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 microtubulemicrotubule 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 ∼100 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.C ells rely on a two-way transport system to deliver important proteins and organelles to their location of function. Kinesin and dynein motors are responsible for long-range transport along microtubules (1). Although dynein walks toward the (−) end of the microtubule (retrograde), carrying cargo toward the cell nucleus, most kinesins walk toward the (+) end (anterograde), carrying cargo toward the cell periphery (1). Microtubules organize into a complex, 3D network inside cells, and the intersections between microtubule filaments or between microtubules and other cytoskeletal filaments (actin, intermediate filaments) likely have important consequences on the efficiency and accuracy of cargo transport (2). For example, microtubule-microtubule intersections can serve as switching points or barriers that disrupt continuous transport in a given direction.The effect of microtubule-microtubule intersections on the movement of individual motors and motor-decorated beads has been studied using in vitro reconstituted microtubules deposited on top of each other (3, 4). These studies showed that although single motors can have varied behavior (passing, dissociation, switching), at high motor densities dynein-decorated beads stop and tether at the intersection (3). On the basis of these results, it was suggested that microtubul...

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.

Planar supported lipid bilayers (PSLB) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signaling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.