title: Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections.
abstract: 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 ∼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.
authors: Institut de Ciències Fotòniques (ICFO), 08860 Castelledefels (Barcelona),  Spain.
authors: Štefan
authors: Š
authors: Bálint
authors: Ione
authors: I
authors: Verdeny Vilanova
authors: Ángel
authors: Á
authors: Sandoval Álvarez
authors: Melike
authors: M
authors: Lakadamyali
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002470:Cell Survival; D008247:Lysosomes / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological
references: Trends Cell Biol. 2009 Nov;19(11):555-65
references: 19836954
references: Curr Opin Cell Biol. 2008 Feb;20(1):41-7
references: 18226515
references: Curr Biol. 2004 Nov 23;14(22):R971-82
references: 15556858
references: Biophys J. 2012 Aug 8;103(3):492-500
references: 22947865
references: Science. 2008 Feb 8;319(5864):810-3
references: 18174397
references: Nat Methods. 2008 Dec;5(12):1047-52
references: 19029906
references: Curr Opin Cell Biol. 2009 Feb;21(1):59-67
references: 19179063
references: Biophys J. 2008 Apr 15;94(8):3115-25
references: 18227130
references: Nat Commun. 2012;3:1030
references: 22929787
references: J Cell Sci. 2010 Oct 15;123(Pt 20):3447-55
references: 20930140
references: Curr Biol. 2007 Jun 19;17(12):R478-86
references: 17580082
references: Proc Natl Acad Sci U S A. 2012 Aug 28;109(35):13978-83
references: 22891300
references: Chemphyschem. 2012 Jan 16;13(1):99-107
references: 22213647
references: Cell. 2010 Dec 23;143(7):1047-58
references: 21168201
references: Science. 1998 Jan 23;279(5350):519-26
references: 9438838
references: Curr Biol. 2010 Apr 27;20(8):697-702
references: 20399099
references: Structure. 2009 Nov 11;17(11):1433-41
references: 19913478
references: Science. 2007 Sep 21;317(5845):1749-53
references: 17702910
references: Nat Methods. 2011 Jun;8(6):499-508
references: 21552254
references: Biophys J. 2010 Oct 6;99(7):2143-52
references: 20923648
references: Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10011-6
references: 18626022
references: Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):87-92
references: 17190808
references: Nat Methods. 2012 Apr 22;9(7):721-3
references: 22522657
references: Nat Methods. 2010 Sep;7(9):717-9
references: 20693998
references: Science. 2007 May 25;316(5828):1153-8
references: 17525330