Effects of Dynactin Disruption and Dynein Depletion on Axonal Microtubules

Abstract

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 ''comets'' observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were intitally slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dyneindepleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

“…Under such a scenario, cytoplasmic dynein could move the MTs either anterogradely or retrogradely depending whether its cargo domain is attached to the actin cytomatrix or to other MTs, though anterograde motion should dominate. Indeed, experiments have confirmed the crucial role of cytoplasmic dynein in MT movement [4].…”

“…The motility is achieved through molecular motors, which are anchored on actin filaments or longer MTs and exert forces on the transported MT by walking on it. It has in fact been shown that dynein and the actin cortex are responsible for half of the anterograde MT transport in axons while the rest depends on kinesin [269,4]. One thus finds two populations of MTs in the axon: short and mobile MTs which are transported and perform bidirectional motion and long and immobile MTs which serve as structures along which the short MTs can be transported.…”

“…These motor proteins usually consist of at least two parts: the motor domain 4 and the cargo-binding domain. The motor domain is able to bind to the polar filament, while the cargo domain binds to intracellular cargos such as organelles or other elements of the cytoskeleton [10,243].…”

“…This conformational change, which is presumably directed by tubulin-GDP, may destabilize lateral contacts between adjacent protofilaments. The polymerization-depolymerization cycle is completed by exchanging GDP of the disassembly products with GTP (part c; (4)). …”

“…These motors can thus carry +TIPs to the plus-end. 4. +TIPs might be added to the MT through a co-polymerization mechanism, i.e., tubulin dimers or oligomers associate in solution with the +TIP before these tubulin subunits are added to the filament.…”