Cytoskeletal requirements in axonal transport of slow component-b.
Slow component-b (SCb) translocates approximately 200 diverse proteins from the cell body to the axon and axon tip at average rates of approximately 2-8 mm/d. Several studies suggest that SCb proteins are cotransported as one or more macromolecular complexes, but the basis for this cotransport is unknown. The identification of actin and myosin in SCb led to the proposal that actin filaments function as a scaffold for the binding of other SCb proteins and that transport of these complexes is powered by myosin: the "microfilament-complex" model. Later, several SCb proteins were also found to bind F-actin, supporting the idea, but despite this, the model has never been directly tested. Here, we test this model by disrupting the cytoskeleton in a live-cell model system wherein we directly visualize transport of SCb cargoes. We focused on three SCb proteins that we previously showed were cotransported in our system: alpha-synuclein, synapsin-I, and glyceraldehyde-3-phosphate dehydrogenase. Disruption of actin filaments with latrunculin had no effect on the velocity or frequency of transport of these three proteins. Furthermore, cotransport of these three SCb proteins continued in actin-depleted axons. We conclude that actin filaments do not function as a scaffold to organize and transport these and possibly other SCb proteins. In contrast, depletion of microtubules led to a dramatic inhibition of vectorial transport of SCb cargoes. These findings do not support the microfilament-complex model, but instead indicate that the transport of protein complexes in SCb is powered by microtubule motors.
Roy
Subhojit
S
Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Winton
Matthew J
MJ
Black
Mark M
MM
Trojanowski
John Q
JQ
Lee
Virginia M-Y
VM
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D020409:Molecular Motor Proteins / Q000378:metabolism*; D016704:Synapsins / Q000378:metabolism; D053778:Thiazolidines / Q000494:pharmacology; D013997:Time Factors; D051844:alpha-Synuclein / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins; D016704:Synapsins; D053778:Thiazolidines; D051844:alpha-Synuclein; D005986:Glyceraldehyde 3-Phosphate; C037067:latrunculin A