Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network
Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membran...
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Actin Cytoskeleton Intracellular Transport Myosin Single-Molecule Super Resolution Molecular Biology Physics |
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Actin Cytoskeleton Intracellular Transport Myosin Single-Molecule Super Resolution Molecular Biology Physics Lombardo, Andrew Thomas Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network |
description |
Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need.
To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system. |
author |
Lombardo, Andrew Thomas |
author_facet |
Lombardo, Andrew Thomas |
author_sort |
Lombardo, Andrew Thomas |
title |
Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network |
title_short |
Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network |
title_full |
Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network |
title_fullStr |
Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network |
title_full_unstemmed |
Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network |
title_sort |
cargo transport by myosin va molecular motors within three-dimensional in vitro models of the intracellular actin cytoskeletal network |
publisher |
ScholarWorks @ UVM |
publishDate |
2018 |
url |
https://scholarworks.uvm.edu/graddis/860 https://scholarworks.uvm.edu/cgi/viewcontent.cgi?article=1859&context=graddis |
work_keys_str_mv |
AT lombardoandrewthomas cargotransportbymyosinvamolecularmotorswithinthreedimensionalinvitromodelsoftheintracellularactincytoskeletalnetwork |
_version_ |
1719272574346592256 |
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ndltd-uvm.edu-oai-scholarworks.uvm.edu-graddis-18592019-10-20T11:29:01Z Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network Lombardo, Andrew Thomas Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system. 2018-01-01T08:00:00Z text application/pdf https://scholarworks.uvm.edu/graddis/860 https://scholarworks.uvm.edu/cgi/viewcontent.cgi?article=1859&context=graddis Graduate College Dissertations and Theses en ScholarWorks @ UVM Actin Cytoskeleton Intracellular Transport Myosin Single-Molecule Super Resolution Molecular Biology Physics |