Dynein heavy chain 64C


Table of contents

Dynactin - a link between Dynein and its cargo and/or the substratum:
(1) Binding of p150Glued/Glued/CLIP-170/BIK1 family subunit of Dynactin to microtubules

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, the microtubule-binding properties of CLIP-170 were characterized to understand the mechanism of its plus end targeting. 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 is 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. It is concluded from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization (Diamantopoulos, 1999).

CLIP-170, a microtubule binding protein that has been implicated in the attachment of endosomes to microtubules, is so far the only microtubule binding protein that has been observed to colocalize with microtubule plus ends. Binding of CLIP-170 to microtubules is regulated by phosphorylation, and it has been speculated that specific phosphorylation may be involved in the targeting of CLIP-170 to microtubule plus ends. Interestingly, CLIP-170 may also interact with the dynactin complex (C. Valetti, submitted, communicated to Perez, 1999), an activator of cytoplasmic dynein that contains a subunit, p150Glued, which has one CLIP-170-related microtubule binding domain per molecule. CLIP-170 may thus play a role in regulating the dynamic properties of microtubule plus ends or may be a capturing device to clip peripheral organelles to microtubules before it hands them over, perhaps via dynactin, to the microtubule minus end-directed motor protein cytoplasmic dynein. To this end, the functional significance of the microtubule plus end localization of CLIP-170 is unclear (Perez, 1999 and references).

A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. It is proposed that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin (Perez, 1999).

The dynamic properties of CLIP-170 in vivo may hint at interesting functional properties of the protein. On the one hand, CLIP-170 may regulate the dynamics of tubulin polymerization. In vitro experiments indicate that CLIP-170 interacts with small tubulin oligomers and stimulates microtubule polymerization. CLIP-170 could thus stimulate polymerization of tubulin at plus ends either by stabilization or by cooperatively recruiting CLIP-170-tubulin oligomers in a way similar to that proposed for XMAP310 The dynamic distribution of CLIP-170, on the other hand, has interesting implications for a putative role of CLIP-170 in microtubule-directed movement of endosomes and other particles in a cell. Since it treadmills on growing microtubule ends, its activity cruises on dynamic microtubules throughout the cytoplasm and thus continuously explores the cytoplasmic space. Interestingly, it has recently been proposed that dynamic microtubules may be preferentially used by cytoplasmic dynein. In addition, it has been shown that CLIP-170 mediates interaction of endosomes with microtubules in vitro, and more recently that it is associated with prometaphase kinetochores. Thus, CLIP-170 could be a capturing device, comparable to the 'Tip Attachment Complex', establishing an initial contact between a particle and a microtubule. Once CLIP-170 has docked cargo to a track for movement, it may hand this cargo over to cytoplasmic dynein for transport. Evidence for an interaction of the dynactin complex, the activator of cytoplasmic dynein, with CLIP-170 has indeed recently been obtained. That growing microtubules could thus be preferred for capturing cargo is particularly interesting, since this may be part of a feedback mechanism regulating microtubule dynamic properties and their spatial arrangement. A microtubule may become transiently stabilized by tethering cargo via CLIP-170 to its dynamic end and by the subsequent interactions with dynactin and cytoplasmic dynein. An accumulation of such events may then indirectly lead to preferential growth of microtubules toward areas of a cell generating cargo at higher frequency for example, the differentiating growth cone, a leading edge of a motile fibroblast, or the site of interaction between a killer cell and its target. The dynamic properties reported for GFP-CLIP170, highlighting growing microtubule ends in vivo, will stimulate further studies both on the regulation of microtubule dynamics, since for the first time growth of microtubules can now be visualized specifically and directly in living cells, and on the role of CLIP-170 in regulating transport along microtubules (Perez, 1999 and references).

Cytoplasmic dynein is a minus end-directed microtubule motor responsible for centripetal organelle movement and several aspects of chromosome segregation. The search for cytoplasmic dynein-interacting proteins has implicated the dynactin complex as the cytoplasmic dynein 'receptor' on organelles and kinetochores. Immunofluorescence microscopy using a total of six antibodies generated against the p150Glued, Arp1 and dynamitin subunits of dynactin revealed a novel fraction of dynactin-positive structures aligned in linear arrays along the distal segments of interphase microtubules. Dynactin staining reveals that these structures colocalize extensively with CLIP-170. Cytoplasmic dynein staining was undetectable, but extensive colocalization with dynactin becomes evident upon transfer to a lower temperature. Overexpression of the dynamitin subunit of dynactin removes Arp1 from microtubules but does not affect microtubule-associated p150Glued or CLIP-170 staining. Brief acetate treatment, which has been shown to affect lysosomal and endosomal traffic, also disperses the Golgi apparatus and eliminates the microtubule-associated staining pattern. The effect on dynactin is rapidly reversible and, following acetate washout, punctate dynactin is detected at microtubule ends within 3 minutes. Together, these findings identify a region along the distal segments of microtubules where dynactin and CLIP-170 colocalize. Because CLIP-170 has been reported to mark growing microtubule ends, these results indicate a similar relationship for dynactin. The functional interaction between dynactin and cytoplasmic dynein further suggests that this these regions represent accumulations of cytoplasmic dynein cargo-loading sites involved in the early stages of minus end-directed organelle transport (Vaughan, 1999).

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, intact and mutant forms of CLIP-170 were transiently expressed in mammalian cells (HeLa and Vero cells). 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 (Pierre, 1994).

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), that are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Colocalization and direct interaction between CLIP-170 and LIS1 is demonstrated in this study. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics (Coquelle, 2002).

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. Mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. LIS1 has been localized to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. The COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. Of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, these data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities (Tai, 2002).

During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs). This study reports the native-level localization of the dynein heavy chain and characterizes the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) results in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein results in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also results in selective accumulation of dynein on the plus ends of anaphase aMTs. It is proposed that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, it is suggested that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors (Sheeman, 2003).

A significant concentration of dynein is found at the plus ends of both polymerizing and depolymerizing aMTs. These results are consistent with observations in animal cells and Aspergillus. These experiments shed light on the mechanism by which dynein is recruited to the plus ends of MTs. Bik1p is a functional component of the dynein mechanism for spindle positioning and Bik1p, specifically its cargo binding domain, has an important role in recruiting dynein to aMT plus ends. These findings are in agreement with recent studies in mammalian cells reporting that the cargo binding domain of human CLIP-170 binds LIS1 and that overexpression of CLIP-170 can recruit LIS1, and potentially other dynein components, onto MTs. Evidence was also obtained for binding of Bik1p to Pac1p by two-hybrid experiments. Thus, the complex between CLIP-170- and LIS1-related proteins appears to be a highly conserved element of the mechanism for targeting dynein to MTs. Recruitment to MTs by a plus-end tracking protein (CLIP-170/Bik1p) may explain how a minus end-directed motor protein can be returned to the plus end (Sheeman, 2003).

p150Glued-related proteins have also been suggested to link dynein to MT plus ends. Surprisingly, loss of Nip100p did not diminish MT-association of dynein, but in fact caused enhanced Bik1p- and Pac1p-dependent localization of dynein to aMT plus ends in anaphase. Therefore, at least in budding yeast, Nip100p does not appear to play the major role in recruiting dynein to aMT plus ends (Sheeman, 2003).

There are differences in the apparent role of LIS1-related proteins in different systems. (1) LIS1-related proteins have also been implicated in recruitment of dynein to the cell cortex in some cell types. One way to reconcile the current findings with previous work is to postulate that the plus ends of aMTs deliver LIS1 to the cell cortex in these cells. If this explanation is correct, then (at least in the Drosophila oocyte), once delivered to the cortex, LIS1 must interact stably with a cortical component, because LIS1 cortical localization is not lost after disruption of MTs by colchicine treatment. (2) In Aspergillus, dynein appears to localize normally to MT plus ends in the absence of the LIS1 ortholog NUDF. At this point, the basis for these species-specific differences is not known; however, in Aspergillus dynactin might assume a more important role in dynein localization than in budding yeast (Sheeman, 2003).

The targeting of dynein to MT plus ends raises interesting questions about the regulation of dynein in vivo. Because of the rapid speed of minus end-directed movement of cytoplasmic dynein, it seems likely that dynein could only accumulate at MT plus ends if the motor activity were inhibited and/or if dynein processivity were extremely low. The results support this idea. (1) Anaphase cells lacking components of the dynactin complex display a marked accumulation of the dynein heavy chain on the plus ends of aMTs. (2) Concomitant with this plus-end enhancement, there was a decrease in the amount of dynein associated with the SPBs in these cells. (3) An inactivating mutation in a dynein P loop domain also enhances dynein localization to aMT plus ends. These findings suggest that dynactin promotes the ability of dynein to translocate from the plus end of an aMT to the minus end (SPB). Additional levels of control on the rate of recruitment of dynein to the MT plus end, on dynein motor activity, or on the activity of dynactin may also exist. Cell cycle regulation at any of these levels might account for the finding that in dynactin mutants, dynein accumulation at aMT plus ends is primarily seen during anaphase (Sheeman, 2003).

Dynactin - a link between Dynein and its cargo and/or the substratum:
(2) Role of p150Glued/Glued/CLIP-170/BIK1 family subunit of Dynactin in neurons

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm were examined. Treatment of axoplasm with antibodies to the p150(Glued) subunit of dynactin results in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. The molecular mechanism of the observed inhibition of motility were examined. Antibodies to p150(Glued) disrupt the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150(Glued) antibodies have no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport (Waterman-Storer, 1997).

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. Human cDNAs encoding p150Glued have been isolated as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. Transfection assays and in vitro microtubule-binding assays were used to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody reveals that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, it is hypothesized that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules (Tokito, 1996).

Dynactin - a link between Dynein and its cargo and/or the substratum:
(3) Miscellaneous p150Glued/Glued/CLIP-170/BIK1 interactions

Human EB1 (see Drosophila Eb1) is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein, a domain of APC that is commonly deleted in colorectal neoplasia. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 and Saccharomyces cerevisiae Bim1p. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle, in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis. Human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 is also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sediments at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex is independent of APC and is preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes (Berrueta, 1999).

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. The C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 forms mutually exclusive complexes with APC and dynactin and a direct interaction between EB1 and p150(Glued). EB1-GFP deletion mutants demonstrate a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibits microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes is also inhibited. None of these effects are seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression does not induce Golgi apparatus fragmentation. It is propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array (Askham, 2002; full text of article).

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. This study reports the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, these studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. It is therefore proposed that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator (Hayashi, 2005).

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, a yeast two-hybrid screen was performed with a HsEg5 stalk-tail construct as bait. A cDNA was isolated coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) is enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 has a phosphorylatable residue at position 927. Phosphorylation also enhances the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors (Blangy, 1997).

Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, it was found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bind to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitate with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 is enriched in rat brain microtubules and comigrates with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalizes with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. It is proposed that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport (Li, 1998).

Table of contents

Dynein heavy chain 64C: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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