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).
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).
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