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Vol. 16, Issue 11, 5127-5140, November 2005
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* Howard Hughes Medical Institute and Institutes of Neuroscience and Molecular Biology, University of Oregon, Eugene, OR 97403;
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455; and
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854
Submitted April 22, 2005;
Revised July 8, 2005;
Accepted August 10, 2005
Monitoring Editor: Erika Holzbaur
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Importantly, sister kinetochores must be attached to MTs originating from opposite spindle poles, a configuration referred to as bipolar attachment, before sister chromatid separation and anaphase onset. A surveillance mechanism termed the "mitotic checkpoint" or "spindle assembly checkpoint" delays anaphase onset and sister chromatid separation until all chromatid pairs are attached and tension is generated between sister chromatids (reviewed in Cleveland et al., 2003; Taylor et al., 2004).
Components of the mitotic checkpoint were first identified in budding yeast and include the Mad1, Mad2, Mad3, Bub1, and Bub3 proteins (Hoyt et al., 1991; Li and Murray, 1991). Although the Mad and Bub proteins are highly conserved from yeast to human, additional checkpoint proteins including members of the Rough-deal/Zeste-white10 (Rod/Zw10) complex have been identified in higher eukaryotes (Starr et al., 1997; Basto et al., 2000; Chan et al., 2000; Scaerou et al., 2001; Williams et al., 2003). Similar to disruption of Mad or Bub protein function, interference with Rod or Zw10 function abrogates mitotic checkpoint function, promoting chromosome segregation defects due to premature anaphase onset before bipolar chromosome attachment (Karess and Glover, 1989; Williams et al., 1992; Basto et al., 2000; Chan et al., 2000).
Several observations revealed the central role of kinetochores in checkpoint signaling. Functional kinetochores are required for generation of the mitotic checkpoint signal (Rieder et al., 1995), and checkpoint proteins such as Mad2 and Rod are localized to unattached kinetochores (reviewed in Cleveland et al., 2003; Taylor et al., 2004). The Rod/Zw10 complex promotes recruitment of Mad2 to unattached kinetochores (Buffin et al., 2005; Kops et al., 2005). At unattached kinetochores Mad2 is thought to be converted into an "active" species and subsequently released into the cytoplasm where it indirectly inhibits the "anaphase promoting complex/cyclosome" (APC/C). Inhibition of the APC/C in turn prevents premature sister chromatid separation and anaphase onset. At the end of metaphase (after correct bipolar MT attachment of all chromatid pairs is achieved), the mitotic checkpoint is inactivated, leading to increased APC/C activity which consequently triggers initiation of sister chromatid separation and anaphase onset (reviewed in Cleveland et al., 2003; Taylor et al., 2004). Insights into the mechanism of checkpoint inactivation in metazoans came from the following observations in mammalian tissue culture and Drosophila cells. After MT-attachment, Mad2 and Rod stream off kinetochores along kinetochore MTs (kMTs), resulting in the reduction of kinetochore-associated Mad2 and Rod levels (Howell et al., 2000; Basto et al., 2004). Interference with the function of dynein, a minus end-directed MT-based motor complex that also localizes to unattached kinetochores, causes accumulation of the Mad2 and Rod proteins on attached metaphase kinetochores by blocking their poleward transport and delays metaphase-to-anaphase transition (Howell et al., 2001; Wojcik et al., 2001). These observations led to the model that dynein-dependent poleward transport of checkpoint proteins contributes to silencing of the mitotic checkpoint signal and thereby promotes timely initiation of anaphase.
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; Smith et al., 2000; Tai et al., 2002). Mutations in the human Lis1 gene have been identified as the cause of lissencephaly (Reiner et al., 1993), a condition characterized by severe neurodevelopmental defects as a consequence of abnormal neuronal migration and perhaps neural precursor cell divisions (reviewed in Feng and Walsh, 2001; Vallee et al., 2001). The role of Lis1 in dynein-dependent cellular processes is not clearly understood. For example, dynein is required for pericentrosomal Golgi positioning (reviewed in Karki and Holzbaur, 1999), yet there are conflicting reports whether Lis1 is involved in this process (Faulkner et al., 2000; Smith et al., 2000). The role of Lis1 in cell cycle timing and mitotic checkpoint signaling is also unclear. Injection of Lis1 function-blocking antibodies in mammalian cells led to kinetochore-MT attachment defects and prolonged prometaphase, but no delay in metaphase-to-anaphase transition was observed (Faulkner et al., 2000). These data suggest that Lis1 regulates chromosome alignment rather than contributing to dynein-dependent mitotic checkpoint inactivation. Thus, although Lis1 protein is highly conserved from yeast to man, functional evidence for a role in mitotic checkpoint signaling is minimal.
The evolutionarily conserved dynactin complex also associates with dynein, increases the processivity of the dynein motor (King and Schroer, 2000) and is thought to mediate dynein attachment to its cargo (reviewed in Schroer, 2004). Dynactin recruits dynein to kinetochores (Echeverri et al., 1996) and is required for poleward Mad2 transport and timely anaphase onset in mammalian cells (Howell et al., 2001). However, the role of dynactin in checkpoint signaling has not been investigated in Drosophila because of lack of suitable mutant alleles.
Here we report a detailed functional analysis of Lis1 and dynactin in Drosophila larval neuroblasts, using an improved time-lapse video microscopy technique that allows live imaging of the neuroblast cell cycle in whole larval brain explants. We show that Lis1 and dynactin are required for centrosome separation and spindle assembly during prophase/prometaphase and contribute to timely metaphase-to-anaphase transition by promoting efficient mitotic checkpoint inactivation. In addition, we describe the subcellular localization of Lis1 and dynactin in mitotic neuroblasts, providing evidence that, in contrast to their homologous proteins in mammals and Caenorhabditis elegans, Drosophila Lis1 and dynactin localize to kinetochores in a codependent manner.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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); Lis1k13209/CyO; l(3)KG07739; Df(3L)fz-GF3b (Bloomington Stock Center); rodH4.8/TM6B (Karess and Glover, 1989); p[w+ GFP-Rod] rodX5 e ca (Scaerou et al., 1999; Basto et al., 2004); p[w+mC = His2AvT:Avic\GFP-S65T]62A expressing His2AvD-GFP (Clarkson and Saint, 1999); the gene trap line G147, which expresses a green fluorescent protein (GFP)-tagged MT-associated protein (Morin et al., 2001). All mutant alleles were rebalanced over CyO actin-GFP or TM3 actin-GFP Ser. Newly hatched mutant larvae were identified based on the absence of GFP expression in the gut. Larvae were aged to the late 2nd instar stage (wild type, Lis1–, Lis1– rodH4.8, rodH4.8 48 h after larval hatching (ALH); Gl mutants: 96–120 h ALH) for all phenotypic analyses.
Generation and Characterization of Glued Alleles
We generated a new Glued allele, Gl
22, by imprecise excision of the P-element l(3)KG07739 inserted into the 5'-UTR encoding region of the Glued gene (Bellen et al., 2004). PCR and sequence analysis revealed that Gl22 contains a 
3.2-kb deletion removing the presumptive transcriptional start site of the Glued gene (Figure 1B). In addition, the molecular nature of the Gl1–3 allele (Harte and Kankel, 1982) was determined by sequence comparison of genomic DNA amplified from homozygous Gl1–3 mutant and Oregon R larvae using standard PCR techniques, revealing a point mutation converting codon 932 into a premature stop codon (Figure 1A). Homozygous Gl1–3, homozygous Gl22, and hemizygous Gl1–3/Df(3L)fz-GF3b animals die as 2nd instar larvae.
Generation of Transgenic Fly Lines Expressing GFP-tagged Lis1 Protein
The complete Lis1 coding sequence was amplified from Drosophila EST RE28987 and subcloned into the pUAST vector downstream of, and in-frame with, three repeats encoding EmeraldGFP (Tsien, 1998). Transgenic flies were generated by standard methods. GFP-Lis1 was expressed in larval neuroblasts by crossing pUAST-3xEmeraldGFP-Lis1 transgenic flies to a worniu-Gal4 driver line (Albertson et al., 2004; see Figure 10 and Supplementary Movie 10). In addition, one of us (R.S.) subcloned the Lis1 coding sequence into the pUASP vector downstream of a single GFP coding sequence; this GFP-Lis1 was ubiquitously expressed in embryos using the maternal nanos-Gal4 driver for the immunoprecipitation experiments (see Figure 8B).
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). Brains were imaged using a Bio-Rad Radiance 2000 laser scanning confocal microscope equipped with a 60x1.4 NA oil immersion objective. For the analysis of spindle assembly (neuroblasts expressing 2 copies of G147-GFP), stacks containing four focal planes spaced by 1.5 µm were acquired at intervals of 10 s. For the analysis of chromosome movements and cell cycle timing (neuroblasts expressing one copy of His2AvD-GFP) or GFP-Rod localization, stacks containing six focal planes spaced by 1.5 µm were acquired at intervals of 15 s. For the analysis of GFP-Lis1 localization, stacks containing four focal planes spaced by 1.5 µm were acquired at intervals of 15 s. Time-lapse image series were converted into movies using MetaMorph (Universal Imaging, West Chester, PA) and ImageJ. All movie frames are maximum intensity projections.
Antibodies and Immunofluorescent Staining
Drosophila EST RE28987 was used to amplify a Lis1 cDNA encoding amino acids 1–90, which was subcloned into bacterial expression vectors to express 6xHis-tagged Lis1 protein. Purified 6xHis-Lis1 was injected into rats to generate polyclonal antibodies.
Larvae were dissected in Schneider's medium (Sigma, St. Louis, MO), fixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, and 1 mM MgCl2 for 25 min and blocked for 1 h in 1x phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.1% Triton X-100 (PBS-BT). For labeling of DNA with propidium iodide, RNase A was added to a final concentration of 1 µg/ml. After blocking, specimen were extensively washed in PBS-BT for 1 h and incubated with primary antibodies in PBS-BT overnight at 4°C. Primary antibodies were: rat anti-Lis1 (1:2500; this study); rabbit anti-Gl (raised against Gl C-terminus, 1:150; Waterman-Storer and Holzbaur, 1996); rabbit anti-Cnn (1:1000; Heuer et al., 1995); rabbit anti-nPKC
(Santa Cruz Biotechnology, Santa Cruz, CA; 1:500); rat anti-Miranda (1:1000; Irion et al., 2004); mouse anti-
-tubulin (DM1A, Sigma, 1:2000); rat anti--tubulin (MCA78S, Serotec, Raleigh, NC; 1:100); rabbit anti-Cid (1:500; Henikoff et al., 2000); mouse anti-
-tubulin (GTU-88, Sigma, 1:2000); rabbit anti-phospho-histone H3 (Upstate Biotechnology, Lake Placid, NY; 1:1000); rabbit anti-Rod (1:200; Scaerou et al., 1999). Primary antibodies were extensively rinsed off with PBS-BT for 1 h at room temperature, and specimens were incubated with fluorescently conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, and Molecular Probes, Eugene, OR) diluted in PBS-BT, followed by extensive rinsing with PBS-BT. For DNA labeling, specimens were mounted in FluoroGuard Antifade Reagent (Bio-Rad, Richmond, CA) containing 2.5 µg/ml propidium iodide. Brains were imaged using a Bio-Rad Radiance 2000 or Leica TCS SP2 laser scanning confocal microscope (Deerfield, IL) equipped with a 60x 1.4 NA or 63x 1.4 NA oil immersion objective, respectively. Figures were assembled in Adobe Photoshop (San Jose, CA).
Cell Cycle Analysis in Fixed Specimens
Larval brains were labeled for phospho-histone H3 (mitotic DNA), -tubulin, and Miranda. Neuroblasts were identified by size and expression of Miranda protein, and scored for cell cycle stage using phospho-histone H3 and -tubulin.
Immunoprecipitation Experiments
Immunoprecipitation experiments were conducted in high-speed supernatants at 4°C, and no microtubules were present. In detail, 0–24-h embryos were homogenized in 2.5 volumes IP buffer (50 mM HEPES, pH 7.2, 150 mM KCl, 0.9 M glycerol, 0.5 mM dithiothreitol, with protease inhibitors 10 µg/ml aprotinin, 1 µg/ml each leupeptin and pepstatin, 0.1 µg/ml each of soybean trypsin inhibitor, n-tosyl-L-arginine methylester, and benzamidine), and then supplemented with Triton X-100, 0.1%. After ultracentrifugation at 25,000 rpm for 20 min in a 50ti rotor, the supernatant was collected and precleared against washed protein A-Sepharose beads (Sigma). Cleared extract, 700 µl, was incubated 2 h at 4 °C with beads that had previously been bound anti-Dhc monoclonal P1H4 (dynein heavy chain; McGrail and Hays, 1997), anti-GFP monoclonal 3E6 (Molecular Probes), or anti-Lis1 antibodies. Beads were washed in IP buffer three times, the last two washes without detergent. Pellets were eluted into 20 µl 2x sample buffer, and samples were analyzed by SDS-PAGE followed by Western analysis using anti-Dhc monoclonal P1H4 (McGrail and Hays, 1997); rabbit anti-Gl C-terminal (Waterman-Storer and Holzbaur, 1996) or anti-GFP monoclonal JL-8 (Clontech, Palo Alto, CA) antibodies.
Drug Treatments
Larval brains were dissected in Schneider's medium (Sigma) and incubated for 2 h at room temperature either in Schneider's medium supplemented with 30 µM colcemid (Sigma) or in Schneider's medium without drugs (controls). Afterward, brains were processed for immunofluorescent antibody staining as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) described a live cell imaging technique to characterize key mitotic events in neuroblasts of wild-type larval brains squashed in Voltalef oil. Two major limitations of this method were that neuroblasts deteriorated relatively quickly in culture (within less than 2 h) and that wild-type neuroblasts contained secondary spindles besides the primary main spindle (in 17% of all observations) or failed to complete cytokinesis (Savoian and Rieder, 2002). Using a modified technique, Fleming and Rieder (2003) were able to prolong viability of neuroblasts in culture, but cytokinesis defects still occurred. We improved the original technique of Savoian and Rieder by making two essential changes: 1) minimizing physiological stress by substituting Voltalef oil with insect culture media, and 2) minimizing physical stress by imaging neuroblasts in whole larval brain explants instead of brain squashes (see Materials and Methods). Using this new technique, we never observed formation of secondary spindles or failure in cytokinesis in wild-type brains within the first 3 h of observation.
For our first set of experiments we imaged the GFP gene trap line G147 (Morin et al., 2001) that expresses an MT-associated GFP-fusion protein labeling spindle poles, spindle MTs, and astral MTs. Henceforth, we will refer to this GFP fusion protein as G147-GFP. The use of the G147 line allowed us to precisely define many key stages of mitosis, including centrosome separation during prophase, initiation of prometaphase (defined by nuclear envelope breakdown [NEB] as judged by penetration of spindle microtubules into the cell center), and anaphase onset (defined by the first sign of widening of the gap between opposing kMTs). Here we used this time-lapse method to analyze the role of Lis1 in spindle assembly and mitotic checkpoint signaling.
Lis1 and Dynactin Are Required for Centrosome Separation and Spindle Assembly
Time-lapse imaging of wild-type second instar larval neuroblasts expressing G147-GFP showed that duplicated centrosomes stay in close proximity to each other until they separate during prophase. By the onset of prometaphase, the pair of centrosomes was always completely separated and positioned on opposite sides within the neuroblast (average separation 171 ± 7°, n = 15; Figure 2, A (0:00) and C, Supplementary Movie 1). As prometaphase progressed, the centrosomes nucleated MTs that formed a straight bipolar spindle (Figure 2A (0:00–6:10), Supplementary Movie 1).
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). In Lis1G10.14/Lis1k13209 (hereafter referred to as Lis1–) larval brains, Lis1 protein was reduced below detectable levels (see below and Figure 9). In contrast to wild-type, time-lapse imaging of Lis1– mutant neuroblasts expressing G147-GFP revealed that centrosome separation was frequently incomplete at the onset of prometaphase (average separation 124 ± 38°, n = 12; Figure 2, B (0:00) and C, Supplementary Movie 2), and MTs emanating from both spindle poles often developed a Y-shaped morphology during prometaphase, presumably because of the close apposition of the centrosomes (Figure 2B (0:00–2:40), Supplementary Movie 2). Strikingly, these Y-shaped spindles generally "recovered" to form seemingly bipolar arrays of kMTs by late metaphase (Figure 2B (17:50–24:10), Supplementary Movie 2). However, despite the apparent bipolar spindle organization, microtubule organizing centers (MTOCs) occasionally detached from spindles or two MTOCs appeared to maintain attachment to the same side of a half-spindle. In few cases neuroblasts contained more than the normal two MTOCs (Figure 2B (17:50–24:10), Supplementary Movie 2). We conclude that Lis1 is required for centrosome separation, proper spindle assembly, and regulation of MTOC number.
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-tubulin) and spindle markers (-tubulin) in fixed neuroblasts. We found that wild-type metaphase neuroblasts had a straight bipolar spindle with each spindle pole tightly focused on a single centrosome (Figure 2, D and I, Table 1). In contrast, Lis1– mutant neuroblasts showed curved spindles, unfocused spindle poles, spindles with one or two unattached centrosomes, or even spindles in which both centrosomes were attached to the same half-spindle (Figure 2, E, F, J, and K; Table 1). Strikingly, some of these metaphase neuroblasts appeared to contain 3–4 centrosomes with multipolar spindles, bipolar spindles with more than one centrosome on each half spindle, or bipolar spindles with 1–2 unattached centrosomes (Figure 2, G and L, Table 1). Because of the lack of suitable Drosophila centriole markers, we were unable to determine whether all ectopic centrosomelike (Centrosomin/-tubulin positive) structures contained centrioles. We conclude that Lis1 has a critical role in correct spindle assembly by regulating centrosome separation, focusing of spindle poles, centrosome attachment, and centrosome number and/or centrosome integrity in larval neuroblasts. The presence of a subset of Lis1– mutant neuroblasts with normal bipolar spindles, however, allowed us to examine these neuroblasts for defects in later steps of the cell cycle (see next section).
Lis1 functions together with dynactin in many cell types, so we wanted to determine if dynactin was also required for spindle formation. Well-characterized loss-of-function dynactin mutants do not exist in Drosophila, so we generated a null mutation in Glued (Gl22), which encodes the largest dynactin subunit, as well as molecularly characterized an extant allele, Gl1–3 (Harte and Kankel, 1982; Figure 1; see Materials and Methods). Both Gl22 and Gl1–3 appeared to comprise protein-null alleles (see below, Figure 9). We found that Gl22 homozygous or Gl1–3 hemizygous mutant neuroblasts phenocopied centrosome and spindle formation defects observed in Lis1– mutants. Defects included metaphase neuroblasts with curved spindles, unfocused spindle poles, spindles with one or two unattached centrosomes, spindles in which both centrosomes were attached to the same half-spindle, or occasionally neuroblasts with three or four centrosomes forming bipolar or multipolar spindles (Figure 2, H and M, Table 1, and unpublished data). Thus, both dynactin and Lis1 promote centrosome separation and proper spindle formation in larval Drosophila neuroblasts.
Lis1 and Dynactin Are Required for Timely Anaphase Onset
In wild-type neuroblasts (expressing G147-GFP), the duration of prometaphase and metaphase (from NEB to anaphase onset) was quite rapid and highly reproducible, lasting 6 min 12 s ± 0 min 49 s (n = 18; Figure 3, A and D, Supplementary Movie 1). In contrast, Lis1– mutant neuroblasts showed dramatic lengthening of the prometaphase/metaphase interval to an average of 46 min 54 s ± 18 min 33s(n = 11, Figure 3, B and D, Supplementary Movie 3). We reasoned that this delay in anaphase onset might be due to extended mitotic checkpoint activity. To test this, we reduced Rod function to bypass the checkpoint (Basto et al., 2000). In Lis1– rodH4.8 double mutant neuroblasts, progression through prometaphase/metaphase only took 11 min 31 s ± 4 min 23 s (n = 11, Figure 3, C and D, Supplementary Movie 4), confirming that the delay in anaphase onset is checkpoint-dependent. Note that centrosome separation and spindle assembly defects were still present in Lis1– rodH4.8 double mutant neuroblasts, indicating that these defects were independent of altered mitotic checkpoint signaling (Figure 3C, Supplementary Movie 4, and unpublished data). In addition, we also calculated mitotic index and metaphase:anaphase ratio in fixed specimens of wild type, Gl single, Lis1– single, and Lis1– rodH4.8 double mutants. Both mitotic index and metaphase:anaphase ratio of neuroblasts were increased in Lis1– and Gl single mutants compared with wild-type and Lis1– rodH4.8 double mutant neuroblasts (Table 2). Thus, the observed delays in anaphase onset in Lis1 and Gl mutant neuroblasts are consistent with extended mitotic checkpoint activity, indicating a role for Lis1/dynactin in satisfying or inactivating the mitotic checkpoint.
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To measure the length of prometaphase and metaphase individually, we expressed a single copy of a GFP-tagged histone variant His2AvD under control of its native promoter (His2AvD-GFP; Clarkson and Saint, 1999) in larval neuroblasts. We defined prometaphase as the interval from NEB (evident by a slight increase of cytoplasmic His2AvD-GFP fluorescence intensity not associated with chromosomes) to the moment of completed chromosome congression on the metaphase plate, and we defined metaphase as the interval between completion of chromosome congression and initiation of poleward chromosome movement. In wild-type neuroblasts, prometaphase and metaphase took 4 min 14 s ± 1 min 31 s and 2 min 24 s ± 1 min 13 s, respectively (n = 12, Figure 4, A and D, Supplementary Movie 5). Throughout metaphase, all chromosomes remained aligned in a tight metaphase plate. In contrast, in Lis1– mutant neuroblasts both prometaphase (mean duration 32 min 18 s ± 15 min 55 s) and metaphase (mean duration 19 min 21 s ± 16 min 35 s) were significantly prolonged (n = 10, Figure 4, B–D, Supplementary Movies 6 and 7). These data indicate that Lis1/Gl affects cell cycle timing in at least two ways. First, Lis1/Gl is required for timely prometaphase progression, possibly by promoting spindle assembly and thereby facilitating efficient MT-kinetochore capturing/chromosome congression. Second, Lis1/Gl is also required for the timely initiation of the metaphase-to-anaphase transition, possibly by contributing to mitotic checkpoint inactivation.
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Lis1 and Gl Are Required to Transport Checkpoint Proteins from Kinetochores and Generate Interkinetochore Tension
Loss of Lis1/dynactin function could cause prolonged mitotic checkpoint activity by several mechanisms, including impairment of MT-kinetochore attachment (Rieder et al., 1995), a reduction of interkinetochore tension (Li and Nicklas, 1995), or a defect in the transport of checkpoint proteins off kinetochores (Howell et al., 2001; Wojcik et al., 2001). Here we tested which, if any, of these mechanisms were affected in Lis1 and Gl mutant neuroblasts.
We first tested whether defective MT-kinetochore attachment was the primary cause of delayed metaphase-to-anaphase transition in Lis1 and Gl mutant neuroblasts. Analysis of fixed preparations stained for spindle, DNA, and/or kinetochore markers revealed that in Lis1 and Gl single mutant metaphase neuroblasts typically all chromosomes had congressed into a tight metaphase plate with kinetochore fibers abutting kinetochores (Lis1–: 72.5%, n = 102; Gl1–3: 75.0%, n = 40; Gl1–3/Df(3L)fz-GF3b: 69.2%, n = 107; Figure 5, C and D). In addition, we followed chromosome movement (visualized with His2AvD-GFP) in Lis1– mutant neuroblasts using time-lapse analysis. We found that during prometaphase chromosomes showed delayed congression to the equatorial plate but eventually aligned into a tight metaphase plate (Figure 4, B and C, Supplementary Movies 6 and 7, n = 10). In two Lis1– mutant neuroblasts, we observed formation of a tight metaphase plate, subsequent chromosome loss, and recongression of the lost chromosome to the metaphase plate (Figure 4C (17:15–40:45), Supplementary Movie 7). Importantly, even after congression of all chromosomes into a tight metaphase plate, Lis1– mutant metaphase neuroblasts showed delayed transition into anaphase (Figure 4B (25:15–33:15) and 4C (40:45–45:30), Supplementary Movies 6 and 7). Although chromosome separation in Lis1– mutant anaphase neuroblasts was slightly slower than in wild-type counterparts, chromosomes were always completely partitioned in both daughter cells in these mutants (n = 10). We conclude that defective MT-kinetochore attachment is unlikely to be the only cause of extended checkpoint activity in Lis1 (and Gl) mutant neuroblasts.
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). Increased tension between sister kinetochores is evident by increased distance between sister centromeres (intercentromere distance). During prophase, in the absence of MT-kinetochore attachment, the measured intercentromere distance reflects the "resting length" corresponding to 0% tension. We found that wild-type, Lis1 mutant, and Gl mutant prophase neuroblasts showed statistically indistinguishable intercentromere resting lengths (wild type: 0.51 ± 0.08 µm, n = 36; Lis1–: 0.51 ± 0.07 µm, n = 20; Gl1–3: 0.55 ± 0.07 µm, n = 5; Student's t test analysis revealed p values > 0.2 in all pair wise combinations, Figure 5, A and E). On entry into metaphase, the wild-type intercentromere length increased to 1.10 ± 0.13 µm (n = 36, Figure 5, B and E), which presumably reflects 100% tension. In Lis1 and Gl mutant metaphase neuroblasts the intercentromere distance was slightly but significantly reduced compared with wild-type metaphase counterparts (Lis1–: 0.97 ± 0.16 µm, n = 58; Gl1–3: 0.93 ± 0.13 µm, n = 25; p < 0.001; Figure 5, C–E). Assuming a linear correlation between measured intercentromere distance and generated tension, we calculated that tension was reduced to 77 and 70% in Lis1 and Gl mutant metaphase neuroblasts, respectively. Thus, it is possible that the observed reduction in interkinetochore tension activates the mitotic checkpoint and delays anaphase onset in Lis1 and Gl mutant neuroblasts (see Discussion).
To test whether checkpoint protein localization was normal in Lis1 and Gl mutant neuroblasts, we assayed the dynamic localization of GFP-Rod. GFP-Rod was expressed under the control of the native Rod promoter in a rod mutant background (Scaerou et al., 1999; Basto et al., 2004). In wild-type neuroblasts, GFP-Rod was excluded from the nucleus during interphase and prophase, but accumulated on unattached kinetochores at the onset of prometaphase (Figure 6A (0:00), Supplementary Movie 8). At metaphase, GFP-Rod moved from kinetochores onto kMTs (Figure 6A (2:15–5:30), Supplementary Movie 8), presumably as a result of poleward streaming along kMTs (Basto et al., 2004). In contrast, Lis1– mutant neuroblasts lacked poleward GFP-Rod streaming and thus showed persistent GFP-Rod kinetochore localization during metaphase and anaphase (Figure 6B (0:00–49:15), Supplementary Movie 9). In addition, we confirmed that GFP-Rod resembled endogenous Rod localization by analyzing fixed preparations. In wild-type neuroblasts, Rod localized to prometaphase kinetochores and redistributed along kMTs during metaphase (Figure 7, B and C). In Lis1, Gl, and dhc (dynein heavy chain) mutant neuroblasts we observed persistent high level of Rod localization at metaphase kinetochores that were seemingly attached to MTs (Figure 7, D–F), consistent with the notion that a Lis1/dynactin/dynein complex is required for timely removal of the Rod checkpoint protein from kinetochores at metaphase.
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Lis1 and Dynactin Coimmunoprecipitate with Dynein and Colocalize with the Checkpoint Protein Rod on Kinetochores
We have shown that Lis1 and dynactin have similar functions throughout the cell cycle, ranging from centrosome separation to generation of interkinetochore tension and checkpoint protein transport. We next determined whether Lis1/dynactin are physically associated in vivo, and we analyzed subcellular localization of Lis1/dynactin throughout the neuroblast cell cycle. Using anti-Lis1 antibodies, we could immunoprecipitate both dynein and dynactin subunits, and in a reciprocal experiment we used anti-Dhc antibodies to immunoprecipitate Gl protein (Figure 8A). We also expressed full-length Lis1 protein fused to GFP (GFP-Lis1) in wild-type embryos and showed it could immunoprecipitate Dhc and Gl proteins; similarly, both anti-Dhc and anti-Gl antibodies could immunoprecipitate GFP-Lis1 (Figure 8B and unpublished data). From these results we conclude that some Lis1, dynactin, and dynein proteins are associated in a complex in Drosophila embryos. However, our immunoprecipitations were not quantitative, and we did not determine the percentage of each protein that exists together in a complex. Our findings support previous observations reported for the interaction of dynein, Lis1, and dynactin in mammalian brain cytosolic extracts (Faulkner et al., 2000; Smith et al., 2000).
We next assayed the subcellular localization of Lis1 and Gl proteins in mitotic neuroblasts. Lis1 and Gl showed spindle pole/centrosome association from late prophase through telophase (Figure 9, A-B and F-G, and unpublished data). Both proteins were colocalized with Rod on prometaphase kinetochores (Figure 7B and unpublished data), and distributed along kMTs during metaphase (Figures 7C and 9, B and G). During anaphase/telophase, Lis1/dynactin staining intensity was diminished on kMTs (unpublished data). A similar localization at centrosomes and kinetochores has been reported for dynein (Pfarr et al., 1990; Steuer et al., 1990; Starr et al., 1998; Gonczy et al., 1999; Wojcik et al., 2001). In Lis1 and Gl mutant neuroblasts, Lis1 and Gl proteins were undetectable at all of these locations, respectively, demonstrating that labeling of these structures with the anti-Lis1 and anti-Gl antibodies was specific (Figure 9, D, J, and K). Furthermore, we observed the same localization of a GFP-tagged full-length Lis1 (GFP-Lis1) protein in live neuroblasts. In prophase neuroblasts, GFP-Lis1 was excluded from the nucleus. With the beginning of prometaphase GFP-Lis1 was strongly associated with kinetochores. During late prometaphase/metaphase, GFP-Lis1 made a transition from kinetochore to centrosomal/spindle localization, whereas during anaphase and telophase there was progressively less GFP-Lis1 associated with the mitotic spindle (Figure 10A, Supplementary Movie 10).
In summary, Lis1/dynactin/dynein coimmunoprecipitate, localize to centrosome/spindle poles, and are transiently colocalized with the checkpoint protein Rod on prometaphase kinetochores before enrichment on kMTs. These results are consistent with a function of Lis1/dynactin/dynein in centrosome separation and spindle assembly as well as kinetochore-based checkpoint function (see Discussion).
Lis1 and Dynactin Are Codependent for Kinetochore Localization
Lis1/dynactin localization to kinetochores is evolutionarily conserved, but appears to be regulated differently in mammalian and C. elegans cell types (Coquelle et al., 2002; Tai et al., 2002; Cockell et al., 2004). Here we investigated the interdependence of Lis1 and dynactin kinetochore localization in Drosophila neuroblasts. We found that after reduction of dynein activity (in dhc6–10 mutants, Gepner et al., 1996; Wojcik et al., 2001), the initial localization of Lis1, dynactin, and the mutant Dhc6–10 proteins (Wojcik et al., 2001) to kinetochores was normal, but Lis1/dynactin failed to become depleted from metaphase kinetochores (Figure 9, C and H). Thus, dynein activity is required for transporting Lis1 and dynactin off the kinetochore along kMTs. In contrast, Lis1 mutant neuroblasts lacked Gl kinetochore localization (Figure 9I), and Gl mutant neuroblasts lacked Lis1 kinetochore localization (Figures 7E and 9E). This was true even when microtubules were depolymerized to block potential dynein-based transport off kinetochores, strongly suggesting that in Drosophila neuroblasts Lis1 and Gl are codependent for kinetochore localization.
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; Gonczy et al., 1999; Robinson et al., 1999; Cockell et al., 2004; Schmidt et al., 2005). However, the exact mechanism by which they promote centrosome separation is unclear. One proposed model suggests that dynein may promote centrosome separation by generating pulling forces on astral MTs attached to the cortex or cytoplasmic structures (Vaisberg et al., 1993; Waters et al., 1993). Alternatively, dynein associated with the nuclear envelope may exert pulling forces on astral MTs to promote centrosome separation (Gonczy et al., 1999; Robinson et al., 1999). We did not detect GFP-Lis1 on the nuclear envelope or at the neuroblast cortex, although it is possible that high cytoplasmic levels masked low levels of Lis1/dynactin at these sites. Thus, it remains unclear how Lis1/Gl promotes centrosome separation in neuroblasts. We found that centrosome separation was not completely blocked in Lis1 or Gl mutant neuroblasts, either because of residual amounts of maternal protein or because of the presence of a Lis1/dynactin/dynein-independent pathway. Interestingly, cortical nonmuscle myosin II has recently been shown to contribute to centrosome separation in some cell types (Rosenblatt et al., 2004), raising the possibility that Lis1/dynactin/dynein and myosin II play partially redundant roles in neuroblast centrosome separation.
Our observations further support a role for centrosomal/spindle pole-associated Lis1/Gl in spindle assembly, spindle pole focusing, and centrosome attachment in prometaphase and metaphase neuroblasts (summarized in Figure 11A). Detachment of centrosomes from the spindle has previously been observed in dynein mutants in Drosophila (Robinson et al., 1999; Wojcik et al., 2001), and in mammalian cells with reduced dynein or dynactin function (Quintyne et al., 1999). These findings, together with our results, show that Lis1 and dynactin act as cofactors for dynein-dependent focusing of spindle poles and attachment of spindle MTs minus-ends to centrosomes. In vertebrate cells dynein/dynactin is thought to contribute to focusing of spindle poles and attaching MT-minus ends to centrosomes by transporting pericentriolar proteins and MT-binding proteins, such as NuMA, to centrosomes (reviewed in Wittmann et al., 2001; Blagden and Glover, 2003). Although no clear NuMA orthologue is encoded in the Drosophila genome, a dynein/dynactin/Lis1 complex may contribute to spindle pole focusing by concentrating other MT cross-linking proteins with NuMA-like function at spindle MT minus ends.
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We found that Gl and Lis1 mutant neuroblasts occasionally formed multipolar spindles and had more than two centrosomelike Centrosomin/-tubulin structures. Because of the lack of Drosophila centriolar markers we were not able to determine whether these extracentrosome-like structures contained centrioles. Multipolar spindles have also been observed in mammalian cells overexpressing Lis1 protein or in which Lis1 function was reduced (Faulkner et al., 2000). Our time-lapse analysis of Lis1 mutant neuroblasts revealed occasional cosegregation of both centrosomes into the neuroblast as a consequence of incomplete centrosome separation and centrosome detachment from the spindle. Such a mis-segregation event may be followed by duplication of both centrosomes during the next cell cycle, leading to supernumerary centrosomes. Alternatively, extracentrosomes in Lis1 and Gl mutant neuroblasts may be due to uncoupling of centrosome duplication from the cell cycle or centrosome fragmentation.
The Role of Lis1/Dynactin in Regulating Cell Cycle Timing and Mitotic Checkpoint Signaling
Our time-lapse imaging experiments showed that loss of Lis1/Gl in neuroblasts results in extension of both prometaphase and metaphase. Prometaphase in Lis1 mutant neuroblasts was characterized by delayed congression of chromosomes to the equatorial plate, which is likely to be largely due to inefficient kinetochore capturing as an indirect result of spindle assembly defects. Importantly, in Lis1 mutant neuroblasts congression of all chromosomes into a tight metaphase plate eventually occurred, suggesting that Lis1/Gl are not absolutely critical for MT/kinetochore attachment per se.
In addition we observed severe delays in metaphase-to-anaphase transition. A few of these neuroblasts showed individual chromosomes that were transiently lost from and recongressed to the metaphase plate. Thus, consistent with findings in mammalian cells (Faulkner et al., 2000), Lis1 appears to play some role in maintaining stable chromosome alignment in metaphase neuroblasts. However, in contrast to Faulkner et al. (2000), we found that loss of Lis1 function caused delays in metaphase-to-anaphase transition even when all chromosomes stayed aligned in a tight metaphase plate. Thus, mitotic checkpoint activity remained high even after apparent bipolar kinetochore attachment. Two defects appear to contribute to prolonged checkpoint activity in Lis1 mutant metaphase neuroblasts: reduced interkinetochore tension and failure to transport checkpoint proteins (e.g., Rod) off kinetochores. Reduced interkinetochore tension may be due to lack of Lis1/dynactin on kinetochores or on spindle pole/MTs (which may affect forces acting on kinetochore pairs as a consequence of altered spindle morphology or MT dynamics). Defects in Rod checkpoint protein transport off kinetochores can be explained as a direct consequence of depletion of kinetochore-associated Lis1/dynactin/dynein motor complex, which in wild-type cells is loaded with Rod at kinetochores. However, previous studies indicated that Rod and Zw10 are removed from kinetochores in response to interkinetochore tension not MT attachment (Williams et al., 1996; Scaerou et al., 2001; Basto et al., 2004). Therefore, in addition to its direct role as a "carrier," Lis1/dynactin/dynein may also play an indirect role in modulating Rod transport by generating the interkinetochore tension required to trigger initiation of Rod streaming.
In summary, our data are consistent with and extends a model recently proposed for dynein function in checkpoint protein transport in Drosophila and mammalian cells (Howell et al., 2001; Wojcik et al., 2001; Basto et al., 2004). According to this model a Lis1/dynactin/dynein-Rod/Zw10 complex, preassembled on unattached kinetochores, is critical for timely anaphase onset by promoting poleward streaming of checkpoint proteins away from kinetochores after correct kinetochore-MT attachment has occurred (Figure 11B). Our data demonstrate that in Drosophila, the Lis1 protein is an obligate component in this process. Although the Lis1-binding proteins NudE/Nudel have been implicated in facilitating dynein-dependent checkpoint protein transport (Yan et al., 2003), it remains to be directly tested whether Lis1 has a similar function in mammalian cells.
What is the link between Rod/Zw10 and Mad2 in mitotic checkpoint function? Two recent studies demonstrated that the Rod/Zw10 complex is required for efficient recruitment of Mad2 to unattached kinetochores in mammalian cells and Drosophila neuroblasts (Buffin et al., 2005; Kops et al., 2005) and that Mad2 and Rod colocalize during poleward transport along kMTs in Drosophila neuroblasts (Buffin et al., 2005). Although a physical link between the Rod/Zw10 complex and Mad2 has not been discovered, an attractive model is that Rod/Zw10 links Mad2 to the Lis1/dynactin/dynein complex during poleward checkpoint protein transport (Buffin et al., 2005; Kops et al., 2005).
Epistasis of Lis1/Dynactin Localization at Kinetochores
Lis1/dynactin localization is regulated differently in worm and mammalian cells. In mammalian cells, dynactin is required for Lis1 kinetochore association, but Lis1 is not required for dynactin localization (Coquelle et al., 2002; Tai et al., 2002), whereas in C. elegans, Lis1 localizes to kinetochores independently of dynactin (Cockell et al., 2004). Surprisingly, we find a third mechanism in Drosophila neuroblasts, where Lis1 and dynactin (Gl) are codependent for their localization to kinetochores. In neuroblasts, Lis1 may have a "structural" role in recruiting dynein/dynactin to the kinetochore, in addition to stimulating dynein/dynactin activity. Thus, despite the conservation of the physical interaction between Lis1/dynein/dynactin, subcellular localization of these proteins can be regulated differently in various organisms.
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