CLIP-170 family members: a motor-driven ride to microtubule plus ends

CLIP-170 family proteins regulate microtubule plus end dynamics. Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins. These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.     

CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).     

Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules

The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends. The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics. Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips. There, tea2p and tip1p accumulate in larger particles. Particle formation requires the EB1 homolog, mal3p. Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.     

LIS1, CLIP-170's key to the dynein/dynactin pathway

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), which 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. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. 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.     

A +TIP for a smooth trip

Is there a cellular mechanism for preventing a depolymerizing microtubule track from “slipping out from under” its cargo? A recent study in budding yeast indicates that when a chromosome is transported to the minus end of a spindle microtubule, its kinetochore-bound microtubule plus end–tracking protein (+TIP) Stu2 may move to the plus end to promote rescue; i.e., to switch the depolymerizing end to a polymerizing end. The possibility that other +TIPs may play a similar role in sustaining a microtubule track during vesicular transport deserves investigation.Microtubule motor proteins such as dynein and kinesins are responsible for transporting cellular cargos along microtubule tracks (Vale, 2003). The net direction and speed of cargo movement, however, are likely to be regulated in a very complicated fashion, especially when a cargo is bound to multiple motors with opposite directionalities (Vale 2003; Mallik and Gross 2004; Levi et al., 2006). The fact that the microtubule track is not very stable further complicates matters. The plus ends of microtubules, which face the cell periphery in most cell types, are highly dynamic, exhibiting alternating periods of polymerization (growth) and depolymerization (shrinkage; Desai and Mitchison, 1997). Such plus end dynamics may be useful for searching and capturing relatively stationary cargos near the cell periphery that need to be transported inward (Vaughan et al., 2002). However, the dynamic nature of the track can also create an obvious problem for the transport process. If a microtubule''s rate of shrinkage is greater than the rate of cargo transport, then the microtubule may shrink past an attached cargo, causing its dissociation from the track. Does this happen in cells, or do cells have a mechanism to prevent it?Although this question has never been directly addressed, a recent study on budding yeast chromosome segregation has shed new light on the issue (Tanaka et al., 2005). In this study, the authors took advantage of a strategy that allowed them to specifically shut off the function of a single kinetochore, thereby preventing it from attaching to a spindle microtubule while, at the same time, permitting other kinetochores to attach to the spindle. After the function of this single kinetochore was switched back on, the behavior of its associated chromosome on a spindle microtubule was subjected to a detailed image analysis. Several important insights from this study on chromosome–microtubule interactions during mitosis have been recently reviewed (Bloom 2005), and, thus, only those observations that pertain to cargo transport will be highlighted here. The chromosome was first seen to undergo a lateral interaction with the microtubule followed by minus end–directed transport toward the pole. The mechanism of the minus end–directed transport is not entirely clear, although a member of the kinesin-14 family, Kar3, may be one of the players in this process (Tanaka et al., 2005). During transport, the attached microtubule can undergo shrinkage with a rate higher than that of the minus end–directed chromosome movement (Tanaka et al., 2005); however, it never shrank beyond the position of the cargo. Such exquisite control over the extent of shrinkage appears to rely on a conversation between the cargo and the plus end of the microtubule that is mediated by the microtubule plus end–tracking protein Stu2 (Bloom 2005; Tanaka et al., 2005).Microtubule plus end–tracking proteins (+TIPs) are a class of proteins that use different structural motifs or specific targeting mechanisms to localize to the dynamic plus ends of microtubules (Carvalho et al., 2003; Akhmanova and Hoogenraad, 2005). Although most +TIPs associate with only the growing ends of microtubules, several +TIPs also localize to the shrinking ends (Carvalho et al., 2003; 2004; Akhmanova and Hoogenraad, 2005; Mennella et al., 2005; Sproul et al., 2005; Molk et al., 2006; Wu et al., 2006). Many +TIPs have been found to impact microtubules by either promoting their growth or promoting dynamic behavior. Stu2 is a member of the XMAP215/TOG/Dis1/DdCP224 family of proteins that have been shown to affect microtubule dynamics in multiple ways depending on different experimental conditions (Ohkura et al., 2001; Popov and Karsenti 2003; Holmfeldt et al., 2004; Akhmanova and Hoogenraad, 2005). In vitro, Stu2 binds to the plus ends of preformed microtubules and promotes catastrophe, which is a switch from growth to shrinkage (van Breugel et al., 2003). In vivo studies using mutants of Stu2, however, indicate that Stu2 promotes microtubule growth (Severin et al., 2001) and the dynamics of both kinetochore and cytoplasmic microtubules (Kosco et al., 2001; Pearson et al., 2003). During anaphase B spindle elongation, Stu2 may antagonize the function of Kip3 (a kinesin-13 family member) to promote the plus end polymerization of overlapping microtubules (Severin et al., 2001). Although it is not fully understood how or why Stu2 is so versatile, it is well recognized that the in vivo interactions among +TIPs are very complicated, and the loss of function of a +TIP in vivo may decrease or increase the accumulation of other +TIPs that also regulate microtubule dynamics (Carvalho et al., 2003; 2004; Lansbergen et al., 2004; Akhmanova and Hoogenraad, 2005; Galjart 2005; Komarova et al., 2005). Tanaka et al. (2005) identified Stu2 as a rescue (a switch from shrinkage to growth) factor based not on phenotypic studies of Stu2 mutants but, instead, on a direct observation of the relationship between microtubule plus end behavior and Stu2 localization. They found that Stu2 was localized at the plus ends of microtubules emanating from the spindle pole body, and, during periods of microtubule shrinkage, Stu2 levels at the plus ends were decreased. Interestingly, Stu2 was also localized at the unbound kinetochore. When the kinetochore subsequently attached laterally to a spindle microtubule and underwent minus end–directed transport, the Stu2 proteins were transported from the kinetochore to the microtubule plus end. The arrival of Stu2 at the plus end closely correlated to the rescue of the shrinking microtubule (Tanaka et al., 2005). These observations strongly suggest that the Stu2 carried by the kinetochore may serve as a rescue factor for the microtubule track, preventing it from vanishing before the migrating chromosome.Could such a scenario exist during microtubule-dependent transport of nonchromosomal cargoes during interphase? We do not yet know the answer. However, based on published studies, it seems reasonable to hypothesize that other +TIPs, especially the cytoplasmic linker protein CLIP-170, may function in a manner similar to yeast Stu2 to ensure a safe trip for a minus end–directed cargo. CLIP-170 contains CAP-Gly microtubule-binding motifs at its NH₂ terminus and was initially identified as a protein required for linking endocytic vesicles to microtubules in vitro (Pierre et al., 1992; Rickard and Kreis 1996). Later, CLIP-170 was identified as a founding member of the microtubule plus end–tracking proteins (Perez et al., 1999). The connection between CLIP-170''s in vitro endosome–microtubule linking property and its in vivo plus end tracking behavior has not been clearly made. Could an endocytic vesicle use its bound CLIP-170 as a rescue factor to prevent the disappearance of the track on which it is traveling?CLIP-170 is indeed considered to be a rescue factor in mammalian cells (Komarova et al., 2002a). Komarova et al. (2002b) have found that in cultured CHO and NRK cells, microtubule dynamics seem to be controlled spatially; catastrophe and rescue occur frequently only near the cell periphery. Although the mechanisms behind catastrophe and rescue are not fully understood, protein factors are required for regulating both events in vivo (Desai and Mitchison, 1997). In CHO cells, a dominant-negative form of CLIP-170 that displaces the endogenous CLIP-170 from microtubule plus ends severely reduces the rescue frequency so that microtubules are more likely to shrink all the way back to the microtubule-organizing center (Komarova et al., 2002a). Moreover, both in vivo and in vitro studies suggest that the rescue activity of CLIP-170 is localized to the NH₂ terminus containing the CAP-Gly motifs (Komarova et al., 2002a; Arnal et al., 2004). How CLIP-170 rescues a shrinking end is not clear. CLIP-170 can promote tubulin oligomerization (Diamantopoulos et al., 1999; Arnal et al., 2004), and it is likely that this property serves to increase the local concentration of tubulin substrate, thereby lowering the entropic barrier for the polymerization reaction. CLIP-170 in mammalian cells has only been found at growing plus ends, most likely as a result of copolymerization with tubulin subunits followed by its release from older segments (Diamantopoulos et al., 1999; Perez et al., 1999; Folker et al., 2005). When a microtubule end shrinks, CLIP-170 falls off. Is there a mechanism to get CLIP-170 close to the depolymerizing end and facilitate its function as a rescue factor? Given the proposed function of Stu2 as a rescue factor for spindle microtubules, one may easily imagine a similar scenario in which vesicle-bound CLIP-170 may be transported to the approaching microtubule end to rescue it from further shrinkage.If vesicle-bound CLIP-170 is transported to the plus end in a manner similar to Stu2, could such transport be mediated by plus end–directed kinesins? Although the kinesin involved in transporting Stu2 toward the microtubule plus end still needs to be identified, detailed image analyses have revealed a role for the Kip2/Tea2 kinesins (members of the kinesin-7 family) in transporting CLIP-170 homologues in fungi (Busch et al., 2004; Carvalho et al., 2004). Bik1 and Tip1 are the CLIP-170 homologues in budding and fission yeasts, respectively, and these proteins are found at microtubule plus ends, where they act as growth-promoting factors or anticatastrophe factors (Berlin et al., 1990; Brunner and Nurse 2000; Carvalho et al., 2004). In both yeasts, the Kip2/Tea2 kinesins bind to and comigrate with the CLIP-170 homologues along the microtubule toward the plus end (Busch et al., 2004; Carvalho et al., 2004). Kinesins have also been implicated in targeting other +TIPs to microtubule plus ends (Jimbo et al., 2002; Maekawa et al., 2003; Zhang et al., 2003; Wu et al., 2006). For example, the mammalian tumor suppressor protein APC (adenomatous polyposis coli) may be targeted to the plus end by KIF3A/KIF3B (a heterotrimeric kinesin II in the kinesin-2 family) as well as by other mechanisms (Jimbo et al., 2002; Nathke 2004; Slep et al., 2005). It will be interesting to see whether a similar transport process for CLIP-170 exists in higher eukaryotic cells. It is possible that such a mechanism would deliver just enough CLIP-170 to the shrinking plus end to initiate rescue. When microtubule growth is resumed, CLIP-170''s intrinsic higher affinity for tubulin subunits and lower affinity for the microtubule wall may allow these proteins to “treadmill” on the growing end (Perez et al., 1999; Folker et al., 2005).The regulation of CLIP-170 activity appears to be rather complex. CLIP-170 is most likely phosphorylated by multiple kinases, including FKBP12–rapamycin-associated protein (mTOR; Choi et al., 2002). Although phosphorylation by mTOR/FKBP 12–rapamycin-associated protein may stimulate CLIP-170''s microtubule binding, phosphorylation by other kinases may cause CLIP-170 to dissociate from microtubules (Rickard and Kreis 1996; Choi et al., 2002). In vivo, CLIP-170 has a closed conformation that is presumably inactive and an open conformation that may interact with microtubules and dynein regulators such as dynactin (Schroer 2004) and LIS1 (Morris et al., 1998; Lansbergen et al., 2004). It is possible that phosphorylation may regulate the conversion between these two forms, but the specific mechanism and the spatial regulation for this conversion have yet to be resolved. If CLIP-170 is indeed released from a membranous cargo to move to the plus end in order to serve as a rescue factor, it would be interesting to know when and/or where such a conformational switch occurs. Finally, other proteins may play redundant roles with CLIP-170 in vesicular trafficking, which may explain why a dramatic defect in vesicle/organelle distribution is not detected when the CLIP-170 level is lowered or when the gene is knocked out (Lansbergen et al., 2004; Akhmanova et al., 2005).+TIPs other than CLIP-170 may play a similar role in rescuing shrinking microtubule tracks. For example, the dynactin complex that links dynein to membranous cargoes and promotes the processive motion of dynein (Schroer 2004) may act as a rescue factor. The p150^Glued subunit of dynactin and CLIP-170 both contain CAP-Gly microtubule-binding motifs at their NH2 termini, although p150^Glued contains one, whereas CLIP-170 contains two such motifs. Dynactin has been shown to behave as a +TIP facilitating the capture of vesicular cargo for minus end–directed transport (Vaughan et al., 1999; 2002). The head domain of the p150^Glued subunit containing the CAP-Gly motif has been shown to promote rescue in vivo in the absence of endogenous CLIP-170, although the effect was much weaker than that caused by the exogenous CLIP-170 head domain (Kamarova et al., 2002a). In vitro studies showed that dynactin may promote nucleation during microtubule assembly (Ligon et al., 2003), which is consistent with it being a potential rescue factor. As shown with CLIP-170, this capacity to bring multiple tubulins together may help to overcome the entropic barrier of the polymerization reaction. Finally, cargo-bound dynactin may also use kinesin to get to the plus end. The p150^Glued subunit of dynactin has been shown to interact directly with the COOH terminus of KAP3, a subunit of the heterotrimeric kinesin II (a member of the kinesin-2 family) that also binds to APC (Jimbo et al., 2002; Deacon et al., 2003; Dell 2003). Although this binding is implicated in dynactin''s role as a cargo adaptor for kinesin II, it is possible, in theory, that a small amount of dynactin may use this connection to move to the plus end.The proposed hypothesis that +TIPs may be released from a membranous cargo to rescue a shrinking microtubule track may apply to both minus and plus end–directed transport. In addition, it is important to point out that this hypothesis does not exclude other mechanisms for rescuing long microtubule tracks. Rescue may occur stochastically, and, sometimes, +TIPs may participate in other ways such as mediating microtubule capture by the actin-rich cortex to stabilize the track (Wen et al., 2004; Galjart 2005). In some situations, microtubule dynamics are modulated by the direct binding of membranous cargo to the growing or shrinking plus ends of microtubules (Waterman-Storer and Salmon, 1998).Currently, the ability of CLIP-170 or other +TIPs to be released from a membranous cargo and to act as a rescue factor for a shrinking microtubule is just a hypothesis. Nevertheless, searching for proteins involved in the communication between a cargo and the approaching shrinking end of its microtubule track is clearly an endeavor worth pursuing.     

Kinesin’s Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin’s processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.     

Regulation of lamellipodia formation and cell invasion by CLIP-170 in invasive human breast cancer cells

Lamellipodia formation necessary for cell invasion is regulated by Rac1. We report here that lamellipodia formation and three-dimensional invasion were significantly promoted by HGF and serum, respectively, in invasive human breast cancer cells. Rac1 formed a complex with CLIP-170, IQGAP1, and kinesin in serum-starved cells, and stimulation of the cells with HGF and serum caused the partial release of IQGAP1 and kinesin from Rac1-CLIP-170 complex. The HGF-induced release of the proteins and promotion of lamellipodia formation were inhibited by an inhibitor of PI3K. Moreover, downregulation of CLIP-170 by siRNA released IQGAP1 and kinesin from Rac1 and promoted lamellipodia formation and invasion, independent of HGF and serum. The results suggest that promotion of lamellipodia formation and invasion by HGF or serum requires PI3K-dependent release of IQGAP1 and kinesin from Rac1-CLIP-170 complex and that CLIP-170 prevents cells from the extracellular stimulus-independent lamellipodia formation and invasion by tethering IQGAP1 and kinesin to Rac1.     

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.     

CLIP-170 facilitates the formation of kinetochore-microtubule attachments

CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore-microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore-microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore-microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore-microtubule attachments, possibly through direct capture of microtubules at the kinetochore.     

Interactions between CLIP-170, tubulin, and microtubules: implications for the mechanism of Clip-170 plus-end tracking behavior

CLIP-170 belongs to a group of proteins (+TIPs) with the enigmatic ability to dynamically track growing microtubule plus-ends. CLIP-170 regulates microtubule dynamics in vivo and has been implicated in cargo-microtubule interactions in vivo and in vitro. Though plus-end tracking likely has intimate connections to +TIP function, little is known about the mechanism(s) by which this dynamic localization is achieved. Using a combination of biochemistry and live cell imaging, we provide evidence that CLIP-170 tracks microtubule plus-ends by a preassociation, copolymerization, and regulated release mechanism. As part of this analysis, we find that CLIP-170 has a stronger affinity for tubulin dimer than for polymer, and that CLIP-170 can distinguish between GTP- and GDP-like polymer. This work extends the previous analysis of CLIP-170 behavior in vivo and complements the existing fluorescence microscope characterization of CLIP-170 interactions with microtubules in vitro. In particular, these data explain observations that CLIP-170 localizes to newly polymerized microtubules in vitro but cannot track microtubule plus-ends in vitro. These observations have implications for the functions of CLIP-170 in regulating microtubule dynamics.     

CLIPR-59, a new trans-Golgi/TGN cytoplasmic linker protein belonging to the CLIP-170 family

The microtubule cytoskeleton plays a fundamental role in cell organization and membrane traffic in higher eukaryotes. It is well established that molecular motors are involved in membrane-microtubule interactions, but it has also been proposed that nonmotor microtubule-binding (MTB) proteins known as CLIPs (cytoplasmic linker proteins) have basic roles in these processes. We report here the characterization of CLIPR-59, a CLIP-170-related protein localized to the trans-most part of the Golgi apparatus. CLIPR-59 contains an acidic region followed by three ankyrin-like repeats and two CLIP-170-related MTB motifs. We show that the 60-amino acid-long carboxy-terminal domain of CLIPR-59 is necessary and sufficient to achieve Golgi targeting, which represents the first identification of a membrane targeting domain in a CLIP-170-related protein. The MTB domain of CLIPR-59 is functional because it localizes to microtubules when expressed as a fragment in HeLa cells. However, our results suggest that this domain is normally inhibited by the presence of adjacent domains, because neither full-length CLIPR-59 nor a CLIPR-59 mutant missing its membrane-targeting region localize to microtubules. Consistent with this observation, overexpression of CLIPR-59 does not affect the microtubule network. However, CLIPR-59 overexpression strongly perturbs early/recycling endosome-TGN dynamics, implicating CLIPR-59 in the regulation of this pathway.     

Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.     

Cytoplasmic linker proteins promote microtubule rescue in vivo

The role of plus end-tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.     

Microtubule binding proteins CLIP-170, EB1, and p150Glued form distinct plus-end complexes

Microtubule plus-end proteins CLIP-170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP-170 binds tubulin dimers and co-assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co-assembly is observed. However, EB1 binds to CLIP-170, and forms a co-complex with CLIP-170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP-170 and EB1 is competitively inhibited by the related CAP-Gly protein p150Glued, which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus-end complexes may differentially affect microtubule dynamics in vivo.     

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.     

Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte

Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. We also show that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex.     

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation

A microtubule-based machine called the mitotic spindle segregates chromosomes when eukaryotic cells divide. In the fission yeast Schizosaccharomyces pombe, which undergoes closed mitosis, the spindle forms a single bundle of microtubules inside the nucleus. During elongation, the spindle extends via antiparallel microtubule sliding by molecular motors. These extensile forces from the spindle are thought to resist compressive forces from the nucleus. We probe the mechanism and maintenance of this force balance via laser ablation of spindles at various stages of mitosis. We find that spindle pole bodies collapse toward each other after ablation, but spindle geometry is often rescued, allowing spindles to resume elongation. Although this basic behavior has been previously observed, many questions remain about the phenomenon's dynamics, mechanics, and molecular requirements. In this work, we find that previously hypothesized viscoelastic relaxation of the nucleus cannot explain spindle shortening in response to laser ablation. Instead, spindle collapse requires microtubule dynamics and is powered by the minus-end-directed motor proteins dynein Dhc1 and kinesin-14 Klp2, but it does not require the minus-end-directed kinesin Pkl1.     

Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.