Cytoplasmic Dynein and Dynactin Are Required for the Transport of Microtubules into the Axon

Abstract

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761–2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617–633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.

Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway

“…Additionally, the cells adopt a rounded morphology as soon as 8 h after transfection. This phenotype is consistent with reported dominant negative and knockdown studies of LIS1 pathway members (6,14,30,31). Notably, the cells expressing myc-C do not detach from the plate even after 72 h; thus, this effect is not likely to be a simple cell death response.…”

“…7 B and C), similar but less severe than that observed with expression of myc-C and representative of disruption of microtubule transport to the cell periphery. Because the LIS1 pathway is critical for this transport process, inhibition of the function of any member of the LIS1 pathway has consistently resulted in accumulation of microtubules around the nucleus (6,30,31). This result thus provides additional evidence that cytLEK1 expression is critical for LIS1 pathway function.…”

“…Additionally, cytLEK1 dysfunction alters the distribution of endogenous LIS1 pathway members, which appear to be decreased at the cell periphery and trapped near the nucleus with myc-C. Because this change is evident even before cells exhibit a rounded morphology, the dominant negative effect of myc-C on microtubule function is partially due to disrupting the movement and localization of these proteins. The tight perinuclear distribution of microtubules observed here with cytLEK1 dysfunction has previously been detected in LIS1 heterozygous fibroblasts and in dynein and Nudel dysfunction studies and is attributed to the role of the LIS1 pathway in dynein-directed outward movement of microtubules (6,30,31). Studies using a dominant negative LIS1 protein have also revealed abnormalities in cell shape (14).…”

Generation of noncentrosomal microtubule arrays

“…A combination of photobleaching and difference imaging in cultured nerve cells showed that a subset of MTs in axons move fast and asynchronously (Wang and Brown, 2002). Injection of recombinant dynamitin arrests the translocation of MTs from the cell body into the axon, implicating cytoplasmic dynein in the process (Ahmad et al, 1998). Depletion of cytoplasmic dynein by RNAi in cultured rat sympathetic neurons has confirmed that cytoplasmic dynein is a key player in the anterograde transport of MTs (He et al, 2005).…”

How does an axon grow?

“…Another axonal MAP named collapsin response mediator protein-2 (CRMP-2) binds tubulin dimers and stimulates axon growth and branching in hippocampal neurons in culture (Fukata et al 2002). Finally, microtubule motors such as dynein actively transport microtubules along the length of the axon, are required for axon growth, and may contribute to the microtubule invasion of growth cones during elongation (Ahmad et al 1998). Together, these data support a role for active regulation of microtubule activity in the control of axon outgrowth.…”