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Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170–dynein interactions and in coordinating dynein cargo-binding and motor activities.
Mutations in the human LIS1 gene are responsible for the autosomal dominant brain developmental disease, type I lissencephaly (Reiner et al., 1993). This condition involves gross disorganization of neurons within the cerebral cortex, and is thought to reflect a failure in normal neuronal migration during early development.
Genetic studies in lower eukaryotes have indicated that LIS1 homologues participate in dynein-mediated nuclear migration (Xiang et al., 1994; Geiser et al., 1997). In vertebrates, LIS1 was initially identified as a noncatalytic subunit of brain platelet-activating factor acetylhydrolase (PAFAH)* (Hattori et al., 1993). We, and others, have more recently found LIS1 to interact biochemically with cytoplasmic dynein and dynactin (Faulkner et al., 2000; Sasaki et al., 2000; Smith et al., 2000). We also obtained evidence for a role for LIS1 in vertebrate cell division, raising the possibility that the errors in neuronal distribution characteristic of lissencephaly arise from defects in the genesis of neuronal progenitors (Faulkner et al., 2000). Overexpression of LIS1, application of LIS1 antisense oligonucleotides, and microinjection of anti-LIS1 antibody all produced potent mitotic defects, including spindle misorientation and chromosome misalignment at the metaphase plate. Consistent with a mitotic function, LIS1 localized prominently to prometaphase kinetochores and to the cell cortex of dividing vertebrate cultured cells, known binding sites for cytoplasmic dynein and its accessory complex, dynactin.
The molecular relationship between dynein, dynactin, and LIS1 is poorly understood. Dynein and dynactin are each large, multisubunit complexes. Dynein consists of a heavy chain (HC), responsible for ATPase activity and force production, plus a variety of accessory subunits (intermediate, light intermediate, and light chains; ICs, LICs, and LCs) implicated in binding to diverse forms of subcellular cargo (Vaughan and Vallee, 1995; Tynan et al., 2000b; Ye et al., 2000; Tai et al., 2001). Dynactin consists of a short filament of the actin-related protein Arp1 and a variety of associated polypeptides. Dynactin has been found to link dynein to the prometaphase kinetochore (Echeverri et al., 1996) and the surface of Golgi membranes (Burkhardt et al., 1997; Roghi and Allan, 1999), suggesting that it participates in dynein cargo binding. Dynactin has also been reported to stimulate dynein processivity (King and Schroer, 2000). The ICs of cytoplasmic dynein interact directly with the p150Glued subunit of dynactin in in vitro assays (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). However, the interaction between the two intact complexes has been observed only at very limited levels (Kini and Collins, 2001), suggesting that it is weak or regulated (Vaughan et al., 2001). LIS1 has been reported to interact with a portion of the cytoplasmic dynein HC in yeast two-hybrid and coexpression/coimmunoprecipitation assays (Sasaki et al., 2000). Whether it interacts with other subunits of dynein, whether it interacts with dynactin independently, and how these interactions relate to dynein function and subcellular organization are poorly understood.
How the structural organization of LIS1 relates to its function is also poorly understood. LIS1 is predicted to consist of two distinct domains. The NH2-terminal 97 amino acids (aa) contains a short region of predicted coiled-coil that has been implicated in self-association (Ahn and Morris, 2001). The COOH-terminal 317 aa contains a series of seven WD repeats, which in the β subunit of the heterotrimeric G proteins and other polypeptides constitute a β-propeller fold (Wall et al., 1995). COOH-terminal deletions of LIS1 are sufficient to cause lissencephaly (Cardoso et al., 2000), indicating that the WD repeat domain is important functionally. This region has been implicated in binding to the catalytic subunit of PAFAH (Kitagawa et al., 2000; Niethammer et al., 2000; Sweeney et al., 2000). Whether this is its sole function, or whether it also functions in the dynein pathway is unknown. LIS1 has been reported to interact with two additional classes of protein in the dynein pathway, NudC and NudE/NudEL (Morris et al., 1998; Efimov and Morris, 2000; Feng et al., 2000; Niethammer et al., 2000), but their role in LIS1 function and their binding sites within LIS1 are uncertain.
We report here that LIS1 associates with kinetochores through its WD repeat domain. Using dominant inhibitory dynactin and LIS1 constructs, we find LIS1 to be linked to the kinetochore through the motor protein complexes, rather than the reverse. We find that the WD repeat domain of LIS1 interacts with three distinct subunits of the dynein and dynactin complexes, representing both motor and cargo-binding regions. Overexpression of full-length LIS1 and its WD repeat fragment specifically displaced CLIP-170 from mitotic kinetochores. Together, these results suggest a direct role for LIS1 in novel forms of dynein/dynactin regulation.
Previous work from this lab implicated LIS1 in mitotic chromosome alignment and spindle orientation (Faulkner et al., 2000). To determine which portion of the LIS1 molecule is involved in these activities, we constructed two cDNAs encoding HA-tagged fragments of LIS1 corresponding to the NH2-terminal 87 aa, comparable to the 89-aa product resulting from a known disease-causing deletion (Cardoso et al., 2000), and the COOH-terminal 327 aa, respectively (Fig. 1 A). The NH2-terminal fragment contains a 28-aa predicted coiled-coil domain, whereas the COOH-terminal fragment contains seven WD repeat motifs. To our surprise, overexpression of each fragment produced a dramatic increase in mitotic index comparable to that observed with full-length LIS1 (Fig. 1, B and C), with a greater number of multipolar spindles and a more dramatic disruption of spindle organization (Fig. 1 B). The more severe phenotype is reminiscent of that produced by overexpression of the dynamitin subunit of dynactin (Echeverri et al., 1996). However, as reported previously for full-length LIS1 (Faulkner et al., 2000) and unlike dynamitin, no effect on Golgi morphology was detectable with the LIS1 fragments (unpublished data).
To gain insight into the manner in which the individual fragments produced these effects, we examined their ability to associate with prometaphase kinetochores, a site at which we had previously observed endogenous LIS1 using immunofluorescence microscopy (Faulkner et al., 2000). We also treated cells with nocodazole, which allows outer kinetochore components to accumulate above their normal levels (Echeverri et al., 1996; King et al., 2000; Hoffman et al., 2001). Full-length HA-tagged LIS1 exhibited dramatic localization to prometaphase kinetochores, identified by costaining with CREST human autoimmune antiserum in both COS-7 (Fig. 2 A, upper row) and HeLa cells (unpublished data), adding strong support to the identification of LIS1 as a kinetochore component. Within the kinetochore, LIS1 staining was slightly peripheral to the CREST signal, consistent with outer kinetochore localization. In cells exposed to nocodazole for prolonged periods (see below), LIS1 exhibited an elongated crescent shape, as observed for dynein and other outer kinetochore components (Echeverri et al., 1996; King et al., 2000; Hoffman et al., 2001). Clear kinetochore localization was also observed with the WD repeat fragment of LIS1 alone. This effect was more pronounced in nocodazole-treated cells (Fig. 2 A, middle row), but was also readily observed in untreated cells (Fig. 2 B). In contrast, specific localization of the NH2-terminal LIS1 fragment (LIS1 N) was not generally observed (Fig. 2 A, lower row), though very weak kinetochore staining could be detected in occasional cells (unpublished data). Together, these results indicate that the WD repeat domain of LIS1 is sufficient for kinetochore targeting.
Despite this difference in kinetochore binding behavior, the two fragments produce phenotypes of comparable severity (see above). The NH2 terminus of the LIS1-related Aspergillus protein NudF has been implicated in self-association (Ahn and Morris, 2001), raising the possibility that the corresponding portion of LIS1 might interact with the endogenous full-length protein and interfere with its behavior. In coexpression experiments, we observed the NH2-terminal fragment coimmunoprecipitating with itself and full-length LIS1, but not with the WD region (Fig. 3 A). In contrast to previous results with NudF, removal of the predicted coiled-coil (HA–LIS1ΔC-C) had no effect on self-association, suggesting that the entire NH2-terminal domain participates in this activity. To determine whether the NH2-terminal domain can displace the endogenous wild-type protein from kinetochores, we examined cells overexpressing the NH2-terminal construct for endogenous LIS1 staining. For this purpose, we used a monoclonal antibody (Sapir et al., 1997) that we determined to react with the WD repeat region of LIS1 (Fig. 3 B). In high expressers, the endogenous LIS1 kinetochore staining was, indeed, dramatically reduced (Fig. 3 C, right column).
We previously reported that dynein and dynactin remain at the prometaphase kinetochore in LIS1-overexpressing cells (Faulkner et al., 2000), though our current data (Fig. 2) indicate that the full-length epitope-tagged LIS1 protein occupies the kinetochore under these conditions. To test the effects of removing endogenous LIS1 from this site, we overexpressed LIS1 N. No effect on the dynein (Fig. 3 D) and dynactin (unpublished data) signals was observed in cells overexpressing the LIS1 fragment versus untransfected cells (unpublished data).
To test whether LIS1 displaced other kinetochore proteins, we stained cells overexpressing full-length LIS1 and the NH2-terminal and WD repeat fragments with antibodies against CENP-E, CENP-F, Bub1, BubR1, Mad2, and CLIP-170. No effect was observed, except in the case of CLIP-170, which was completely displaced by the full-length and WD repeat LIS1 constructs (Fig. 4; unpublished data).
To test whether LIS1 requires dynein/dynactin for kinetochore binding, we overexpressed the dynamitin subunit of dynactin, which dissociates dynactin into smaller subcomplexes and releases dynein from the kinetochore (Echeverri et al., 1996). We now find that epitope-tagged dynamitin persists at the kinetochore under these conditions (Fig. 5 A). This behavior suggests an additional effect of dynamitin overexpression: competition for binding to the dynein/dynactin anchoring protein ZW10 (Starr et al., 1998). As expected, Arp1, a dynactin subunit, was displaced from the kinetochore (Fig. 5 B, upper left). The LIS1 signal was also greatly diminished (Fig. 5 B, upper right). Together, our data indicate that the localization of LIS1 to the kinetochore requires dynein and dynactin rather than the reverse.
The dependence of LIS1 kinetochore targeting on dynein and dynactin supports a physiological interaction with the two complexes. To define the molecular basis for these interactions further, we explored whether LIS1 can interact with subunits in each complex. LIS1 was coexpressed with cytoplasmic dynein and dynactin subunits, and interactions were assessed by coimmunoprecipitation.
Both the HCs and ICs of cytoplasmic dynein coimmunoprecipitated with LIS1, as did the dynamitin subunit of dynactin (Fig. 6 A). LIC1 and LIC2 and LCs (Tctex-1, RP3, and LC8) of the dynein complex, and the p150Glued, Arp1, and p62 subunits of the dynactin complex were all negative in this assay. These results indicate that LIS1 interacts with each complex independently and, in the case of cytoplasmic dynein, through multiple subunit interactions. We also examined these interactions by immunoprecipitation from [35S]methionine-labeled total cell extracts. In each case (HC, IC2C, and dynamitin), LIS1 was the major nondynein/dynactin polypeptide pulled down, with minor background bands also visible (unpublished data).
We also used the yeast two-hybrid assay as a further test for interaction specificity. A positive reaction was observed between LIS1 and the cytoplasmic dynein HCs and ICs and dynamitin (Fig. 6 B). In the case of the HC, three fragments were analyzed for interactions. HC N649C907 and HC N1874C2124 showed positive reactions, whereas HC N547C649 did not (see next section for further detail). Thus, together our data support a direct interaction between LIS1 and each of three distinct dynein and dynactin subunits.
To gain further insight into the molecular basis for the observed interactions, we investigated which region of LIS1 is involved. No interaction was detected using LIS1 N. Instead, each of the LIS1-interacting subunits coimmunoprecipitated with the WD repeat domain of LIS1 alone (Fig. 7). These results are consistent with a role for the WD repeat domain in linking LIS1 to the kinetochore through dynein and dynactin (see above).
The dynein HC consists of two major functional regions. The NH2-terminal aa 1100–1300 are thought to constitute the “stem” of the molecule, and contain the known sites for interaction with the cargo-binding ICs, LICs, and LCs (Habura et al., 1999; Tynan et al., 2000a; Mok et al., 2001). The remainder of the molecule represents the motor domain. The site for microtubule binding is located at the tip of a projecting stalk (aa 3160–3506; Gee et al., 1997), which resides within a series of recently predicted AAA ATPase domains (Neuwald et al., 1999). Photocleavage (Gibbons and Gibbons, 1987) or mutation (Gee et al., 1997) of the P-loop of the first AAA domain abolishes ATPase activity, implicating this site most clearly in ATP hydrolysis and force production.
The identity of the site within the HC with which LIS1 interacts should provide important insight into LIS1 function. To investigate this issue systematically, we coexpressed LIS1 with a series of motor domain and stem fragments and subfragments (Fig. 8). Surprisingly, clear binding was observed with HC fragments corresponding to each major domain (e.g., N1137flag corresponding to the entire motor domain and C1140myc corresponding to the stem domain). Analysis of subfragments of the motor domain indicated that one LIS1 binding site is located toward the NH2 terminus of this region. During the course of this study a LIS1-binding fragment from the same region encompassing part, but not all, of the first two AAA domains was also identified in a yeast two-hybrid screen (Sasaki et al., 2000). Recently, the same fragment was also found to interact with the Aspergillus LIS1 homologue NUDF (Hoffmann et al., 2001). To determine whether the LIS1 binding site corresponded to a predicted HC functional domain, we designed a fragment (N1874C2124flag) corresponding to the boundaries of the first AAA repeat (Neuwald et al., 1999). Clear binding with LIS1 was observed. To determine whether LIS1 binding is affected by ATP binding state, full-length HC and AAA domain constructs containing K to E mutations in the first P-loop element were tested (Fig. 8, A and B, K1910Eflag and N1874C2124K1910Eflag). No effect on LIS1 binding was detected, indicating that the LIS1–HC interaction does not require a functional ATPase site.
LIS1 also bound to a single discrete site within the stem region close to the binding sites for the other dynein accessory subunits (Fig. 8, A and C, and Fig. 6 B). This site is independent of that for IC binding and overlaps with that for LIC binding. To test whether LIS1 competes with the LICs for HC binding, we immunoprecipitated the LICs. LIS1 was present in the precipitates (unpublished data), indicating that LIS1 and the LICs can coexist within the same dynein complex.
As for full-length LIS1, overexpression of the LIS1 N and WD repeat domains produced pronounced increases in mitotic index. Neither fragment affected Golgi organization (compare with Smith et al., 2000), again indicating a role in a restricted range of dynein functions (Faulkner et al., 2000). The NH2-terminal region is involved in self-association (Fig. 3 A; Ahn and Morris, 2001), but, so far, not in interactions with dynein, dynactin (Fig. 7), α-PAFAH, or NudEL (Kitagawa et al., 2000; Niethammer et al., 2000; Sweeney et al., 2000). The NH2-terminal domain failed to bind to kinetochores, but displaced endogenous full-length LIS1 (Fig. 3). We suggest that this behavior is likely to reflect a weakened affinity of the of LIS1 N/full-length LIS1 heterodimer for kinetochores. The resulting loss of endogenous LIS1 from this and, potentially, other sites seems likely to contribute to the mitotic phenotype.
In contrast, the WD repeat fragment showed no evidence of self-association, mediated all of the heterologous LIS1 interactions identified in this study, and targeted to kinetochores very efficiently (Fig. 2 A). The mechanism by which this region affects mitosis may involve competition with full-length LIS1 for dynein and dynactin binding, and displacement of CLIP-170 from the kinetochore (see below). Although α-PAFAH interacts with the WD repeat region of LIS1 (Kitagawa et al., 2000; Niethammer et al., 2000; Sweeney et al., 2000), it was undetectable in our cell lysates (unpublished data) and fails to associate with dynein (Niethammer et al., 2000; unpublished data). It cannot, therefore, account for the mitotic effects we observed. The precise sites of LIS1 interaction with NudE, NudEL, and NudC are not known, nor has their fate been determined in LIS1-overexpressing cells.
As in the case of full-length LIS1 (Faulkner et al., 2000), the fragments failed to displace dynein and dynactin from the kinetochore despite the efficient displacement of endogenous LIS1 by the NH2-terminal fragment. The latter result argues strongly against a role for LIS1 in anchoring dynein and dynactin at the kinetochore. Conversely, LIS1 was displaced by dynamitin overexpression, which removes dynein and dynactin from the kinetochore. Together, our results are most clearly consistent with a model in which LIS1 is linked to the kinetochore through either dynein, dynactin, or both (Fig. 9 A).
CLIP-170 was displaced from kinetochores by overexpression of LIS1 or its WD domain (Fig. 4). Because CLIP-170 can also be displaced by dynamitin overexpression, it is likely to associate with the kinetochore indirectly through dynein or dynactin (Dujardin et al., 1998). LIS1 overexpression appears to interfere with the latter link (Fig. 4), suggesting that LIS1 may mediate the interaction between CLIP-170 and the motor protein complexes. Although this is an appealing model, it is disputed by the effects of LIS1 N overexpression, which displaces endogenous LIS1 from the kinetochore (Fig. 3 C) without affecting CLIP-170 (Fig. 4).
All three LIS1 constructs produce more severe accumulation of cells in mitosis and more dramatic alterations in spindle morphology than are generated by a dominant-negative CLIP-170 fragment (Dujardin et al., 1998), suggesting a greater overlap in LIS1 and dynein functions. Loss of CLIP-170 almost certainly contributes to the LIS1 or LIS1 WD overexpression phenotypes, but is unlikely to explain fully the greater range of LIS1 phenotypic defects. It is uncertain whether this difference reflects a role for LIS1 in a wider range of mitotic dynein functions than CLIP-170. Our evidence for an interaction between LIS1 and subunits of dynein and dynactin suggests a direct form of regulation in which CLIP-170 is unlikely to participate. In this regard, we have already found that LIS1 displaces the p150Glued subunit of dynactin from growing microtubule ends (Fig. 9 A; Faulkner et al., 2000), an effect that may weaken the interaction between kinetochores and microtubules during mitosis. It remains to be seen whether displacement of CLIP-170 from the kinetochore, which has similar microtubule-binding properties to those of p150Glued, has redundant or additive effects (Fig. 9 A).
We have found LIS1 to interact with three different polypeptides, two in the dynein complex and a third in the dynactin complex. The ability of LIS1 to interact with subunits of both cytoplasmic dynein and dynactin suggests an important new role in linking the two complexes together. Despite extensive genetic and biochemical evidence for an interaction between the dynein IC and p150Glued subunits (Karki and Holzbaur, 1995; McGrail et al., 1995; Vaughan and Vallee, 1995), only limited evidence for the copurification of the complexes has been obtained (Paschal et al., 1993; Kini and Collins, 2001). Phosphorylation of the ICs has recently been reported to regulate this interaction (Vaughan et al., 2001), and the current study suggests that LIS1 acts as a protein cofactor that may also contribute to the stability of the cocomplex.
The association of LIS1 with the first AAA domain of the dynein HC strongly predicts an additional role in regulating motor activity. Although the mechanism by which the multiple dynein AAA domains coordinate their behavior to produce force and transmit it through the stalk is not understood, the importance of the first AAA domain has been clearly demonstrated (Gibbons and Gibbons, 1987; Gee et al., 1997).
Thus, LIS1 interacts with both cargo-binding and force-producing regions of dynein itself, as well as with dynactin, suggesting a role for LIS1 in complex and novel forms of regulation (Fig. 9 B). Conceivably, LIS1 might serve to signal whether the dynein complex is associated with cargo (such as the mitotic kinetochore) and regulate motor activity accordingly. Alternatively, it could serve to stiffen the linkage between the stem and motor domains of dynein via a mechanism reminiscent of the role of LCs in stabilizing the myosin neck region (Lowey et al., 1993). Further study of the functional properties of LIS1 is likely to provide important new insight not only into mechanisms of brain development, but into dynein regulation and function as well.
Most of the HC tagged constructs are described elsewhere (Mazumdar et al., 1996; Gee et al., 1997), except N907C1138–myc, N1141C1405–flag, N1406C1669–flag, N1670C1873–flag, N1874C2124–flag, and N1874C2124K1910E–flag constructs, which were made by adding a NotI site, Kozak sequence, myc tag, or flag tag at the appropriate location by PCR, using VENT DNA polymerase (New England Biolabs, Inc.). They were then cloned into the NotI site of pCMVβ (CLONTECH Laboratories, Inc.). LIS1 fragments were created by applying the same strategy with a hemagglutinin (HA) tag added at the NH2 terminus of each fragment. The NH2-terminal 87 aa of LIS1 was used as the NH2-terminal construct, aa 88–414 was used as the WD repeat domain construct, and an internal deletion of aa 56–78 of LIS1 was used as the coiled-coil deletion construct. Dynein IC and LIC constructs were described previously (Vaughan and Vallee, 1995; Tynan et al., 2000b), and LC constructs were obtained from Stephen King (University of Connecticut Health Center, Farmington, CT). Untagged p150Glued (Vaughan and Vallee, 1995), dynamitin (Echeverri et al., 1996), Arp1 (Clark and Meyer, 1999), and p62–myc (Garces et al., 1999) were described elsewhere. All PCR-amplified constructs were sequence-confirmed by the University of Massachusetts Medical School Nucleic Acid Facility.
Anti-myc polyclonal antibody has been described elsewhere (Gee et al., 1997). Anti-HA polyclonal and monoclonal and anti-myc monoclonal antibodies were purchased from Covance. Human CREST autoimmune antiserum was provided by Bill Brinkley (Baylor College of Medicine, Houston, TX). Anti-LIS1 monoclonal antibody was provided by Dr. Orly Reiner (Weizmann Institute, Rehovot, Isreal) and has been characterized previously (Faulkner et al., 2000). Anti–CLIP-170 monoclonal antibody was provided by Holly Goodson (University of Notre Dame, Notre Dame, IN) and anti-tubulin monoclonal antibody was from Amersham Pharmacia Biotech (clone DM1A). Antibodies against Bub1, BubR1, and CENP-E were provided by Tim Yen (Fox Chase Cancer Center, Philadelphia, PA), and anti-Mad2 antibody was provided by Bonnie Howell and Edward Salmon (University of North Carolina, Chapel Hill, NC). Secondary HRP-conjugated donkey anti–mouse and anti–rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories. For immunofluorescence, Cy3-conjugated donkey anti–mouse and anti–rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories, and Alexa 488 anti–mouse and anti–rabbit antibodies were purchased from Molecular Probes, Inc.
COS-7 and HeLa cells were grown in DME with 10% FBS and penicillin/streptomycin (GIBCO BRL). For transient transfections, cells were plated either on a 10-cm petri dish or onto 18-mm coverslips to 70–80% confluency, and then were transfected with the appropriate amount of DNA mixed with lipofectAMINE (for 10-cm dish) or lipofectAMINE PLUS (coverslips) (GIBCO BRL). Transient expression was allowed for 40–48 h.
The coimmunoprecipitation procedure was described previously (Tynan et al., 2000b). COS-7 cells were transfected with plasmid DNA, purified from either Qiagen Maxiprep or QIAprep Spin miniprep kits (QIAGEN), and LipofectAMINE reagents overnight at 37°C. 48 h after the addition of the DNA–liposome mixture, cells were washed twice with PBS and lysed with modified RIPA buffer (100 mM NaCl, 1 mM EGTA, 50 mM Tris, pH 8.0, 1% IGEPAL CA-630), containing a protease inhibitor mixture (2 μg/ml of aprotinin and leupeptin [Sigma-Aldrich] and 1 mM AEBSF [Roche Molecular Biochemicals]). Cells were then incubated on ice for 20 min and spun at 13,000 g for 10 min in a microfuge (Eppendorf). Protein G beads (Amersham Pharmacia Biotech) or flag M2 affinity resin (Sigma-Aldrich) were used for all immunoprecipitations. Extracts were mixed with RIPA-washed beads and incubated at 4°C overnight with gentle rotation. Beads were then washed three times with RIPA at room temperature, 10 min each, and boiled in 5× SDS-PAGE sample buffer for 5 min. The entire eluates with 4% supernatant were loaded into 5–15% SDS-PAGE gels and transferred onto Immobilon-P membranes (Millipore). Immunoblotting was performed with various antibodies against tags or specific proteins.
The LexA-based two-hybrid assay (Gyuris et al., 1993) was used to assess interactions between LIS1 fragments and various dynein/dynactin subunits, including IC2C, dynamitin, HC N547C649, HC N748C907, and HC N1874C2124. All LIS1 fragments were cloned into the pEG202 bait vector by PCR and tested for auto-activation of the reporter. The LIS1 N bait construct activated the reporter in the absence of prey and was omitted in the two-hybrid assay. IC2C, dynamitin, and HC fragments were cloned into pJG4-5 prey vector by PCR. Interactions were scored using the β-galactosidase filter assay.
For kinetochore staining, cells were either unextracted (Fig. 4 A) or extracted with 0.5% (Figs. 2 and and3)3) or 0.01% Triton X-100 (Fig. 5 B) (Sigma-Aldrich) for 1 min and fixed in 3% paraformaldehyde (EMS) in PHEM buffer (120 mM Pipes, 50 mM Hepes, 20 mM EGTA, 4 mM magnesium acetate, pH 6.9) for 20 min at room temperature. Cells were then permeabilized with −20°C methanol for 4 min. All primary and secondary antibody incubations were performed at 37°C for 1 h. After all antibody incubations, cells were incubated with 0.1 μg/ml of DAPI (Sigma-Aldrich) for 10 min before mounting with Prolong Antifade solution (Molecular Probes, Inc.). Immunofluorescence images were obtained using a Leica DM IRBE microscope equipped with a Hamamatsu ORCA 100 CCD camera and processed by MetaMorph software (Universal Imaging Corp.). Confocal microscopy was performed with a Nikon Diaphot 200 microscope, using a Bio-Rad Laboratories MRC1000 system with a Kr/Ar laser. Each image was obtained from the total projection (20–24 stacks, 0.3 μm/step), and background subtraction was processed by the MetaMorph software after the image was pseudocolored and cropped by Adobe Photoshop® 5.5 (Adobe Systems, Inc.) and Corel Draw 9.0 (Corel Corp.).
We thank Drs. Atsushi Mikami, Peter Pryciak, and Peter Hook for helpful comments, Dr. Orly Reiner for anti-LIS1 monoclonal antibody, Dr. Bill Brinkley for CREST auto-antiserum (University of California at Los Angeles School of Medicine, Los Angeles, CA), Dr. David Meyer for Arp1 cDNA, Dr. Stephen King for dynein LC cDNA clones, and Dr. Sharon Tynan for extensive technical advice.
This work was supported by National Institutes of Health grants GM47434 and HD61982, the March of Dimes Birth Defects Foundation, the Human Frontier Science Program, and the H. Arthur Smith Charitable Foundation.
D.L. Dujardin's and R.B. Vallee's present address is Department of Pathology, Columbia University, College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032-3702.
N.E. Faulkner's present address is Genzyme Genetics, 5 Mountain Rd., Framingham, MA 01710.
*Abbreviations used in this paper: aa, amino acid(s); HA, hemagglutinin; HC, heavy chain; IC, intermediate chain; LC, light chain; LIC, light intermediate chain; LIS1 N, NH2-terminal LIS1 fragment; PAFAH, platelet-activating factor acetylhydrolase.