Our data provide new insights into the roles of dynein, dynactin, and CLIP-170 in endosome function. The clear but limited effects of dynamitin overexpression on endocytic trafficking refine our understanding of the contributions of microtubule-based movement to this process. CLIP-170 and dynactin colocalize at microtubule plus ends, and binding is found to be CLIP-170 dependent, suggesting a hierarchy of binding. We have also identified the highly conserved dynamitin N terminus as a novel potential dynein-binding element. On the basis of our findings, we propose a series of molecular interactions that may underlie endosome docking and movement. Endosome-associated CLIP-170 provides the initial link to microtubules and recruits dynactin, which then binds dynein. Once dynein is bound and/or activated, CLIP-170 releases its grip on the microtubule, and long-range endosome motility begins.
The discovery that overexpression of the dynamitin N terminus perturbs endomembrane dynamics without affecting dynactin structure was unexpected. Although p150Glued
is the only dynactin subunit that has been shown to bind dynein directly (reviewed by Allan, 1996
; Schroer, 1996
; Holleran et al., 1998
), our results suggest that dynamitin may also play a role. p150Glued
and dynamitin are tightly associated within the projecting dynactin sidearm (Eckley et al., 1999
) that is proposed to serve as the dynein-binding site. Dynamitin may stabilize the dynein–dynactin interaction by binding dynein directly. We find (Quintyne et al., 1999
) that overexpression of different dynactin subunits can have a variety of effects, including Golgi complex and endosome dispersion, disorganization of the interphase microtubule array, and disruption of dynactin structure. Dynamitin induces all these effects, whereas other dynactin subunits such as p150Glued
disrupt Golgi structure and microtubule organization without affecting dynactin integrity. These overexpressed dynactin subunits, as well as the p150Glued
released by dynamitin overexpression, most likely perturb dynactin function by competing for binding sites on dynein or cargo. The dynamitin N terminus may act in a similar manner.
We suspect that the immediate effect of dynamitin overexpression is to inhibit dynein-based movement selectively, allowing plus end-directed movement to predominate for a short time, in support of a previous hypothesis (Burkhardt et al., 1997
). However, we observe no plus end-directed particle movements in dynamitin-overexpressing cells, suggesting that, at steady state, kinesin and/or kinesin-related protein-based motility is also inhibited. Late endosomes and lysosomes might possess a latent microtubule plus end binding activity (e.g., CLIP-170) that would explain their microtubule-dependent retention at the periphery (Burkhardt et al., 1997
; Harada et al., 1998
). Regardless of the mechanism for endosome relocalization, our results suggest that cells overexpressing dynamitin have arrived at a new steady-state condition in which endosome movement has stopped. Under normal conditions, overall membrane flux is kept in balance so that export parallels import (Steinman et al., 1976
). The bidirectional movement of individual organelles (e.g., lipid droplets) is also held in balance, because motility in both directions is altered in parallel when cells are subjected to physiological (Hamm-Alvarez et al., 1993
) or genetic manipulation (Welte et al., 1998
). Endosome movements may be subject to similar controls. Current models of the mechanism underlying coordinated organelle movement invoke a shared motor receptor (Sheetz et al., 1989
; Vallee and Sheetz, 1996
), although other mechanisms are possible. The use of dynamitin overexpression and other dynein-selective inhibitors should prove useful in further studies of this important question.
Our results suggest the additional intriguing possibility that endosome function is governed by a control mechanism that links trafficking with compartment architecture. Precedent is seen in two trafficking-defective Chinese hamster ovary cell lines that exhibit distinct endocytosis phenotypes (McGraw et al., 1993
; Daro et al., 1997
). Both show peripheral accumulations of early and late endosomes without any obvious alteration to microtubules. Similar endosome rearrangements are seen in chloroquine-treated chick embryo fibroblasts (Lippincott-Schwartz and Fambrough, personal communication). Apparently, disruption of endocytic traffic and endosome rearrangement are tightly coupled. Although the primary defect is different from cells overexpressing dynamitin (e.g., the ldlF cell line encodes a mutant ε-COP; Daro et al., 1997
), mutant Chinese hamster ovary cells show alterations in late endosome function similar to those we observe. Endocytic cargoes such as VSV (Daro et al., 1997
) and ricin (McGraw et al., 1993
) do not pass from acidic endosomal compartments to the cytoplasm, and delivery of epidermal growth factor to lysosomes is impaired (Daro et al., 1997
). In dynamitin-overexpressing cells and in mutant cell lines, short-range cycling of material between early endosomes and the plasma membrane continues. Late endosomal membranes may be induced to cycle in parallel when traffic is disrupted. The perturbation of normal mechanisms for forward or inward movement (e.g., budding or dynein-driven motility) might then allow the endosomal compartments participating in these loops to accumulate in the cell periphery near sites of uptake.
Early events in the endocytic pathway, such as ligand uptake and receptor recycling, are found to occur normally in dynamitin-overexpressing cells. However, trafficking to late endosomes is slowed. Under normal conditions, microtubules have been proposed to expedite transfer of material from early to late endosomes, perhaps via endocytic carrier vesicles (Gruenberg et al., 1989
). Why then, in cells in which these compartments are near each other, should endocytic traffic be impaired? One possibility is that dynein-based motility is required to transport endocytic vesicles in the periphery through actin-rich cortex (Marsh and Bron, 1997
). The spatial segregation that results from microtubule-based movement may also be required to maintain the distinct functions of different endocytic compartments (Gruenberg and Maxfield, 1995
; Mellman, 1996
). Endosomes that have been relocated to the cell periphery may fuse promiscuously with each other, which would result in membrane mixing unless balanced by sorting and retrieval mechanisms. Early and late endosome markers remain distinct in dynamitin-overexpressing cells, indicating that the two compartments are not completely randomized. However, inappropriate exchange of functionally important components or inhibitory factors not examined here might lead to the trafficking delays we observe.
Early and late endosomes are both highly pleiomorphic organelles, yet no clear relationship between structure and function has been established. The membrane deformations induced by microtubule motor activity may, in fact, contribute to membrane fusion. In pure lipid bilayer systems, high degrees of membrane curvature facilitate fusion (Chernomordik, 1996
). The enhancement of early and late endosome content mixing in vitro seen in the presence of microtubules (Aniento et al., 1993
) may be another reflection of this phenomenon.
Whatever other roles it may play in trafficking, it is clear that the dynein/dynactin motor is critical for the translocation of endosomal membranes on microtubules. What remains an open question is how endosomes switch from the short-range, oscillatory movements seen early on to the long-range, bidirectional translocations seen at later times. The discovery that dynactin colocalizes with CLIP-170 at microtubule ends suggests an order of assembly of the microtubule–endosome complex. Microtubules extending into the periphery may contact an endosome and become docked via CLIP-170. Once the endosome is tethered in this manner, CLIP-170 can recruit dynactin. At this point, dynactin may simply provide a dynein-binding site, or it may transiently stabilize the endosome–microtubule assembly via its own cargo- and microtubule-binding sites. To switch from this stable binding configuration to one that allows motility, the CLIP-170-microtubule link must be severed, perhaps by phosphorylation. This model provides many hypotheses to be tested in future studies.