See the embryonic expression pattern of Lis1 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
To study at which developmental stages Lis1 is expressed, developmental Northern analysis was performed and Lis1 mRNA was found to be present throughout the Drosophila life cycle with high levels in the ovary. Four major transcripts of Lis1 are detected in all developmental stages. From an ovarian cDNA library, cDNAs were isolated corresponding to each of the mRNAs; sequence analysis determined that they share the same coding region and 5' untranslated region. Thus the difference in size is likely to arise from the usage of alternative polyadenylation sites or alternative splicing in the 3' untranslated region. Mouse antiserum raised against the N-terminal 164 amino acids of Lis1 recognizes a single band at 45 kDa in extracts from every developmental stage of flies, as indicated by Western analysis. Unfortunately, the antiserum fails to recognize the native protein in whole-mount ovaries or embryos (Liu, 1999). Nevertheless, Lis1 is reported to be strongly expressed in the central nervous system in Drosophila (Liu, 1999).
To further define which cell types express Lis1 in the ovary, whole-mount mRNA in situ hybridization was performed using a Lis1 antisense probe. Lis1 transcripts are detected in all germline and somatic cells in the germarium except the terminal filament cells and in the egg chambers. From stage 4 to stage 7 egg chambers, LIS1 RNA accumulates in the oocytes. Only low levels of LIS1 RNA are detected in stage 8 and 9 egg chambers. The RNA was strongly expressed in nurse cells of stage 10 egg chambers, indicating that Lis1 may function in early embryogenesis. Indeed, LIS1 RNA is uniformly distributed in early embryos before the onset of zygotic gene expression, indicating maternal derivation. The results from Northern analysis and mRNA in situ hybridization experiments suggest that Lis1 may well be required throughout Drosophila development (Liu, 1999).
During oogenesis LIS1 mRNA is expressed primarily in germ-line cells. Low levels of LIS1 mRNA can be detected in the germarium in regions 1 and 2 and in both nurse cells and the oocyte in stage 2-4 egg chambers. LIS1 message is enriched in the oocyte during stages 5-7; however, in stage 8-10 egg chambers LIS1 mRNA accumulates at much higher levels in nurse cells. In embryos LIS1 transcript is most abundant at precellular blastoderm stages, suggesting the presence of a maternal contribution. LIS1 mRNA is ubiquitously distributed in early embryos; however, the levels decline after the onset of gastrulation. Following germ-band retraction it is enriched in the developing central nervous system. In third-instar larvae, low levels of LIS1 transcript can be detected in the brain hemispheres and imaginal discs (Lei, 2000).
To reveal its function, mutations were generated in the Lis1 gene. Based on the observation that Lis1 is expressed throughout development and that a null Lis1 mutation in the mouse is lethal, it was expected that Lis1 should be an essential gene also in Drosophila. Lis1 homozygous embryos hatch normally and the first instar larvae look indistinguishable from their heterozygous siblings. At the L2 stage, the development of the homozygotes is clearly retarded and they die 5-6 days later without phenotypically entering the L3 stage. The survival of Lis1 mutants through embryonic and early larval development is probably due to the contribution of maternal wild-type Lis1 protein, as directly demonstrated by Western analysis. When homozygous larvae were picked at L1 stage or early L2 stage, wild-type Lis1 protein was clearly detectable in the extract. But Lis1 protein is reduced or not detectable when the extracts are made from homozygotes close to death (Liu, 1999).
The expression pattern of Lis1 during oogenesis and early embryogenesis suggests that Lis1 may have important functions in the ovary and embryo. To investigate such a possibility, mosaic egg chambers whose germline lacks wild-type Lis1 were produced using the FLP/DFS technique. The germline phenotype in clones of all four Lis1 mutants were similar and failed to produce any eggs. Egg chambers specifically lacking Lis1 in germline cells and control egg chambers retaining Lis1 in both germline and follicle cells were compared. Control egg chambers invariably contain 15 nurse cells and one oocyte. Lis1 egg chambers often contain fewer than 16 cells and usually lack an oocyte. All the cells in the mutant Lis1 cysts appear to develop as nurse cells, as indicated by their polyploid nuclei. Lis mutation results in a 59-fold reduction in the frequency of oocyte formation. As a result of synchronized cystocyte division, there are either 1, 2, 4, 8 or 16 cells in developing wild-type cysts. However, in Lis1 mutant cysts, the cell numbers range consecutively from 1 to 16, indicating that the synchrony of germline cell divisions is disrupted in the absence of Lis1 germline function (Liu, 1999).
Quantitative differences in the number of cells in the Lis1 mutant cysts were observed, dependent on when the germline clones were induced. Fewer cells in the mutant cysts were observed the earlier the induction. When clones were induced at embryonic or first instar larval stages, the average nurse cell number in Lis1 egg chambers was 7.2. The average cell number increased to 11.9 with egg chambers containing mostly even numbers of nurse cells, when the induction was done at the third instar larval stage. The frequency of mutant clone formation was also increased significantly with late induction. However, the frequency of oocyte differentiation was similar in early and late induction. These results suggest that oocyte differentiation is more sensitive to the perturbation of Lis1 function than is cystocyte division. The weaker phenotype observed with later induction may be a reflection of the perdurance of wild-type Lis1 protein in germline stem cells. The relatively low frequency of clones resulting from early induction may be due to the death and growth arrest of germline stem cells or cystoblasts. Lis1 protein level in these cells is expected to be lower in early induced clones than in those induced later (Liu, 1999).
Mosaic egg chambers that contain wild-type germline cells surrounded by only mutant follicle cells produce normal germline cysts of 15 nurse cells and one oocyte. Thus, Lis1 function is not required in the follicle cells for normal cyst formation. These results clearly demonstrate that Lis1 is required in the germline but not in somatic follicle cells to regulate cystocyte division and oocyte differentiation (Liu, 1999).
The cyst defects observed in Lis1 mutants are similar to those of hts and alphalpha spectrin mutants. The hts and alpha spectrin genes encode membrane skeletal proteins that are components of the fusome, and mutations in either gene abolish fusome formation. The absence of a fusome is thought to block the rapid and synchronous cystocyte divisions in both mutants. The morphology of fusomes was examined in cysts mutant for Lis1 by double-staining the egg chambers with anti-beta-galactosidase antibody and monoclonal anti-Hts antibody 1B1. In a wild-type germarium, a spherical fusome, also called spectrosome (ss), is found in germline stem cells. The fusome (f) grows from a spherical fusome in the cystoblast into a polarized branched structure in 2-, 3-, 8- and 16-cell cysts (Liu, 1999). Several major defects in Lis1 mutant cysts have been observed. (1) Some mutant cystoblasts fail to divide, while their fusomes continue to grow to a larger sphere and their nuclei sometimes become polyploid, suggesting that Lis1 is required for cystoblast division (2) When all germline cells in a germarium are mutant for Lis1, the number of developing cysts is dramatically reduced compared with a wild-type germarium, suggesting that the division of germline stem cells is also retarded in Lis1 mutants. (3) Branched fusomes in the Lis1 mutant cysts are often thinner and fragmented as compared to the intact, smoothly branched fusomes in wild-type cysts. The DNA staining of these mutant cysts indicates that the nuclear appearance of most cystocytes is normal, although some cystocytes appear arrested in cell division and the nuclei become prematurely polyploid. (4) Finally, fusomes in more posterior regions of the germarium are sometimes absent and, in all these cysts, the nuclei are often polyploid or apoptotic as determined by DNA staining. Most likely, these cysts are growth-arrested based on their location in the more posterior region of the germarium, eventually degenerating the fusomes (Liu, 1999).
Sometimes, cysts with apparently normal fusomes are also detected. However, they probably do not function normally, since virtually no mutant egg chambers with a normal complement of 15 nurse cells and one oocyte were observed. It is concluded that the fusome is defective in the absence of Lis1 protein. The wide spectrum of fusome phenotypes may result from the different levels of perdurance of Lis1 protein in germline stem cells after the clone is induced (Liu, 1999).
Lis1mutant egg chambers have packaging defects About 5% of the mutant cysts are compound egg chambers that contain a wild-type cyst and some mutant cells. These hybrid cysts are almost always found in association with a mutant cyst. This condition occurred even in egg chambers that were surrounded by wild-type follicle cells. There were usually fewer than 16 combined Lis1 cells in the two adjacent mutant cysts. The mutant cells in the compound egg chamber are not transient clones generated during cystocyte division because the size of the mutant nuclei is the same in the two neighboring mutant egg chambers and differs in size from the wild-type nurse cells in the same egg chamber. Also, the wild-type cyst in the hybrid egg chamber contains a full complement of 15 nurse cells and one oocyte (Liu, 1999).
The likely explanation for this packaging defect is that the Lis1 cysts are unstable and break into smaller groups of interconnected cells during or shortly after the cystocyte divisions. It is possible that Lis1 is required for normal functions of the cyst cytoskeleton, important for maintaining the integrity of a cyst. Alternatively, the formation of compound egg chambers is due to mis-signaling between the Lis1 germline cells and the wild-type follicle or wild-type germline cells (Liu, 1999).
Animals homozygous for EMS-induced DLis1 alleles isolated in a screen, as well as the P-element mutant DLis113209, are late larval or early pupal lethals. However, flies bearing these alleles in trans to the weak DLis111702 allele are viable, but male and female sterile. Eggs laid by such transheterozygous females show defects in the location and morphology of the dorsal appendages. Dorsal appendages are paired paddle-shaped structures that arise from the dorsal-anterior side of the eggshell and are used by the embryo for respiration. Defects in dorsal-ventral as well as anterior-posterior patterning during oogenesis are often associated with alterations in the morphology or placement of the dorsal appendages (Lei, 2000).
Eggs laid by females carrying a copy of DLis111702 in trans to three different EMS alleles were examined. Based on the severity of the dorsal appendage phenotype the mutant eggs were placed in five categories. Class 1 comprises eggs with apparently normal dorsal appendages, in both their structure and their position. In Class 2 eggs the appendages are located closer than normal but remained separate. A third class consists of eggs in which the dorsal appendages are partially fused. In Class 4 eggs the dorsal appendages are fused along their entire length to form a single structure. In a small fraction of the eggs laid by DLis1 mutant mothers, the fused dorsal appendages are severely reduced in size and displaced posteriorly. These eggs constitute Class 5. All three DLis1 alleles tested in trans to DLis111702 show similar defects, although the severity of the phenotype varies in an allele-specific manner. In all cases, greater than 68% of the eggs show fused dorsal appendages typical of Classes 4 and 5. By comparison, females carrying the EMS alleles or DLis111702 in trans to wildtype lay morphologically normal eggs (Lei, 2000).
In normal development, the follicle cells provide spatial cues that are required to establish the dorsal-ventral axis of the embryo. Thus many mutations that alter eggshell polarity also perturb patterning of the embryo. In order to determine whether reduction in DLis1 activity results in ventralization of the embryos, DLis13.1.2/DLis111702 transheterozygous females were crossed to wild-type males and the development of the eggs were monitored under halocarbon oil. Fewer than 20% of the eggs laid undergo cellularization and gastrulation. Of these, about half (10% of the total) show obvious signs of ventralization. During early gastrulation the cephalic furrow that normally occupies a lateral position is displaced dorsally, indicating that the dorsal cells have acquired a more ventral identity. DLis1 mutants also show defects in germ-band extension consistent with a shift in cell fate along the dorsal-ventral axis. Germ-band extension is delayed in mutant embryos with respect to wildtype and does not extend as far anteriorly. At later stages, the germ-band fails to extend completely and instead invaginates into the interior of the embryo. However, the majority of these embryos fail to complete embryogenesis. This was confirmed by examining cuticle preparations of egg lays from DLis1 mutant mothers. Only a small fraction of the embryos differentiate a cuticle, all of which are wild-type in appearance (Lei, 2000).
The defects observed in DLis1 transheterozygotes are similar to those caused by reduction in EGF signaling. Genetic interactions between DLis1 and components of the EGF signaling pathway, such as grk, torpedo (top), and cornichon (cni), were tested. A strong enhancement of the eggshell phenotype is observed in flies double heterozygous for DLis111702 and the strong grkHK36 allele. About 68% of the eggs laid by such females showed completely fused dorsal appendages typical of Classes 4 and 5. Similar phenotypes have been observed with other Lis1 alleles in trans to grkHK36. Flies double heterozygous for DLis1 and topCO, a strong loss-of-function mutation in the Egf receptor, also display a significant increase in the frequency of fused dorsal appendages in comparison to the control females. In contrast, only weak interactions have been detected between DLis1 and the cniAR and cniAA alleles (Lei, 2000.
The ventralized chorion phenotype resulting from reduction in Lis1 activity, as well as the genetic interactions observed between Lis1 and components of the Egf signaling pathway, raised the possibility that Lis1 is required for localization of the Grk ligand. In order to test this, the distribution of GRK mRNA was examined in ovaries from females transheterozygous for DLis111702 and different Lis1 alleles. In wild-type egg chambers from stages 1-7, GRK transcript is localized in a crescent between the oocyte nucleus and the posterior of the oocyte. At stage 8, the microtubule cytoskeleton undergoes a reorientation and the oocyte nucleus migrates to the anterior. Consequently GRK mRNA accumulates transiently along the anterior margin of the oocyte and from late stage 8 onward is tightly localized around the dorsal anterior side of the oocyte nucleus. In Lis1 mutant ovaries, no abnormalities are observed in GRK mRNA localization during stages 1-7. However, from stage 8 onward GRK message is mislocalized in 10%-20% of mutant egg chambers, depending on the strength of the allelic combination. Most frequently, the transcript is less tightly confined to the anterior cortex compared to wildtype. In about 5% of the egg chambers, GRK mRNA is detected in the middle of the oocyte rather than at the anterior, in association with an abnormally positioned oocyte nucleus. Ovaries from Lis1 trans-heterozygotes were also stained with polyclonal antisera against Grk protein to determine the extent to which localization of the ligand was perturbed. Consistent with the effects on GRK transcript, Grk protein distribution is essentially normal in early egg chambers. However, from stage 8 onward, Grk protein appears to be less tightly localized or mislocalized in Lis1 mutant egg chambers. In addition, the level of staining is lower than in comparably staged wild-type oocytes (Lei, 2000).
The preceding results indicate that the altered distribution of GRK mRNA could be a consequence of the mislocalization of the oocyte nucleus. Therefore the position of the germinal vesicle was directly examined in Lis1 mutant egg chambers. A P-element enhancer trap line that drives beta-galactosidase expression in the nuclei of germ-line cells was crossed into a Lis1 mutant background. Ovaries from DLis111702/DLis13.1.2 flies were dissected and lacZ expression in the oocyte nucleus was visualized by activity staining. In ~8% of stage 9-10 egg chambers, the nucleus was misplaced and located approximately midway along the anterior-posterior axis of the oocyte. The frequency of nuclear mislocalization correlates well with the most severe fused dorsal appendage phenotype and the instances of mislocalization of GRK mRNA to the center of the oocyte. An additional 13% of the egg chambers showed less severe but detectable displacement of the oocyte nucleus. Similar effects were observed in eggs from females of the genotype DLis111702/DLis18.25.3 and DLis111702 /DLis111.4.13 (Lei, 2000).
The mislocalization of GRK mRNA in Lis1 transheterozygotes could result from defects in the machinery involved in general transcript localization during oogenesis. In this event one might expect that other localized mRNAs would be aberrantly distributed in Lis1 ovaries. To test this idea the distributions of Bicoid and Oskar, two well-characterized transcripts that are normally localized to opposite ends of the mature oocyte, were examined. In wild-type stage 8-10 egg chambers, BCD mRNA is confined to the anterior margin of the oocyte, while OSK mRNA is present at the posterior pole of the oocyte. No difference in the abundance or localization of either OSK or BCD transcripts was observed in ovaries from Lis1 transheterozygotes (Lei, 2000).
In order to determine whether loss of Lis1 activity affects some global aspect of the microtubule cytoskeleton the distribution of a protein containing the motor domain of the plus-end-directed microtubule motor kinesin fused to beta-galactosidase (Kin:betagal) was examined. Kin:betagal protein accumulates at the plus ends of microtubules in the oocyte as well as in neurons and columnar epithelial cells, and its localization has been used as a sensitive assay for the polarity and integrity of the microtubule network. In wild-type egg chambers Kin:betagal is transiently localized to the posterior of the oocyte during stages 8-9. This localization is lost at stage 10, with the initiation of cytoplasmic streaming in the oocyte. Stage 9 egg chambers from DLis111702/DLis13.1.2 females were examined and Kin:betagal was found tightly focused to the posterior in a majority of the egg chambers (92%). In the remaining cases the fusion protein was present at the posterior of the oocyte but less tightly localized. In comparison, Kin:betagal was found at the posterior in 100% of wild-type stage 9 egg chambers examined, suggesting that the microtubule cytoskeleton and kinesin activity are largely unaffected in DLis111702/DLis13.1.2 egg chambers (Lei, 2000).
The minus-end-directed motor cytoplasmic dynein has been implicated in nuclear migration in several fungal systems. In addition, genetic epistasis data suggest that nudF, the Aspergillus homolog of Drosophila Lis1, acts upstream of dynein in mediating nuclear migration. Lis1 egg chambers were examined to determine whether the level or distribution of Dhc64C protein was affected. In wild-type ovaries high levels of Dhc64C can be detected in the oocyte from region 2 in the germarium onward. Later, in stage 9 egg chambers Dhc64C is enriched at the posterior of the oocyte and also outlines the oocyte nucleus. In Lis1 mutants Dhc64C localization appears unperturbed through early oogenesis. However, in stage 9 mutant egg chambers localization to the posterior of the oocyte is strongly reduced, suggesting that Lis1 activity is required for dynein localization or activity (Lei, 2000).
The dependence of dynein localization on Lis1 activity suggests a functional interaction between these two genes. A test was performed to see whether the Lis1 phenotype is affected by mutations in the Gl and Dhc64C genes that encode structural components of the dynein-dynactin microtubule motor. The Gl1 allele causes a synthetic lethality with strong heteroallelic combinations of Lis1 mutations, while in combinations with weaker Lis1 alleles Gl1 causes a reduction in viability. Adult escapers from the latter crosses generally do not survive for more than a few days. More interestingly, they display defects in eye morphology, bristles, and abdominal tergites. Flies carrying the dominant Gl1 allele have small, rough eyes with irregularly positioned bristles. A reduction in Lis1 activity results in an enhancement of the Gl1 eye phenotype. In addition, the scutellar bristles of escaper flies are reduced in size and frequently lost. Most adult escapers lack bristles on the ventral side of the abdomen, and in many animals abdominal tergites are completely absent in one or more segments. Lis1 mutants show similar but weaker interactions with Dhc64C alleles. A deficiency for Dhc64C was viable in combination with Lis1 mutants. However, the adults display defects in scutellar and abdominal bristle morphology resembling those seen in DLis18.25.3/DLis111702 ;Gl1/+ adults. Thus the genetic interactions between Dhc64C, Gl and Lis1 are consistent with a functional relationship between these genes (Lei, 2000).
Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. This study generated and/or characterized mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, an improved time-lapse video microscopy technique was developed that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, GFP-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: initially promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both C. elegans and mammals. It is concluded that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release (Siller, 2005).
This study shows that both Lis1 and Gl are enriched on centrosomes/spindle poles in wild type neuroblasts, and Lis1/Gl are required for centrosome separation in prophase neuroblasts. A role for centrosome separation has been reported for dynein in Drosophila embryos, dynein in mammalian cells, and dynein/dynactin/Lis1 in C. elegans blastomeres. 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. Alternatively, dynein associated with the nuclear envelope may exert pulling forces on astral MTs to promote centrosome separation. No GFP-Lis1 was detected on the nuclear envelope or at the neuroblast cortex, although it is possible that high cytoplasmic levels mask low levels of Lis1/dynactin at these sites. Thus, it remains unclear how Lis1/Gl promotes centrosome separation in neuroblasts. Centrosome separation is not completely blocked in Lis1 or Gl mutant neuroblasts, either due to residual amounts of maternal protein or due to the presence of a Lis1/dynactin/dynein-independent pathway. Interestingly, cortical non-muscle myosin II has been shown to contribute to centrosome separation in some cell types, raising the possibility that Lis1/dynactin/dynein and myosin II play partially redundant roles in neuroblast centrosome separation (Siller, 2005).
These 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. Detachment of centrosomes from the spindle has been observed in dynein mutants in Drosophila, and in mammalian cells with reduced dynein or dynactin function. These findings show that Lis1 and dynactin act as cofactors for dynein-dependent focusing of spindle poles and attachment of spindle MTs minusends 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. 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 (Siller, 2005).
Gl and Lis1 mutant neuroblasts occasionally form multipolar spindles and have more than two centrosome-like Centrosomin/gamma-tubulin structures. Due to the lack of Drosophila centriolar markers it was not possible to determine whether these extra centrosomelike structures contained centrioles. Multipolar spindles have also been observed in mammalian cells overexpressing Lis1 protein or in which Lis1 function was reduced. Time-lapse analysis of Lis1 mutant neuroblasts reveal occasional co-segregation 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, extra centrosomes in Lis1 and Gl mutant neuroblasts may be due to uncoupling of centrosome duplication from the cell cycle or centrosome fragmentation (Siller, 2005).
Time-lapse imaging experiments show that loss of Lis1/Gl in neuroblasts results in extension of both prometaphase and metaphase. Prometaphase in Lis1 mutant neuroblasts is characterized by delayed congression of chromosomes to the equatorial plate: this 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 occurs, suggesting that Lis1/Gl are not absolutely critical for MT/kinetochore attachment per se (Siller, 2005).
In addition, severe delays were observed 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, Lis1 appears to play some role in maintaining stable chromosome alignment in metaphase neuroblasts. However, in contrast to mammalian studies, it was found that loss of Lis1 function causes delays in metaphase-to-anaphase transition even when all chromosomes stay aligned in a tight metaphase plate. Thus, mitotic checkpoint activity remains high even after apparent bipolar kinetochore attachment. Two defects appear to contribute to prolonged checkpoint activity in Lis1 mutant metaphase neuroblasts: reduced inter-kinetochore tension and failure to transport checkpoint proteins (e.g., Rod) off kinetochores. Reduced inter-kinetochore 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 have indicated that Rod and Zw10 are removed from kinetochores in response to inter-kinetochore tension not MT-attachment. 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 inter-kinetochore tension required to trigger initiation of Rod streaming (Siller, 2005).
In summary, the data is consistent with and extends a model recently proposed for dynein function in checkpoint protein transport in Drosophila and mammalian cells. According to this model a Lis1/dynactin/dyneinRod/Zw10 complex, pre-assembled 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. The 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, it remains to be directly tested whether Lis1 has a similar function in mammalian cells (Siller, 2005).
What is the link between Rod/Zw10 and Mad2 in mitotic checkpoint function? Two recent studies demonstrate that the Rod/Zw10 complex is required for efficient recruitment of Mad2 to unattached kinetochores in mammalian cells and Drosophila neuroblasts, and that Mad2 and Rod colocalize during poleward transport along kMTs in Drosophila neuroblasts. 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 (Siller, 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. Whereas in C. elegans, Lis1 localizes to kinetochores independently of dynactin. Surprisingly, a third mechanism is found in Drosophila neuroblasts, where Lis1 and dynactin (Gl) are co-dependent 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 (Siller, 2005).
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date revised: 25 November 2008
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