In this study, we explored the function and organization of dynactin's pointed-end complex. This part of the dynactin molecule contains three subunits, p62, p27, and p25, not present in yeasts and thus presumed not to be required for dynactin's most fundamental role as a dynein activator. The fourth pointed-end complex subunit, Arp11 (Arp10p in yeast), binds Arp1 directly and serves to cap the end of the filament (Eckley and Schroer, 2003
; Clark and Rose, 2006
). p62 has also been reported to bind Arp1 (Garces et al., 1999
; Karki et al., 2000
). p27 and p25 have been proposed to contribute to, and perhaps specify, the association of dynactin with dynein cargoes (Schroer, 2004
). We find that the pointed-end complex comprises separable entities that contribute in distinct ways to dynactin integrity and function. Arp11 and p62 are required for dynactin stability, and thus all dynactin-dependent behaviors. They also allow dynactin to bind the nuclear envelope just prior to mitosis, which contributes to dynein recruitment and efficient nuclear envelope breakdown. The p27/p25 heterodimer “fine-tunes” dynein/dynactin activity. It can be removed without affecting dynactin stability and most dynactin-dependent, dynein-based motile phenomena. However, the binding of dynactin to a subset of endocytic membranes, and movement of these same membranes, is impaired. Although p27 and p25 may not be required for assembly of mitotic spindles, they are necessary for full recruitment of the important cell cycle kinase Plk1 to kinetochores in mitotic cells. Plk1 binding to p27 is required for proper execution of the spindle assembly checkpoint (Yeh et al.
, unpublished data).
Our protein cross-linking results provide new insights about the intermolecular interactions that occur among pointed-end complex subunits and allow us to speculate on how they associate with other dynactin subunits. The findings that p62 contacts Arp11 and p27 contacts p25 are consistent with the results of siRNA and overexpression experiments in which loss or overabundance of either binding partner phenocopies the other. Both Arp11 and p62 have been reported to interact with Arp1 (Garces et al., 1999
; Karki et al., 2000
; Eckley and Schroer, 2003
). If these proteins favor Arp1 stability, this would explain why loss of either leads to loss of the entire Arp1 minifilament. p27 and p25 are not essential for dynactin integrity, but instead appear to depend on association with dynactin for their own stability. Our cross-linking data suggest that the p27/p25 heterodimer is bound to the rest of the dynactin molecule via an interaction between p25 and Arp11. It is not uncommon for components of protein complexes to show interdependent stability. Depletion of cytoplasmic dynein heavy chain results in loss of other dynein subunits (Palmer et al., 2009
), and the Arp2/3 complex (Steffen et al., 2006
), retromer (Seaman, 2004
), and septins (Estey et al., 2010
) show a similar behavior.
A well-known consequence of dynactin or dynein inhibition is the fragmentation and/or mislocalization of endomembrane compartments (Burkhardt et al., 1997
; Valetti et al., 1999
; King et al., 2003
). We were surprised to find that many compartments whose steady-state localizations are widely assumed to depend on dynactin (e.g., the Golgi complex, late endosomes, the TfR-positive recycling compartment) were insensitive to the loss of p150Glued
or the entire dynactin molecule. This may reflect the fact that a considerable subset of the structures that comprise these organelles is not motile, as observed when they are tagged with GFP reporters (Wassmer et al., 2009
). This observation is also consistent with the notion that some compartments, such as the Golgi complex, have dynactin-independent mechanisms for dynein binding (Yadav et al., 2012
). Additional motile and/or tethering activities may also contribute to steady-state morphology. The dynactin associated with these organelles may also be exceptionally stable; it is known that motor complexes that have been assembled onto membranes do not readily exchange with the cytosolic pool (Niclas et al., 1996
), which itself can have a half-life of a day or more (Brown et al., 2005
). If the membrane-associated pool is even more long-lived, it would be largely insensitive to RNAi.
The full range of mechanisms by which dynactin and dynein bind membranes is still being determined, and it is clear that binding modes vary among organelles. Associations between Arp1 and Golgi-associated spectrin have been reported to contribute to Golgi targeting (Holleran et al., 1996
; Muresan et al., 2001
); other spectrin isoforms may allow dynactin to bind elsewhere. Dynactin–Golgi binding has also been suggested to involve Rab6 (Short et al., 2002
), and dynein can bind the Golgi complex directly via golgin-160 (Yadav et al., 2012
, which has been identified as a binding partner for peripherally associated membrane proteins in a number of studies, is thought to associate with Rab7-RILP on late endosomes (Jordens et al., 2001
; Johansson et al., 2007
; Rocha et al., 2009
), Jip4/Arf6 on recycling endosomes (Montagnac et al., 2009
), the sorting nexin SNX6 (Wassmer et al., 2009
), and huntingtin and its associated protein HAP1 (Engelender et al., 1997
; Caviston et al., 2007
). These proteins all bind p150Glued
in the same vicinity, suggesting a generic and possibly mutually exclusive mode of interaction.
The pointed-end complex subunits p27 and/or p25 provide an additional binding mode that may involve interaction with integral components of the membrane. Although these proteins are not found in yeasts, the dependence of organelle movement and distribution on p25 in the filamentous fungi A. nidulans
and N. crassa
(Lee et al., 2001
; Zhang et al., 2011
) suggests that membrane binding is a highly conserved function. It is not clear from our work whether p27, p25, or both are most important, because depletion of either results in loss of the other. A common feature of all p25 sequences is an abundance of hydrophobic residues, suggesting that this protein might interact with membranes directly.
The present study examines the dynein-targeting mechanism that brings the nascent spindle toward the nucleus to facilitate nuclear envelope breakdown. Although we have pinpointed the dynactin subunits p62 and Arp11 as the components that allow cell cycle–dependent binding, the nuclear envelope binding partner(s) remains unknown. Dynactin perturbation or depletion does not completely prevent dynein binding to the nuclear envelope, indicating that dynein can be recruited in multiple ways. Cell cycle–dependent recruitment of the dynein/dynactin complex to the nucleus has been reported to involve binding between the nuclear pore complex component, RanBP2/Nup358, and BICD2, a scaffolding protein that stabilizes the dynein/dynactin complex via interactions with subunits other than p62 and Arp11 (Splinter et al., 2010
). The dynein pathway components NudE/Nde1 and Lis1 also contribute to dynein and dynactin recruitment to the nuclear envelope in prophase (Hebbar et al., 2008
), once again via interactions with the nuclear pore complex (Bolhy et al., 2011
). In all cases, the molecular basis of cell cycle–dependent binding remains undefined, although it is presumed to involve cell cycle phosphorylation of dynactin and/or its binding partner. Although both p62 and Arp11 have been reported to be phosphorylated (Hoffert et al., 2006
; Rigbolt et al., 2011
), this is not at Cdk1 consensus sites and neither protein appears in published mitotic phosphoproteomes. Regardless, phosphorylation may contribute to the regulation of these proteins’ binding activities. More analysis will be needed to clarify how dynactin mediates the dynein-based motile activities that underlie membrane organization and dynamics.