Cytoplasmic Linker Protein Research Articles

Dynamic localization of CLIP-170 to microtubule plus ends is coupled to microtubule assembly.

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.

The MurineCYLN2Gene: Genomic Organization, Chromosome Localization, and Comparison to the Human Gene That Is Located within the 7q11.23 Williams Syndrome Critical Region

Cytoplasmic linker proteins (CLIPs) have been proposed to mediate the interaction between specific membranous organelles and microtubules. We have recently characterized a novel member of this family, called CLIP-115. This protein is most abundantly expressed in the brain and was found to associate both with microtubules and with an organelle called the dendritic lamellar body. CLIP-115 is highly homologous to CLIP-170, or restin, which is a protein involved in the binding of endosomes to microtubules. Using the rat cDNA as a probe we have isolated overlapping cosmids containing the complete murine and part of the humanCYLN2(cytoplasmic linker-2) genes, which encode CLIP-115. The murine gene spans 60 kb and consists of 17 exons, and its promoter is embedded in a CpG island. MurineCYLN2maps to the telomeric end of mouse chromosome 5. The humanCYLN2gene is localized to a syntenic region on chromosome 7q11.23, which is commonly deleted in Williams syndrome. It spans at least 140 kb at the 3′ end of the deletion. HumanCYLN2is very likely identical to the previously characterized, incompleteWSCR4andWSCR3transcription units.

Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment.

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.

A class VI unconventional myosin is associated with a homologue of a microtubule-binding protein, cytoplasmic linker protein-170, in neurons and at the posterior pole of Drosophila embryos.

Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)-170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.

CLIP-115, a Novel Brain-Specific Cytoplasmic Linker Protein, Mediates the Localization of Dendritic Lamellar Bodies

Intracellular localization of organelles may depend in part on specific cytoplasmic linker proteins (CLIPs) that link membranous organelles to microtubules. Here, we characterize rat cDNAs encoding a novel, brain-specific CLIP of 115 kDa. This protein contains two N-terminal microtubule-binding domains and a long coiled-coil region; it binds to microtubules and is homologous to CLIP-170, a protein mediating the binding of endosomes to microtubules. CLIP-115 is enriched in the dendritic lamellar body (DLB), a recently discovered organelle predominantly present in bulbous dendritic appendages of neurons linked by dendrodendritic gap junctions. Local microtubule depolymerization leads to a temporary reduction of DLBs. These results suggest that CLIP-115 operates in the control of brain-specific organelle translocations.

Molecular characterization of two functional domains of CLIP-170 in vivo.

CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected antibodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal microtubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.

Nematode (Nematoda) Associates and Parasites of Pseudoscorpions (Arachnida)

Cytoplasmic linker proteins (CLIPs) have been proposed to mediate the interaction between specific membranous organelles and microtubules. We have recently characterized a novel member of this family, called CLIP-115. This protein is most abundantly expressed in the brain and was found to associate both with microtubules and with an organelle called the dendritic lamellar body. CLIP-115 is highly homologous to CLIP-170, or restin, which is a protein involved in the binding of endosomes to microtubules. Using the rat cDNA as a probe we have isolated overlapping cosmids containing the complete murine and part of the humanCYLN2(cytoplasmic linker-2) genes, which encode CLIP-115. The murine gene spans 60 kb and consists of 17 exons, and its promoter is embedded in a CpG island. MurineCYLN2maps to the telomeric end of mouse chromosome 5. The humanCYLN2gene is localized to a syntenic region on chromosome 7q11.23, which is commonly deleted in Williams syndrome. It spans at least 140 kb at the 3′ end of the deletion. HumanCYLN2is very likely identical to the previously characterized, incompleteWSCR4andWSCR3transcription units.

CLIP-170 links endocytic vesicles to microtubules

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.