Dynactin, which is necessary for dynein-mediated vesicular transport, is a complex molecule consisting of at least seven polypeptides. Previous studies on the molecular characterization of the p150Glued
, Arp1, and p50 (dynamitin) subunits of dynactin have provided important insights into how this complex may function within the cell. p150Glued
was found to contain a microtubule-binding domain (Waterman-Storer et al., 1995
) and a binding site for Arp1 (Waterman-Storer et al., 1995
), as well as a cytoplasmic dynein intermediate chain-binding domain (Karki and Holzbaur, 1995
; Vaughan and Vallee, 1995
). Molecular analysis of Arp1 revealed that it is an actin-related protein (Clark and Meyer, 1992
; Lees-Miller et al., 1992
) that does not copolymerize with actin (Holleran et al., 1996
). Overexpressed Arp1 was found to associate with spectrin, suggesting a possible linking mechanism for dynactin with membranous organelles (Holleran et al., 1996
). Studies on p50 have demonstrated that disruption in dynactin function blocks mitosis (Echeverri et al., 1996
) as well as ER-to-Golgi transport (Presley et al., 1997
). However, little is known about the functions of other subunits of dynactin.
In this study, we isolated the previously uncharacterized 22-kD subunit of dynactin and obtained a human cDNA encoding this protein. We have characterized this polypeptide as a bona fide subunit of dynactin and have found that it is a novel protein, with no identifiable homology to previously characterized proteins or structural or functional domains. Secondary structural analysis using the DNASTAR software package predicts that p22 is highly α-helical with very little, if any, coiled coil domains. Northern blot analysis shows that the p22 transcript is enriched in muscle tissues and the pancreas as compared with brain or liver (Fig. a). The significance of high levels of p22 transcript in these tissues is currently unknown.
We have characterized p22 as a tightly associated dynactin subunit by several different criteria. First, like other dynactin subunits, p22 is enriched in taxol-polymerized brain microtubules; the association of p22 with microtubules is sensitive to Mg-ATP and NaCl. Second, p22 exists exclusively as a part of the 20-S dynactin complex as determined by sedimentation through a linear sucrose gradient. Third, p22 from cytosol binds to a dynein intermediate chain column as a part of the dynactin complex; this binding can be specifically blocked by p150Glued pretreatment of the column. Fourth, anti-p22 antibody precipitates the same dynactin subunits that are coprecipitated by an anti-p150Glued antibody. Immunolocalization studies revealed a dense perinuclear and punctate cytoplasmic distribution for p22, consistent with the cellular localization of other dynactin subunits. Finally, we have demonstrated a direct binding interaction between p22 and p150 Glued. These data characterize p22 as a tightly associated dynactin subunit, as has been previously shown for p150Glued, dynamitin, and Arp1. This behavior is in contrast to the observation that the α and β subunits of capping protein are found as integral subunits of the dynactin complex, as well as in association with the cellular actin cytoskeleton.
More detailed immunolocalization studies revealed that p22 localizes to the cleavage furrow and the midbody of dividing cells. This localization was also seen with antibodies raised against p150Glued and cytoplasmic dynein, raising the possibility that dynactin may recruit dynein to these structures. The dynactin staining at the cleavage furrow early in cytokinesis is reminiscent of actin filaments at the contractile ring. Careful analysis of staining later in cytokinesis indicates that dynactin is associated with the cell cortex surrounding the intracellular bridge (see Fig. , h and k).
We have previously suggested that dynactin may associate with membrane-bound spectrin (Holleran et al., 1996
). We therefore investigated whether spectrin was also present at the midbody and the cleavage furrow. Antibodies to fodrin (nonerythroid spectrin) and human spectrin did not localize spectrin to the cleavage furrow or at the midbody. It is possible that spectrin is localized to this region but is not accessible to antibodies. The midbody consists of interdigitating antiparallel microtubules surrounded by an amorphous, electron-dense matrix material (McIntosh and Landis, 1971
; Mullins and Biesele, 1977
). Microtubules at the midbody are inaccessible to antitubulin antibody, which results in a dark appearance when cells undergoing cytokinesis are stained with antitubulin antibody and visualized by immunofluorescence (Saxton and McIntosh, 1987
; Sellito and Kuriyama, 1988
). We have also noted that antibodies to the dynactin subunit Arp1 do not stain the midbody. Alternatively, the antispectrin antibodies we tested may not recognize the spectrin isoform involved, or the localization of dynein and dynactin to the cleavage furrow and later to the midbody and the persistent ring may be mediated by a mechanism independent of an association between dynactin and spectrin.
The localization of dynactin at the cleavage furrow and the midbody raises several interesting questions. First, why would dynein/dynactin be required at these structures? And second, what is the mechanism that targets dynein/dynactin there in a cell cycle–dependent manner? While the actomyosin-based contractile ring drives cytokinesis (for reviews see Schroeder, 1981
; Satterwhite and Pollard, 1992
; Fishkind and Wang, 1995
), it has been suggested that the interdigitating spindle microtubules (that eventually make up the microtubules of cleavage furrow and the midbody) are involved in the bipolar flow of surface receptors and in the organization of the cortical cytoskeleton to initiate the contractile ring (Fishkind et al., 1996
). Cooperative interactions between the central spindle and the contractile ring have recently been observed by Giansanti et al. (1998)
in their analysis of the chickadee
, and KLP3A
mutations in Drosophila
. Our observations that dynein and dynactin accumulate at the cleavage furrow and the midbody may suggest that dynein/dynactin activity is required for the flow of cytoskeletal elements or the interdigitation of polar microtubules,
or for the apparent cross talk between the spindle and the contractile ring. Dynein/dynactin may potentially cross-link the interdigitating polar microtubules at the cleavage furrow and the midbody as both dynein and dynactin have microtubule-binding domains. Or, dynein and dynactin may link the interzonal microtubules to cortical actin at the cleavage furrow, in an interaction mediated by the actin-like filament that forms the base of the dynactin complex.
A kinesin-like protein in Drosophila
(KLP3A) was observed to localize to the cleavage furrow and midbody and was suggested to produce signals to initiate cleavage furrow formation in eukaryotic cells (Williams et al., 1995
). Mutations in the KLP3A gene were shown to disrupt the interdigitation of microtubules at the midzone of spindles, which resulted in a failure of cytokinesis (Williams et al., 1995
). Besides KLP3A, there have been a number of reports indicating localization of other kinesin-like proteins at the cleavage furrow and/or the midbody: Eg5 (Sawin and Mitchison, 1995
(Henson et al., 1995
), Xklp1 (Vernos et al., 1995
), CENP-E (Brown et al., 1994
; Yao et al., 1997
), and pavarotti (Adams et al., 1998
). It is of interest to note that the human homologue of Eg5, HsEg5, has recently been reported to associate with dynactin via an interaction with p150Glued
(Blangy et al., 1997
). Together, these observations suggest that microtubule-based motor activity may be required for proper cytokinesis as well as for the mitotic events preceding cytokinesis; potentially oppositely directed microtubule motors may be required to supply counterbalancing forces at multiple stages during cell division. In the future, it will be critical to determine which dynactin subunit targets dynactin/ dynein to the cleavage furrow, which cell cycle determinants may direct this localization, and what specific role or roles dynein and dynactin play in cytokinesis.
Overexpression of p22 in transient transfection analysis indicates that a high cellular concentration of this protein is lethal, although this may be due to nonspecific effects of protein aggregation. More specifically, at moderate levels of overexpression we did not detect any perturbation induced by p22 in the cellular distribution of p150Glued
or cytoplasmic dynein, or in the morphology of the Golgi or the organization of the microtubule cytoskeleton. In contrast, in previous studies the overexpression of the dynactin subunits Arp1 and dynamitin have been shown to lead to the disruption of the Golgi (Holleran et al., 1996
; Burkhardt et al., 1997
). It is possible that high cellular levels of p22 may induce a mitotic block because cells overexpressing this polypeptide did not undergo cell division; a mitotic block was previously observed upon overexpression of the dynamitin subunit of dynactin (Echeverri et al., 1996
In summary, we have cloned and characterized the smallest subunit of dynactin, and studies on this subunit have revealed a novel localization for dynein/dynactin at the cleavage furrow and at the midbody. This novel localization suggests a possible new role for dynein and dynactin during cytokinesis. It will be of considerable interest to see how the microtubule-dependent motor system is involved in a cellular process where actomyosin is thought to provide the contractile force.