Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Abstract

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.

Figure. 1.

Phenotypic analysis of LIS1 fragments. (A) Schematic diagram of LIS1 domains, showing a predicted coiled-coil region (filled box) from aa 51 to 78 and a series of seven WD repeats (shaded boxes) between aa 97 and 408. (B) Effects of overexpression of LIS1 fragments on mitotic spindle morphology. Spindle defects (untransfected, 4%; LIS1 FL, 36%; LIS1 WD, 65.4%; LIS1N, 61.2%) included multiple spindle poles, detached poles, and unfocused poles. (C) Perturbation of mitosis by LIS1 fragment overexpression. Cell cycle stage was scored for nontransfected control cells (control), cells overexpressing full-length LIS1 (Ox LIS1 FL), and the NH₂- and COOH-terminal fragments (Ox LIS1 N and Ox LIS1 WD). Full-length LIS1 and NH₂- and COOH-terminal fragments all produced similar pronounced increases in mitotic index. Values are means ± SD from four independent experiments with ∼2,000 total cells counted in each case. A, anaphase; I, interphase; M, metaphase; PM, prometaphase; T, telophase.

Figure. 2.

Kinetochore localization of LIS1 fragments. (A) Transfected COS-7 cells were treated with nocodazole before fixation for immunofluorescence. Cells were triple-labeled for LIS1 and its fragments using anti-HA monoclonal antibody (green), inner kinetochore antigens using CREST human autoimmune antiserum (red), and DNA using DAPI (blue). (B) Kinetochore localization in untreated cells. Merged images of DNA (blue) and anti-HA antibody–labeled LIS1 signals (green) in COS-7 cells transfected with either HA–LIS1 FL or HA–LIS1 WD constructs. Bars, 5 μm.

Figure. 3.

Effects of LIS1 fragments on kinetochore composition. (A) Self-association of LIS1 N. Myc-tagged LIS1 full-length cDNA was coexpressed with different HA-tagged LIS1 fragments (Fig. 1) and an HA-tagged LIS1 coiled-coil deletion construct (HA–LIS1 ΔC-C), and anti-HA immunoprecipitates were immunoblotted with anti-myc antibody. Clear coimmunoprecipitation was observed only between constructs including the NH₂-terminal region of LIS1. (B) Specificity of LIS1 monoclonal antibody for WD repeat region. COS-7 cells were transfected with HA-tagged LIS1 constructs. Cell lysates were loaded into 5–15% gradient gels and analyzed by immunoblotting with anti-HA monoclonal antibody, which recognized all LIS1 constructs, and monoclonal anti-LIS1 (Sapir et al., 1997; Faulkner et al., 2000). The latter recognized endogenous and full-length HA–LIS1 (arrow), as well as the slightly smaller WD repeat fragment, but not the 10-kD LIS1 N (open arrowhead). (C) Effect of LIS1 N on endogenous LIS1 localization. In contrast to nontransfected cells, endogenous LIS1 protein detected with the LIS1 monoclonal antibody was displaced from kinetochores by LIS1 N overexpression. (D) Effect of LIS1 N and LIS1 WD on endogenous dynein IC localization. Dynein staining at the kinetochore persisted in cells overexpressing either fragment. Bars, 5 μm.

Figure. 4.

Displacement of CLIP-170 by overexpression of LIS1 fragments. HeLa cells were transfected with LIS1 constructs and treated with 10 μM nocodazole for 1 h before fixation for immunofluorescence. (A) CLIP-170 staining in LIS1 overexpressers. Both LIS1 FL and WD constructs clearly displaced CLIP-170 from kinetochores, whereas LIS1 N had no effect. (B) CENP-E staining in LIS1 overexpressers. CENP-E kinetochore signal was unaffected by overexpression of any of the LIS1 constructs. Bar, 10 μm.

Figure. 5.

Displacement of LIS1 by dynamitin overexpression. (A) Confocal images of COS-7 cells transfected with dynamitin–myc and stained with polyclonal anti-myc antibody (green) and CREST human auto-antiserum (red). In addition to its disruptive effect on the dynactin complex, overexpressed dynamitin associates with kinetochores. (B) Nocodazole-treated COS-7 cells transfected with dynamitin–green fluorescence protein (GFP) and stained with anti-Arp1, anti-LIS1, and DAPI. Arp1 and LIS1 were clearly reduced. Bars, 10 μm.

Figure. 6.

Interaction of LIS1 with multiple dynein and dynactin subunits. (A) Coimmunoprecipitation of LIS1 with dynein and dynactin subunits. Full-length HA– or myc–LIS1 was cotransfected with HC–FLAG, IC2C–myc, LIC2–FLAG, LC8-VSVG, Tctex-1–HA, RP3–HA, p150^Glued, dynamitin, Arp1, and p62–myc. Immunoprecipitation was performed with anti-HA or anti-myc antibody and blotted with antibodies against the epitope tag of individual dynein subunit or anti-p150^Glued, -dynamitin, or -Arp1. Interactions were observed with the dynein HC and IC and dynamitin. IC1A was also positive in this assay (unpublished data). (B) Two-hybrid assay of LIS1 with dynamitin, HC, and IC. LIS1 fragments were cloned into the LexA-based bait vector and full-length dynamitin, IC2C, HC N547C649, HC N748C907, and HC N1874C2124 fragments (see Fig. 8) were cloned into the prey vector. Dynamitin, IC, HC N748C907, and HC N1874C2124 were all positive in this assay. The negative HC fragment (HC N547C649) was also negative in the coimmunoprecipitation assay (see Fig. 8).

Figure. 7.

WD repeat domain of LIS1 mediates dynein and dynactin subunit interactions. COS-7 cells were singly- or doubly-transfected with HA–LIS1 FL, HA–LIS1 WD, HA–LIS1 N versus (A) IC2C–myc, (B) dynamitin–myc, (C) HC C1140–myc, and (D) HC N1874C2124–FLAG constructs. Immunoprecipitations were performed by using (A and B) anti-myc antibody, (C) anti-HA antibody, or (D) FLAG M2 beads. The immunoprecipitates (right) were immunoblotted with either anti-myc antibody (C) or anti-HA antibody (A, B, and D). Supernatants (left) were used to monitor protein expression. Each dynein and dynactin subunit immunoprecipitated with HA–LIS1 FL and HA–LIS1 WD, but not with HA–LIS1 N.

Figure. 8.

Two distinct sites of HC interact with LIS1. The HA–LIS1 FL construct was cotransfected with myc-tagged dynein stem region and FLAG-tagged motor domain constructs. HC fragment immunoprecipitates were immunoblotted for HA–LIS1. (A) Diagrammatic representation of dynein HC at top, showing IC– (orange), LIC– (yellow), and HC–HC (green) interaction sites (Tynan et al., 2000a), as well as the microtubule-binding stalk (gray, coiled-coil; red, microtubule-binding site) (Gee et al., 1997) and AAA domains (blue) (Neuwald et al., 1999). Filled bars represent positive interactions with LIS1 and empty bars represent negative interactions. White line in constructs represents K to E mutation at aa 1910. Deduced interaction regions are marked by dotted lines and correspond to aa 649–907 and aa 1874–2124. (B and C) Anti-HA immunoblots of HC fragment immunoprecipitates showing coprecipitating LIS1.

Figure. 9.

Schematic representations of LIS1 interactions. (A) Effect of LIS1 overexpression at kinetochores and microtubule plus ends (see text for detailed discussion and references). For clarity, dynactin is represented only by its dynamitin (p50) and p150^Glued subunits, and dynein only by its HCs and ICs, and the interaction of the HC with microtubules is omitted. CLIP-170 associates with the kinetochore through dynein and dynactin, but the specific link is unknown. LIS1 is found in the current study to interact with p50, HC, and IC. Overexpression of dynamitin (dotted orange line) removes all polypeptides depicted downstream. Overexpression of either full-length LIS1 or LIS1 WD (dotted green line) has no effect on the association of dynein and dynactin with the kinetochore, but dissociates CLIP-170 from this site and interferes with the interaction of p150^Glued with microtubules (Faulkner et al., 2000). Further potential direct effects of overexpressed LIS1 or LIS1 fragments on dynein and dynactin subunits are discussed in the text. (B) Interaction of LIS1 with two dynein HC sites. (I) LIS1 is shown interacting independently with the cargo-binding dynein stem domain or the first AAA unit within the motor domain. (II) The proximity of these two sites within the folded dynein molecule may allow LIS1 to interact with both simultaneously.

Figure. 9.

Schematic representations of LIS1 interactions. (A) Effect of LIS1 overexpression at kinetochores and microtubule plus ends (see text for detailed discussion and references). For clarity, dynactin is represented only by its dynamitin (p50) and p150^Glued subunits, and dynein only by its HCs and ICs, and the interaction of the HC with microtubules is omitted. CLIP-170 associates with the kinetochore through dynein and dynactin, but the specific link is unknown. LIS1 is found in the current study to interact with p50, HC, and IC. Overexpression of dynamitin (dotted orange line) removes all polypeptides depicted downstream. Overexpression of either full-length LIS1 or LIS1 WD (dotted green line) has no effect on the association of dynein and dynactin with the kinetochore, but dissociates CLIP-170 from this site and interferes with the interaction of p150^Glued with microtubules (Faulkner et al., 2000). Further potential direct effects of overexpressed LIS1 or LIS1 fragments on dynein and dynactin subunits are discussed in the text. (B) Interaction of LIS1 with two dynein HC sites. (I) LIS1 is shown interacting independently with the cargo-binding dynein stem domain or the first AAA unit within the motor domain. (II) The proximity of these two sites within the folded dynein molecule may allow LIS1 to interact with both simultaneously.