passing through the intersection. Interestingly, there is a large tendency to maintain the polarity of motion (anterograde or retrograde) after the intersection, suggesting a high degree of regulation of motor activity to maintain transport in a given direction. These results give insights into the effect of the cytoskeletal geometry on cargo transport and have important implications for the mechanisms that cargo-bound motors use to maneuver through the obstructions set up by the complex cytoskeletal network.
