Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends

CLIP-170 was the first microtubule plus-end-tracking protein to be described, and is implicated in the regulation of microtubule plus-ends and their interaction with other cellular structures. The cell-cycle-dependent mechanisms which localise the sole Drosophila melanogaster homologue CLIP-190 have been studied. During mitosis, CLIP-190 localises to unattached kinetochores independently of spindle-checkpoint activation. This localisation depends on the dynein-dynactin complex and Lis1 which also localise to unattached kinetochores. Further analysis revealed a hierarchical dependency between the proteins with respect to their kinetochore localisation. An inhibitor study also suggested that the motor activity of dynein is required for the removal of CLIP-190 from attached kinetochores. In addition, CLIP-190 association to microtubule plus-ends is regulated during the cell cycle. Microtubule plus-end association is strong in interphase and greatly attenuated during mitosis. Another microtubule plus-end tracking protein, EB1, directly interacts with the CAP-Gly domain of CLIP-190 and is required to localise CLIP-190 at microtubule plus-ends. These results indicate distinct molecular requirements for CLIP-190 localisation to unattached kinetochores in mitosis and microtubule ends in interphase (Dzhindzhev, 2005; full text of article).

The CLIP family of proteins is implicated in regulating microtubule dynamics and linking microtubule plus-ends with other cellular structures. To understand these functions, it is crucial to elucidate where and how these proteins localise within cells. This study investigated the molecular mechanisms of CLIP-190 localisation using RNAi in Drosophila cells, rather than using the expression of dominant proteins, to gain a clearer and more comprehensive view. The study revealed that distinct, cell-cycle-dependent mechanisms localise CLIP-190 to microtubule plus-ends and unattached kinetochores (Dzhindzhev, 2005).

CLIP and EB1 proteins are two major families of microtubule plus-end-tracking proteins. This study is the first demonstration of both a physical interaction and localisation-dependency between CLIP and EB1 proteins in higher eukaryotes. CLIP-190 requires EB1 to localise to microtubule plus-ends, and CLIP-190 directly interacts with EB1 through its CAP-Gly domain, which also binds to microtubules. Considering that this has previously been reported for fission yeast homologues, these findings demonstrate that this interaction and dependency are conserved among eukaryotes. The most obvious interpretation would be that EB1 simply bridges microtubule plus-ends and CLIP-190. However, this is thought not be the case. First, CLIP-170 has been shown to bind directly to microtubule plus-ends in vitro. Secondly, localisation studies show incomplete overlapping of the two proteins on microtubule plus-ends. Also, other EB1 interacting proteins, such as RhoGEF2 and the spectraplakin Short stop, which both require EB1 for their localisation to microtubule plus-ends (Rogers, 2004; Slep, 2005), show distinct localisation from that of EB1. Therefore, it is more likely that EB1 acts as a loading factor for these proteins, rather than a simple bridge. In addition, it seems that multiple microtubule plus-end-binding proteins, such as CLIPs, CLASPs, p150Glued, EB1, Lis1, Dynein, Short stop, APC and RhoGEF2, directly interact with each other and with microtubules. It is an exciting challenge to understand the regulatory network acting on microtubule plus-ends (Dzhindzhev, 2005).

In addition, it was found that the association of CLIP-190 to microtubule plus-ends is greatly reduced during mitosis. This is in contrast to EB1, which is associated with plus-ends throughout the cell cycle. This cell-cycle-regulation has not been described in other systems, possibly because of a lack of co-examination with EB1, and leads to the conclusion that it is not the consequence of a change in microtubule dynamics. It might be possible that CLIP-190 is modified during the cell cycle. Since EB1 is essential for CLIP-190 localisation to microtubule ends, EB1 activity or interaction between EB1 and CLIP-190 might also be regulated. Alternatively, other inhibitory proteins might be activated during mitosis to attenuate CLIP-190 association with microtubule ends. Phosphorylation of the EB1 homologue mal3p and its inhibitory effects on the interaction with the CLIP-190 homologue tip1p have been shown in fission yeast (Busch, 2004). It remains to be examined whether this phosphorylation is cell-cycle-regulated. Cell-cycle regulation might be important for releasing CLIP-190 for kinetochore function or preventing the plus-end-binding activity from interfering with CLIP-190 function at kinetochores. This report is the first to describe the cell-cycle regulation of the plus-end-binding of CLIP proteins. Elucidation of the precise mechanism and significance of this regulation may lead to further understanding of the temporal and spatial regulation of microtubules in cells (Dzhindzhev, 2005).

CLIP-190 localises to unattached kinetochores in mitosis. The localisation of CLIP proteins to kinetochores has been shown in mammalian, Drosophila and budding-yeast cells. Studies of CLIP-170 localisation to kinetochores in mammalian cells suggest an intricate physical and functional relationship with the dynein-dynactin complex. CLIP-170 binds directly to and requires Lis1 for kinetochore localisation. In turn, Lis1 interacts with multiple subunits of dynein-dynactin and is displaced from kinetochores when the motor complex is disrupted. Most of these studies relied on the overexpression of dominant-negative proteins (Dzhindzhev, 2005).

A previously unreported dependency was found, namely the requirement of Lis1 for dynein localisation to kinetochores. In mammalian cells, dynein is required for Lis1 localisation, whereas the overexpression of full-length or truncated Lis1 does not prevent dynein localisation to kinetochores. These results lead to the idea that Lis1 might be an auxiliary protein that bridges the dynein complex to cargo proteins. The RNAi results clearly indicate that Lis1 is required for dynein localisation to kinetochores. Combined with previous results, Lis1 and dynein seem to depend on each other for their localisation. This is the first report of such dependency in any eukaryote, and it gives the Lis1 protein a more integral part in dynein function (Dzhindzhev, 2005).

The results also suggest that microtubule attachment directly removes CLIP-190 from kinetochores rather than through spindle-checkpoint signalling. Dynein seems to be responsible for the removal of CLIP-190 from kinetochores in addition to its role in localising CLIP-190 to kinetochores. It has been shown that dynein removes several kinetochore proteins along microtubules upon the attachment of microtubules. This study provides the first evidence that a member of the CLIP family also utilises dynein-motor-activity to leave attached kinetochores. Interestingly, it was found that unlike in interphase, EB1 is not required for the mitotic localisation of CLIP-190 to unattached kinetochores. This is intriguing in the light of recent evidence (Tirnauer, 2002) that EB1 associates with attached kinetochores when the kinetochore microtubules are polymerizing (Dzhindzhev, 2005).

In conclusion, these results indicate that CLIP-190 localisation is regulated during the cell cycle and requires distinct mechanisms in mitosis and interphase. Spatial and temporal regulation of CLIP-190 localisation probably play crucial roles in the regulation of microtubule dynamics and their interaction with other cellular structures (Dzhindzhev, 2005).

Protein Interactions

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits that account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex genomic data, mining was undertaken in search of related proteins in lower eukaryotes. The Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and both genes were cloned. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. This study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit (Sheffield, 2000).

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, independent movement of both apical and basal spindle poles is detected, suggesting that forces act on both poles. Reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. It is proposed that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation (Siller, 2008).

This study shows that spindle/cortical polarity alignment is established at prophase in Drosophila larval neuroblasts, and that both apical and basal spindle poles move independently, as if spindle/cortex forces are applied to both poles. Reducing astral microtubule number reduces the frequency of spindle pole movements, but that maximum spindle pole velocity is unaffected, suggesting that maximum velocity may occur when only one or a few microtubules are simultaneously contacting the cortex. Yhe Lis1/dynactin complex is required for spindle pole movement; reducing Lis1/dynactin complex activity reduces the maximum and average spindle velocity, even though astral microtubules still contact the cortex. This suggests that Lis1/dynactin is required to translate microtubule-cortex contact into spindle movement. Finally, this study shows that Lis1/dynactin is required for spindle orientation at metaphase but not at telophase (Siller, 2008).

Lis1-dependent dynamic microtubule-cortex interactions were observed at both apical and basal spindle poles, as well as asynchronous movements of apical and basal spindle poles. What are the candidate apical or basal cortical proteins that might regulate spindle pole movement? Insight into the role of cortical proteins in regulating spindle movement has been made in both C. elegans and mammals, and can be used to model spindle dynamics in Drosophila. Apical proteins in neuroblasts known to regulate spindle force in C. elegans include Gαi, Pins and Mud. During the first division of the C. elegans zygote, enrichment of the Gα-binding and activating Pins-related GPR1/2 proteins at the posterior cortex leads to increased Gα activity, resulting in higher cortex-spindle force generation, spindle pole rocking, and posterior spindle displacement. This suggests that Gαi/Pins/Mud may promote movement of the apical spindle pole in Drosophila neuroblasts, which is supported by the finding that reducing Gαi can decrease spindle rocking (Siller, 2008).

In C. elegans, Lin-5 mediates the physical interaction of Lis1/dynein/dynactin with the cortical Gα and the Pins-related GPR1/2 proteins, and reduction of dynein or Lis1 function also reduces spindle pole rocking and posterior spindle displacement. Furthermore, in mammalian tissue culture cells Gαi overexpression can induce robust spindle rocking that requires LGN (a Pins/GPR-related protein) and NuMA (a Mud/Lin-5-related protein that binds dynein/dynactin). An attractive model is that Gαi/LGN activates NuMA, which interacts with dynein/dynactin/Lis1-loaded astral microtubules. In Drosophila neuroblasts, Gαi, Pins (LGN-related) and Mud (NuMA-related Pins-binding protein) are all enriched at the apical cortex and required for proper metaphase spindle orientation. Thus, it is tempting to propose that apical Gαi/Pins/Mud interacts with dynein/dynactin/Lis1-loaded astral microtubules to center the apical spindle pole with the apical cortical domain. Identifying a physical link between Mud and dynein/dynactin/Lis1, and determining its functional importance in spindle orientation, would be a good test of this model (Siller, 2008).

Surprisingly, it was found that third instar larval neuroblasts have more vigorous basal spindle pole rocking than apical spindle pole rocking, revealing a Gαi-independent spindle force generation mechanism at the basal cortex. Basal cortical proteins include Armadillo, DE-cadherin, β-catenin, APC2, and Mud. Components of the APC2/DE-cadherin/α-catenin/β-catenin complex physically interact with the dynein complex in mammalian cells, and are required for spindle positioning in the Drosophila pre-cellular embryo, epithelial cells, and germline stem cells. Previous studies indicated no spindle positioning defects in neuroblasts after reduction of APC2 function, however these findings do not rule out a role for APC2 in spindle orientation because it may function redundantly with an apical cue, such as the Gαi/Pins/Mud pathway (Siller, 2008).

This study has demonstrated that both apical and basal spindle pole movements are greatly diminished in Lis1 mutant larval neuroblasts (even in those with well-formed bipolar spindles and asters), providing first evidence that Lis1/dynactin is a critical component in the regulation of both apical and basal cortex-spindle forces. How does Lis1 regulate cortex-spindle forces? One possibility is that translocation of cortically associated motor proteins towards microtubule(-) ends results in movement of the microtubule towards the cortex. Consistent with this hypothesis, Lis1 colocalizes with and binds the microtubule minus-end motor dynein/dynactin complex. Specifically, the budding yeast Lis1 homologue (Pac1) targets dynein to astral microtubule plus-ends where it promotes movement of astral microtubules towards the cortex, resulting in translocation of the spindle apparatus through the bud neck. By analogy, Lis1 may regulate spindle pole movement in neuroblasts by promoting dynein-dependent movement of astral microtubules towards the cortex. Alternatively, Lis1 may modulate the polymerization/depolymerization cycle (dynamic instability) of cortically-attached astral microtubules or the duration of astral microtubule-cortex interactions. In support of this latter hypothesis, loss of Lis1 or dynein function in Aspergillus nidulans or budding yeast results in reduced microtubule catastrophe and/or decreased shrinkage rates, thereby promoting assembly of overly long microtubules. Currently, it was not possible to visualize astral microtubule plus-ends with sufficient spatial and temporal resolution to distinguish between these models for Lis1 function (Siller, 2008).

Both models for Lis1 function described above would require association of Lis1 protein with astral microtubules and/or the neuroblast cortex. Indeed, Lis1/dynactin complex proteins have been detected on astral microtubule plus-ends or at the cortex in mammalian, nematode, and yeast cells. The localization of HA-tagged Lis1, GFP-tagged Lis1, endogenous Lis1, and endogenous dynactin protein distribution were analyzed using various fixation and live imaging methods in embryonic and larval neuroblasts, but no enrichment of Lis1/dynactin at the cortex or at astral microtubule plus-ends was found. The most likely explanation is that Lis1/dynactin at these sites is masked by the high level of cytoplasmic protein present in neuroblasts (Siller, 2008).

The Lis1/dynactin complex is required for reliable spindle orientation with the apical/basal polarity axis in metaphase neuroblasts. These spindle orientation defects may be due in part to failure in anchoring one centrosome at the apical cortex during interphase, as reported for wild type neuroblasts; this study observed mis-positioned interphase centrosomes in Lis1 mutants, but this this phenotype was not analyzed in detail. It was surprising to find that spindle orientation was essentially normal at telophase in Lis1 and dynactin (Gl) mutant neuroblasts, despite severe defects at metaphase. This indicates that there are two pathways for regulating spindle orientation: an early Lis1/dynactin-dependent pathway (prophase/metaphase), and a late Lis1/dynactin-independent pathway (anaphase/telophase). There are several models consistent with these findings: (1) Lis1 and dynactin mutants have a delay in anaphase onset which allows sufficient time for 'telophase rescue' to occur. (2) A spindle orientation checkpoint -- analogous to the yeast spindle orientation checkpoint -- may delay cytokinesis until proper spindle orientation has occurred. These first two hypotheses are disproven by the finding that Lis1 rod double mutants have normal metaphase progression but still show metaphase defects and 'telophase rescue' of spindle orientation. (3) The cleavage furrow may be positioned by cortical polarity cues, such that cell elongation at early anaphase may mechanically re-orient the spindle along the long axis of the neuroblast. This model is unlikely because it is commonly accepted that the position of the cleavage furrow is determined by the position of the mitotic spindle and not by cortical cues. (4) Additional microtubule-cortex regulators unrelated to Lis1/dynactin promote telophase spindle orientation (Siller, 2008).

The fourth model is the most likely, except that microtubule-cortex regulators unrelated to Lis1/dynactin have not yet been identified in Drosophila neuroblasts. Help may come from analysis of budding yeast spindle orientation pathways, where Lis1/dynactin-dependent and -independent pathways have been identified. Several components of the yeast Lis1/dynactin-independent pathway are evolutionarily conserved, including the microtubule plus-end binding protein Bim1p, called EB1 in Drosophila. It is tempting to speculate that these proteins may regulate the Lis1/dynactin-independent pathway in Drosophila neuroblasts (Siller, 2008).

Lissencephaly-1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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