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- Materials and methods
Centrosomes serve as microtubule-organizing centers. However, centrosome function depends on microtubule organization and protein transport because the formation, positioning and maintenance of centrosomes require microtubule-dependent retrograde transport. Linker proteins that associate with the motor protein dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and thus contribute to the composition and placement of the centrosome and other juxtanuclear protein complexes. Members of the hook family of proteins may function as adaptors to link various organelle cargos to dynein for transport and have also been implicated directly in centrosome positioning. Here, we show that mammalian hook2, a previously uncharacterized member of the hook family, localizes to the centrosome through all phases of the cell cycle, the C-terminal domain of hook2 directly binds to centriolin/CEP110, the expression of the C-terminal domain of centriolin/CEP110 alters the distribution of endogenous hook2 and mislocalized wild-type or mutant hook2 proteins perturb endogenous centrosomal and pericentrosomal proteins in cultured mammalian cells. In addition, interference with hook2 function results in the loss of the radial organization of microtubules and a defect in regrowth of microtubules following their nocodazole-induced depolymerization. Thus, we propose that hook2 contributes to the establishment and maintenance of centrosomal structure and function.
Positioning of the centrosome, as other organelles, depends on microtubules that serve as tracks for directional protein transport and as scaffolds to maintain the shape and structural integrity of cells. Centrosomes have microtubule-nucleating and microtubule-anchoring activities (1,2) and thus ultimately regulate organelle distribution and protein trafficking. The molecular motor protein dynein (3) moves membrane-bound organelles (4) as well as nonmembranous protein complexes (5) in retrograde transport toward the centrosome. As dynein also transports centrosomal proteins (6) and microtubules (7,8), there is an intricate interplay between the activities of the dynein complex, anchoring and stabilization of the microtubule array and positioning of organelles (9–11). Retrograde transport depends on the integrity of the microtubule cytoskeleton and the association of dynein with the cargo-binding dynactin complex (12,13). Linker proteins that associate with dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and contribute to the active maintenance of the juxtanuclear localization of organelles (14).
Hook proteins are candidates for serving as linker proteins because they structurally resemble other adaptors and can associate with both microtubules and organelles (15,16). For example, mammalian hook3 binds to isolated Golgi membranes and interference with hook3 function leads to dispersal of the Golgi complex from its normal juxtanuclear placement (15). Furthermore, hook1 is located at the end of microtubules in the manchette of developing spermatocytes (17). Loss of hook1 function in the abnormal spermatozoon head shape mutant mice results in the ectopic positioning of microtubules in the developing spermatid, consistent with a role for hook1 in anchoring microtubule arrays (17). Further genetic support for a role of hook proteins in the positioning of cellular structures comes from studies on Caenorhabditis elegans. A mutation in zyg-12, which encodes a C. elegans homologue of hooks, causes misplaced centrosomes in embryonic cells (18). Zyg-12 participates in the dynein-dependent transport of the centrosome to a position next to the nucleus and also in the attachment of the centrosome to the nucleus (16,18). Therefore, hook proteins are emerging as potential regulators of organelle positioning both as facilitators in the loading of cargos for microtubule-based retrograde transport and as anchors for organelles (11).
In this study, we assessed the function of hook2, a previously uncharacterized member of the hook family. Subcellular localization in cultured cells suggested a function at the microtubule-organizing center (MTOC). Colocalization of hook2 with centrosomal proteins and the identification of the centrosomal protein centriolin/CEP110 as a direct binding partner for hook2 substantiated the idea that hook2 functions at the centrosome. To examine the effects of hook2 on centrosomal function, we explored the organization and regrowth of microtubules after their nocodazole-induced depolymerization in cells that overexpressed wild-type or mutant hook2. The results of these experiments suggest that hook2 participates in the maintenance of the structure and function of the centrosome.
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- Materials and methods
In this study, we present a series of experiments that show an association between mammalian hook2 and centrosomal proteins and suggest that hook2 is a regulator of centrosomal composition, positioning and function: first, endogenous and overexpressed hook2 is enriched at centrosomes throughout the cell cycle by stable interactions that allow it to resist detergent extraction; second, hook2 directly interacts with the C-terminal region of centriolin/CEP110; third, overexpression of the C-terminus of centriolin/CEP110 led to mislocalization of endogenous hook2; fourth, hook2 mutants perturbed the characteristic juxtanuclear distribution of endogenous centrosomal proteins and finally, the expression of truncated hook2 proteins disrupted the radial organization of microtubules and attenuated the regrowth of nocodazole-depolymerized microtubules, suggesting that hook2 may contribute to the MTOC activity of centrosomes that include nucleation and anchoring of microtubules (2,20). Taken together, these experiments establish hook2 as a component of mammalian centrosomes and implicate a regulatory role in microtubule organization.
Centrosomal targeting is not a unique feature of hook2 among members of the hook family of proteins. Small fractions of endogenous hook1 and hook3 were retained at centrosomes of RAW cells after detergent extraction (data not shown). Furthermore, Zyg-12, a distant C. elegans homologue of hook, is necessary for the positioning of centrosomes close to nuclei, as a mutation in zyg-12 caused centrosome displacement in embryonic cells (18). Zyg-12 has been proposed to participate in the dynein-dependent transport of the centrosome to the perinuclear region, and a splice form that contains a transmembrane domain is critical for the attachment of the centrosome to the nucleus (16,18). Interestingly, even Drosophila hook, originally identified based on its endocytic trafficking defect (28), is transiently enriched at centrosomes in developing spermatocytes (H. Krämer, unpublished data). Similarly, in developing spermatocytes of mice, hook1 is enriched at the manchette that functions in the anchoring of microtubules as the sperm tail extends (17).
The existence of centrosome targeting signals shared between different hook proteins was also supported by our studies of hook chimeras (Figure 6). While truncation of the N- and C-termini of hook2 inhibited its accumulation at centrosomes, the corresponding regions of hook3 were sufficient to restore that function. This is reminiscent of the multiple domains in ninein that have been identified to contribute to its targeting to centrosomes (20,30). Together, these data indicate that some of the signals that target hook proteins to the centrosome are conserved.
In addition to this conserved mechanism, hook2, but not hook1 or hook3, specifically interacts with the centrosomal protein centriolin/CEP110. Centriolin and CEP110, which may represent a centriolin splice variant, are preferentially localized to mother centrioles (24,25). Interference with their function disrupts the MTOC (25) and also blocks abscission during cytokinesis (26). Our data indicate that hook2 may participate in the first of these functions (see below). However, hook2 is unlikely to play a role in abscission. Unlike centriolin (26), hook2 is not enriched at the midbody, and interference with its function by the overexpression of dominant-negative forms did not result in the specific defects in abscission observed after knockdown of centriolin/CEP110 (26).
To assess whether hook2 had a role in the functioning of the centrosome we expressed truncated forms of hook2. We relied on this approach because so far our attempts to knock down expression of hook2 in RAW cells by RNA interference based methods have been unsuccessful. Truncated hook proteins may exert their function by dimerizing with endogenous hook2 and inactivating it (28) or by binding to hook2-interacting proteins such as centriolin/CEP110. Importantly, corresponding terminal truncation mutants of the Drosophila hook protein dimerized with endogenous hook, and expression of these mutants mimicked in vivo the phenotype of a hook null allele, indicating that truncated hook proteins can act as dominant-negative inhibitors of hook function (27,28).
As a first assessment of the role of hook2 in centrosomal function, we examined effects of mislocalized hook2 truncation mutants on the distribution of endogenous centrosomal proteins. We found that expression of ΔN-hook2 altered the distributions of ninein and PCM-1 (Figure 7). In cells that expressed ΔN-hook2, ninein staining was reduced and scattered instead of being focused at a single juxtanuclear position, but we did not see colocalization of ΔN-hook2 particles and ninein. PCM-1 staining was not only scattered, but in this case, we did see a partial colocalization of PCM-1- and ΔN-hook2-positive particles (Figure 7), suggesting that these proteins are in the same complex or that hook2 affects sequestering of other centrosomal proteins. Alternatively, ΔN-hook2 may affect the localization of centrosomal proteins indirectly, considering that transport of ninein and PCM-1 to centrosomes is known to depend on microtubule-dependent dynein function (31,32). ΔC-hook2 had no effect on the localization of centrosomal proteins; this may be due to its lack of a centriolin/CEP110-binding domain. Full-length hook2 expressed at the centrosome also had an effect on centrosomal proteins: it caused excessive recruitment of ninein (Figure 7A). Again, this effect may be due either to a direct interaction with centrosomal proteins or to an indirect effect on transport.
Because centrosomes serve as MTOC, we examined effects of hook mutant proteins on microtubule growth and organization. Centrosomes have both microtubule-nucleation and microtubule-anchoring activities (2,32). Expression of hook2 truncations more than doubled the number of cells that lacked clear radial microtubule organization compared with controls (Figure 9). This effect was even more pronounced when the regrowth of microtubule asters was assayed after their depolymerization by nocodazole. This defect in aster formation paralleled the disruptive effect of ΔN-hook2 on ninein and PCM localization. Significantly, ninein was shown to have a role in microtubule nucleation in addition to microtubule anchoring (20) and has a similar distribution to centriolin/CEP110 (24,25). Together these data indicate a role of hook2 in microtubule nucleation or anchoring at centrosomes.
Even though effects observed after interfering with hook function may result from a direct disruption of centrosomal functions, it is also possible that some of the described effects are due to alteration in protein transport. A function of hook proteins in dynein-mediated transport is consistent with several previous observations. The dispersal of the Golgi complex after interference with hook3 function (15) is similar to the overexpression of the dynamitin subunit of the dynein/dynactin complex (4,33). Furthermore, Zyg-12 is necessary for the recruitment of dynein and for the dynein-associated lis-1 and arp-1 to the nuclear membrane in C. elegans embryos (18). The failure of the N-terminally truncated ΔN-hook2 to localize to the centrosome was consistent with the idea that this region in hooks is required for microtubule-based retrograde transport. This part of mammalian hooks is homologous to the region of Zyg-12 that was shown to bind dynein light intermediate subunit, suggesting that hook proteins may play a direct role in retrograde transport (18), although our attempts to demonstrate a direct interaction between hook2 and cytoplasmic dynein have not been successful (B. Hall and H. Krämer, unpublished data). Alternatively, hook proteins may indirectly participate in retrograde transport by functioning as linker proteins or attachment factors during the loading of cargo onto the dynein/dynactin complex, as was recently suggested for hook3, among several other proteins (11,14,34). Such linker proteins may also contribute to the anchoring and stabilization of microtubules (34), functions traditionally attributed to centrosomes.
Because the functions and the location of centrosomes ultimately control vectorial protein transport and dynein mediates the transport of centrosomal proteins and microtubules (6–8), it is not straightforward to distinguish direct effects on centrosomes from indirect effects on microtubule structure and protein transport. This report shows that hook2 is likely to function at the centrosome. The identification of centriolin/CEP110 as a direct binding partner for hook2 opens new avenues to unravel molecular mechanisms by which hook2 dynamically associates with the centrosome and contributes to its function.