Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles

Abstract

SUMMARY Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo but their overall composition on axonal vesicles and whether this composition directly modulates transport activity, is unknown. Here we characterize the intracellular transport and steady state motor subunit composition of mammalian prion protein (PrPC) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrPC vesicle motor complexes, and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment, and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model where PrPC vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.

Subpixel colocalization reveals amyloid precursor protein-dependent kinesin-1 and dynein association with axonal vesicles

“…S4E). The magnitude in observed signal reduction was analogous to decreases in protein levels assessed previously by Western blot (31), and confirmed the specificity of the selected DHC1 antibody (SI Materials and Methods). To validate the specificity of the APP antibody, cells were transfected with fluorescently labeled versions of APP (APP-YFP) and subsequently stained with anti-APP.…”

“…DHC1 antibody specificity was previously assessed by transfecting N2a cells with DHC1 shRNA, which resulted in an 80-90% decrease in message levels (31). Transfected hippocampal neurons exhibited marked decreases in DHC1 staining (Fig.…”

“…For all analyses, the cutoff for subpixel colocalization was set as a 300-nm radius based on the optics, resolution limit, and relevant physical size of the vesicles and motor subunits (31). An analysis of colocalization distance suggested that the percentage of motor association with vesicles plateaus at ∼300 nm and begins to pick up motor subunit signals from adjacent APP vesicles at cutoffs greater than this selected threshold.…”

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

“…Another mechanism, which is also based on the selective activation of the different motor species, is provided by molecules which are potentially able to coordinate the motor activity [112]. Dynactin, for example, usually turns the minus-directed motors off if the plus-end directed motors are active [150].…”

“…Molecular motors transport various types of cargos [388,112,237] of different sizes, such as filament precursors, mRNA granules [377], lipid droplets [389], viral capsids [232], lysosomes, or even objects as large as mitochondria [162,21,322].…”