The effect of acrylamide and other sulfhydryl alkylators on the ability of dynein and kinesin to translocate microtubules in vitro.
Chronic exposure to acrylamide leads to a dying-back axonopathy afflicting the longest axons of all tested mammalian and avian species. Prior to the onset of acrylamide-induced axonal degeneration, alterations in axonal fast transport have been consistently reported to be more severe for the retrograde than the anterograde direction. The putative retrograde motor protein, dynein, is compromised by exposure to the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) at concentrations far below those required to inactivate kinesin, the putative anterograde motor protein. Since acrylamide is capable of alkylating protein sulfhydryl moieties, we tested whether a direct exposure of purified kinesin or dynein to acrylamide would result in an impairment of either enzyme's ability to translocate microtubules. Motor activity was assayed by sequentially adsorbing either kinesin or dynein to acid-washed coverslips, treating with an alkylating agent or control solution, adding microtubules and ATP, and finally imaging and quantifying the binding and gliding of microtubules using video-enhanced differential interference contrast (VE-DIC) microscopy. In comparison to controls, incubation of dynein with NEM, ethacrynic acid, or iodoacetic acid resulted in dose-dependent decreases in the amount and rate of microtubule gliding, but increases in irreversible high-affinity microtubule binding. In contrast, exposure of dynein to 1-100 mM solutions of acrylamide did not significantly alter either the binding or gliding of microtubules (a molar/hour exposure to acrylamide equivalent to 50 times that which causes retrograde transport deficits in vivo). Likewise, kinesin motility parameters were not significantly affected by acrylamide concentrations up to 100 mM while NEM solutions > 100 microM led to significant losses in the ability of kinesin to bind MT. These data indicate that acrylamide does not significantly interact with bound (adsorbed) kinesin or dynein, implying that the mechanism by which acrylamide interferes with fast axonal transport in vivo is by interaction with other factor(s) that govern the movement of vesicles.
Martenson
C H
CH
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Odom
A
A
Sheetz
M P
MP
Graham
D G
DG
D020106:Acrylamide; D000178:Acrylamides / Q000633:toxicity*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004398:Dyneins / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D016547:Kinesins / Q000494:pharmacology*; D008870:Microtubules / Q000187:drug effects*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013552:Swine
D000178:Acrylamides; D007461:Iodoacetates; D013439:Sulfhydryl Reagents; D020106:Acrylamide; D004398:Dyneins; D016547:Kinesins; D005033:Ethylmaleimide; D019807:Iodoacetic Acid