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...

Light microscopy has undergone a revolution with the advent of superresolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. Microsc. Res. Tech. 77:502-509,

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The atomic resolution structure of tropomyosin bound to actin in the 'switched off' state shows that tropomyosin makes contact with actin at only two points: one of which is a cluster of basic amino acids: actin K326, K328 and R147. We have demonstrated that two Tpm2.2 mutations, DE139 and E181K, and the actin K326N mutation destabilize this actintropomyosin interface. We predicted that equivalent charge loss mutations at Tpm3.12 EE 218-219, EE 224-224, or ED 257-258 would also destabilise the interaction with actin leading to a partial switch-on of the muscle. The two newly discovered stiff patient mutation are located at two of the three predicted gain of function sites. We used the quantitative in vitro motility assay and skeletal muscle thin filaments containing recombinant mutant Tpm3.12 expressed in a Baculovirus/ sf9 system. DE218 led to a 2.5-fold increase in Ca 2þ -sensitivity (EC50 ratio DE218/WT = 0.40 5 0.07, ). DE224 also showed an increase in Ca 2þ -sensitivity by 2.2-fold (EC50 ratio DE224/WT for = 0.46 5 0.09). It has been previously shown that there was a 2.5 fold increase in Ca2 þ sensitivity for ACTC K326N mutation (EC50 ratio K326N/WT = 0.4 5 0.05, p=0.07). The increased Ca 2þ -sensitivity indicates that both mutations cause a gain of function that was predicted from the structural analysis and that can account for the stiff patient syndrome.