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Keywords:

  • cell cycle;
  • centriolin/CEP110;
  • centrosome;
  • microtubule organization;
  • organelle positioning

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References

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.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References

Hook2 is widely expressed in different tissues although at different levels

To gain insight into the function of hook2, we examined its tissue and subcellular distributions. We detected hook2 in all analyzed rodent tissues although relative abundance differed between tissues (Figure 1A) and with the developmental stage of a single organ (Figure 1B). In whole brain and in cerebellum, hook2 levels rose sharply at the time when most neuronal precursors stop dividing and start to differentiate as neurons [for a comparison with other markers in these samples, see Szebenyi et al. (19)]. In comparison, the 83-kDa variant of hook3 was more evenly expressed in different tissues [Figure 1A,B; (15)].

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Figure 1. Hook2 is ubiquitously expressed although levels vary between tissues. A) Hook2 and hook3 immunoreactivities were detected in homogenates of the indicated tissues from mature mice; as a loading control, an identically loaded gel was stained with Coomassie Blue. B) Whole brains (brain) or cerebellum (Cbl) homogenates from rats of the indicated ages were probed for hook2 and, as a control for loading, hook3. C) Extracts of the indicated cell lines were probed for hook2 (see Materials and Methods for tissue origins); for some cell lines samples from two parallel cultures are included. Equal amounts of tissue protein (12 μg for hook2 and 6 μg for hook3) or cell homogenates were loaded in each lane. D) The specificity of hook2 antibodies was confirmed on immunoblots of homogenates from HEK cells transfected with hook1, 2 or 3. The endogenous hook2 protein is seen as a faint band in cells transfected with hook1 or hook3 compared with cells transfected with hook2. The arrows in B–D point to the band that is identical in size, 83 kDa, to the hook2 recombinant protein and is seen consistently in all samples. MW, molecular weight.

Consistent with its wide tissue expression, hook2 was detected in all cell lines that we examined (Figure 1C), including cells derived from B lymphocytes (RAW), kidney (HEK and Vero), glia (C6), neurons and neuroblastomas (CAD (mouse brain catecholaminergic neurons) and SH-SY5Y). In addition to the most prevalent ∼83-kDa form of hook2, some tissues and cell lines exhibited other immunoreactive bands whose molecular identities are not known. Nonetheless, recombinant human hook2 comigrated with the most prominent band in adult tissues and cell lines. As a further test for the specificity of the polyclonal antibody against hook2, the three human hook proteins were overexpressed in Vero cells and cell lysates were probed with the anti-hook2 and hook3 antibodies; in hook1- and hook3-transfected cells, only endogenous hook2 was detected, while in hook2-transfected cells, a robust 83-kDa band comigrated with the endogenous hook2 protein (Figure 1D).

A detergent-insoluble fraction of hook2 is enriched at the centrosome during all stages of the cell cycle

Next, we examined the subcellular distribution of hook2. For immunofluorescence studies on endogenous hook2 (Figure 2), we focused on those cell lines that exhibited a single hook2-reactive band observed by immunoblotting, such as RAW and CAD cells (Figure 1C). Hook2 distributed throughout the cells, but staining was more intense at the juxtanuclear area (Figure 2A–E, K,L). Staining for α-tubulin showed that hook2 was enriched at the center of the radial array of microtubules at the centrosome (Figure 2A). This staining was specific for hook2 as it was abolished after preincubation of hook2 antibodies with purified hook2 fusion proteins (Figure 2G,H), but preincubation with a corresponding hook1 fusion protein had no effect on centrosomal hook2 staining (Figure 2F).

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Figure 2. Endogenous and overexpressed hook2 localize to the centrosome. RAW lymphocytes were immunostained to visualize hook2 (red in merged images of A–H) and A) α-tubulin, B and F–H) γ-tubulin, C) ninein, D) dynein or E) GM130 (green in merged images). Hook2 is enriched at the MTOC, from where the microtubule array fans out and closely colocalizes with markers for the centrosome (γ-tubulin and ninein). The Golgi (GM130) is found around the hook2-positive centrosome, but there is no overlap. When centrosome doublets were visible by γ-tubulin or ninein staining, in most cases they were both also positive for hook2 (see insets in B and C). The staining for hook2 at centrosomes was specific as it was still present when hook2 antibodies were preincubated for 1 h with a His6–hook1 fusion protein (F) but was abolished after preincubation with corresponding His6–hook2 (G) or glutathione S-transferase (GST)–hook2 fusion protein (H). Arrows point to the position of centrosomes defined by γ-tubulin staining. In contrast to hook2, distributions of hook1 (I) and hook3 (J) were broader around centrosomes, with hook3 distribution resembling GM130 localization (compare with E). CAD neurons were kept either as dividing, undifferentiated cells (K) or as morphologically differentiated neurons (L). Hook2 staining in the soma is pronounced compared with that in the long axons that are stained for α-tubulin. Similar to endogenous hook2, also overexpressed hook2 in Vero (M) and RAW cells (N) localized to the centrosome. Double staining for hook2 (red in merged images) and α-tubulin (green in merged images) shows hook2 accumulation at the MTOC. Hoechst staining of DNA in N affirmed the juxtanuclear position of overexpressed hook2. Scale bars represent 5 μm.

Hook2 colocalized with γ-tubulin and ninein (Figure 2B,C), centrosomal proteins involved in microtubule nucleation and anchoring (2,20). Dynein distributed throughout the RAW cells in a punctuate pattern that followed microtubules and had a modest centrosomal enrichment that colocalized with hook2 (Figure 2D). The Golgi marker, GM130, highlighted a small area around the centrosome (Figure 2E), a staining pattern that was distinct from that for hook2 but closely resembled the staining pattern for hook3 (Figure 2J), as shown previously in other cell types (15). Hook2 localization in RAW cells was different from what we previously observed in Vero cells (15); however, in Vero cells, there are several hook2 immunoreactive bands of unknown identities (Figure 1C) that may explain the difference. In RAW cells, hook2 localization differed from that of both hook1 and hook3 (Figure 2I,J). Hook2 localization was juxtanuclear also in CAD cells (Figure 2K,L). In differentiating CAD cells, hook2 was enriched at the beginning of microtubule arrays in extending neural processes (Figure 2L, merged).

The juxtanuclear localization of overexpressed hook2 at the center of the microtubule array (Figure 2M,N) and its colocalization with centrosomal proteins (see below) resembled the distribution of endogenous hook2 (Figure 2A–C,K,L) in an accentuated form. Centrosomal accumulation of overexpressed hook2 was seen as early as 6 h after transfection (Figure 2M). For overexpression studies, we used Vero cells because these cells are larger and better spread than RAW or CAD cells. Even though in Vero cells, studying the distribution of endogenous hook2 was complicated by spurious immunoreactivity (Figure 1C,D), these were negligible under the conditions used to detect overexpressed hook2.

The observed centrosomal localization of endogenous hook2 led us to investigate whether the distribution of hook2 was cell cycle dependent. Stages in the cell cycle were assigned based on DNA and α- or γ-tubulin staining (Figure 3). Hook2 colocalized with centrosomes in interphase cells (Figure 3A–C), and in most cases when two distinct centrosomes were seen, hook2 localized to both (insets in Figures 2B,C and 3C). However, in some cells, hook2 was detected in only one of two closely positioned centrosomes, or hook2 localization was slightly offset compared with ninein localization (Figure 3J). In addition, hook2 clearly localized to the spindle poles in metaphase cells (Figure 3F,G). At other phases of the cell cycle, prophase (Figure 3D,E), anaphase (Figure 3H) and telophase (Figure 3I), hook2 was also discernible at centrosomes (arrow in Figure 3I) but appeared less enriched than in interphase or in metaphase. In telophase, the midbody was outlined by immunostaining for α-tubulin (arrowhead in 3I) but did not stain for hook2. In general, hook2 localization to the centrosome was less sharp than γ-tubulin or ninein localization. The centrosomal pool of hook2 was emphasized, however, after detergent extraction of soluble proteins (see below).

image

Figure 3. Endogenous hook2 localizes to centrosomes and spindle poles through all stages of the cell cycle in Raw cells. Hook2 and α-tubulin (A, E, G, and I) or γ-tubulin (B–D, F and H) was visualized by immunostaining. Stages in the cell cycle were assigned based on the nuclear and chromatin morphology (Hoechst staining for DNA), the number and position of centrosomes and the position and shape of the microtubule spindles. Hook2 was enriched at centrosomes throughout the cell cycle. Centrosomal hook2 localization was sharp during interphase before centrosome duplication (A and B) and also after when closely positioned centrosome pairs could be distinguished (C) and in metaphase (F and G). At prophase (D and E), anaphase (H) and telophase (I), hook2 localization appeared less distinct but still discernible at centrosomes (arrow in I). While hook2 was weakly detected in intracellular bridges highlighted with α-tubulin during telophase (I), no staining was visible at the midbody (arrowhead) where centriolin/CEP110 is detected (26). High magnification images (J) of centrosome pairs show that in some cases hook2 localization to centrosomes is more restricted compared with localization of ninein to centrosomes. Scale bar in I represents 5 μm in A–I and 1 μm in J.

In addition to immunostaining, we examined the subcellular localization of hook proteins by differential detergent extraction of live RAW cells followed by immunoblotting (Figure 4A,B). Silver staining of eluates confirmed that a different set of proteins was extracted with each concentration of detergent (Figure 4A). Soluble proteins elute from cells in 0.1% Triton-X-100, but many cytoskeletal and centrosomal proteins remain bound even after incubation in 1% Triton-X-100 (21–23). A fraction of both hook2 and hook3 was extracted from cells with 0.1% Triton-X-100, but some remained in cells even after extraction with 1% Triton-X-100 (Figure 4B) and was found in the 1% Triton-X-100-insoluble fraction solubilized in 5% sodium dodecyl sulfate (SDS). The 0.1% soluble- and the 1% Triton-X-100-insoluble hook2 fractions were distinct as most hook2 that remained insoluble after 0.1% Triton-X-100 was not extracted with 1% Triton-X-100. This profile was similar to that of dynein (Figure 4B). Besides hook2 and dynein, the 1% Triton-X-100-insoluble fraction also contained α-tubulin, γ-tubulin and pericentrin. In contrast, only a very small fraction of hsc-70, a cytosolic protein, was in the insoluble fraction. Immunostaining of 1% Triton-X-100-extracted cells showed that the detergent-insoluble hook2 was retained at the centrosome where it colocalized with γ-tubulin (Figure 4C); the inset shows detergent-insoluble hook2 retained following detergent extraction at the spindle poles in a dividing cell. Overexpressed hook2 in Vero cells was also retained in a juxtanuclear position after extraction with 1% Triton-X-100 (Figure 4D) in contrast to the C-terminally truncated ΔC-hook2, which was efficiently extracted. Staining for α-tubulin showed that detergent-extracted cells retained their normal morphologies and microtubule cytoskeletal network.

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Figure 4. Hook2 at the centrosome is resistant to extraction with 1% Triton-X-100. Live RAW cells were extracted with increasing concentrations of Triton-X-100 (0.01, 0.05, 0.1 and 1.0% and ‘IS’ stands for 1% Triton-insoluble fraction). Extracted cells were scraped and solubilized in 5% SDS for SDS–PAGE. A) The same volume was loaded from each fraction, and the total protein profile was visualized by silver stain. B) Immunoblots show that hook2 fractionates into distinct soluble and insoluble fractions. The profile for detergent solubility of hook2 is most similar to that of dynein that is enriched in the same fractions. The 1% Triton-X-100 insoluble fraction also contained γ-tubulin and pericentrin in addition to hook2. By contrast, hsc-70 is extracted with more dilute detergents. C) Immunostaining shows endogenous hook2 at the γ-tubulin-positive centrosome of RAW cells extracted with 1% Triton-X-100. The inset shows detergent-insoluble hook2 at the spindle poles in a dividing cell. D) Overexpressed hook2 in Vero cells was also retained in a juxtanuclear position after extraction with 1% Triton-X-100. In contrast to full-length hook2 and scattered ΔN-hook2 accumulations, ΔC-hook2 was extracted from cells with detergents; tubulin staining showed that these detergent-extracted cells retained their morphologies and cytoskeletal network, indicating that this construct lacks a tight association with cytoskeletal proteins. Scale bars represent 20 μm. MW, molecular weight.

In summary, endogenous hook2 is expressed in all tissues and cell types we examined and a fraction of both endogenous and overexpressed hook2 accumulated at the centrosome at all phases in the cell cycle. This centrosomal fraction of hook2 was resistant to detergent extraction based on both biochemical and microscopic analysis, indicating that hook2 may associate with centrosomal proteins.

Centriolin/CEP110 binds the C-terminus of hook2

To identify the proteins that directly interact with hook2 we screened a yeast two-hybrid human brain library with the C-terminal domain of hook2 [amino acids (aa) 584–719]. Based on previous work on the structurally similar hook3 (15), the C-terminal region of hook proteins was predicted to contain the organelle-binding region. The hook2 bait was designed to exclude the coiled-coil region to avoid spurious interactions with other coiled-coil-containing proteins. More than 1 million clones were screened. After retransformation, we identified 15 candidate proteins that bound to hook2 but not the negative control p53. Three of these 15 candidates encoded amino acids 1985–2326 of a centrosomal protein known as centriolin (24) or CEP110 (25). CEP110 may be a splice variant that corresponds to the C-terminal part of centriolin, a centrosome-associated protein that has been implicated in both MTOC activity and the abscission step during cytokinesis (25,26).

We further tested the interaction of hook2 with candidate proteins using coimmunoprecipitation. Coexpression of myc-tagged centriolin/CEP1101985–2326 resulted in hook2 pull down by anti-myc antibodies (Figure 5A). No precipitation was observed in the absence of myc-tagged proteins or after coexpression with several other myc-tagged candidates; zinc finger protein 536 (znf536) is shown as an example in Figure 5A. Furthermore, anti-hook2 antibodies coimmunoprecipitated centriolin/CEP1101985–2326 when expressed together with hook2 but not without it (Figure5B). This interaction was specific as anti-hook2 antibodies did not pull down myc-tagged znf536. In the two-hybrid screen, centriolin/CEP110 was identified based on its interaction with the C-terminal domain of hook2. Thus, we tested whether this region was necessary for the coimmunoprecipitation. As predicted, the C-terminally truncated myc-tagged ΔC-hook2 did not pull down coexpressed centriolin/CEP1101985–2326 (Figure 5B), while the C-terminal region of hook2 (aa 401-719) was sufficient (Figure 5C). Because we had noticed that small, detergent-insoluble fractions of hook1 and hook3 also localized to the centrosome (data not shown), we wondered whether they also interacted with centriolin/CEP110. However, neither hook1 nor hook3 interacted with coexpressed centriolin/CEP1101985–2326 unlike hook2 (Figure 5D).

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Figure 5. The C-terminus of hook2 interacts with centriolin/CEP110. For coimmunoprecipitation experiments myc-tagged proteins and hook constructs were expressed in Vero cells as indicated. In A–D input samples are shown in the top panels and were detected with antibodies (indicated on the right side of each panel); 5% of the amount used for immunoprecipitation was loaded as ‘input’; α-tubulin is shown as loading control. A) Hook2 was pulled down with anti-Myc antibodies when coexpressed with myc-tagged centriolin/CEP1101985–2326 (myc-CEP110) but not with myc-tagged znf536, a control protein. B) In the reverse experiment, myc-CEP110, but not znf536, was pulled down with anti-hook2 antibodies when coexpressed with full-length hook2. In contrast, myc-ΔC-hook21–532 did not bind CEP1101985–2326. C) Similarly, myc-CEP110 was pulled down with anti-hook2 antibodies only when coexpressed with constructs that included the C-terminal domain of hook2 (myc-hook2401–719). D) To assess the specificity of the interaction between hook2 and centriolin/CEP110, hook1, hook2 and hook3 were coexpressed with myc-CEP110 and each hook detected with specific antibodies. Pull down of myc-CEP110 was detected only with hook2 but not with hook1 or hook3. Levels of endogenous hook2 were insufficient to be detected in these Western blots. E) RAW cells transfected to express myc-CEP110 were stained for the myc epitope (green), endogenous hook2 (red) and γ-tubulin (blue in merged images). White arrows in the upper panel point to myc-CEP110 and hook2 colocalizing at a centrosome marked by γ-tubulin, and white arrowheads in the lower panel point to myc-CEP110 and hook2 colocalizing in the absence of detectable γ-tubulin. For comparison, magenta arrows point to centrosomes of untransfected cells. Scale bars represent 5 μm.

The interaction between hook2 and centriolin/CEP110 was also detected by immunofluorescence in cells expressing myc-CEP1101985–2326 (Figure 5E). Centriolin/CEP1101985-2326 colocalized with endogenous hook2 in small aggregates, which in some instances formed at the centrosome (identified with γ-tubulin, Figure 5E upper panel), but in most cases were at ectopic locations (Figure 5E).

Taken together, these data showed a specific interaction between the C-terminal domains of hook2 and the centriolin/CEP110 and thus provide a molecular mechanism for the localization of hook2 to centrosomes.

We expressed several truncation mutants and chimera to further analyze the structural requirements in hook2 for localization to centrosomes (Figure 6A,B). In Drosophila, the truncation mutations ΔN-dhook and ΔC-dhook can form dimers with endogenous hook and act as dominant-negative repressors of hook function in fly eyes. The expression of these truncation mutants in wild-type Drosophila resulted in phenotypes similar to those of hook 11 null flies (27). Therefore, hook2 truncations and chimeras to be tested in mammalian cells were constructed following the structure–function relationships (Figure 6A), as they were understood based on previous studies on Drosophila hook (28) and hook3 (15). In transfected cells, proteins of the expected sizes were detected with either hook2 or hook3 antibodies, depending on the presence of respective epitopes (Figure 6B). Consistent with a requirement for the hook2 C-terminal domain in binding to centriolin/CEP110, we found that C-terminally truncated hook2 was never targeted to centrosomes. In contrast, full-length hook2 was enriched at the centrosome in 50% of the transfected Vero cells (Figure 6C,E). ΔC-hook2 localized diffusely, filling the whole cell, including the nucleus; nuclear staining was seen only with this hook2 construct. In addition, ΔC-hook2 was extracted with 1% Triton-X-100 (Figure 4D), again suggesting a lack of association with insoluble centrosomal matrix proteins. These effects were specific for the removal of the C-terminus. Deletion of a portion of the coiled-coil domain next to the C-terminus (ΔCl-hook2) greatly increased the intensity and incidence (close to 100% of transfected cells) of juxtanuclear hook2 staining although ΔCl-hook2 appeared to pack more loosely around the centrosome than hook2. This association was maintained during mitosis, for example in metaphase cells, ΔCl-hook2 was enriched at spindle poles (Figure 6D). By contrast, ΔN-hook2 formed granules throughout the cell (Figure 6C) that were insoluble in 1% Triton-X-100 (Figure 4D). Therefore, the N-terminal region of hook2 contains information for the localization of hook2 to the centrosome and perhaps ΔN-hook2 forms a complex with other detergent extraction-resistant centrosomal proteins prior to delivery to the centrosome (see below). The N-terminal region of hook2 is most similar to the region of zyg-12 required for its interaction with dynein (18). Taken together, these data indicate that removal of either the N- or C-terminal of hook2 changed both the detergent solubility (Figure 4D) and the localization (Figure 6) of hook2.

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Figure 6. Properties of hook2 deletion mutants and hook2 and hook3 chimeras. A) Schematic representation of cDNA constructs containing hook2 (hk2, gray), hook3 (hk3, black), chimeras of hook2 and hook3, hook2 truncation mutants ΔC-hook2, ΔN-hook2 and ΔCl-hook2, which lacks the C-terminal part of the coiled-coil domain. Arrows indicate the location of peptides that were used for generating antibodies. Construct boundaries were chosen to maintain putative functional domains in hook proteins, as shown at the bottom. B) Hook2 and hook3 immunoblots show expression of constructs in transfected Vero cells. Endogenous hook2 and hook3 proteins appear as lighter bands at ∼83 kDa. Staining for γ-tubulin shows that each lane contains a similar amount of cell homogenate. C) Immunostaining for the indicated overexpressed hook proteins in Vero cells. All hook2 containing constructs, except ΔC-hook2, are excluded from the nucleus. Clear juxtanuclear staining is seen for 322 and 223, besides hook2. ΔN-hook2 exhibited many strong foci in transfected cells that distributed throughout the cells. ΔCl-hook2 accumulated at centrosomes although less tightly than hook2, 322 and 223. Arrowheads point to examples of juxtanuclear hook2. D) Double staining of cells 6 h after transfection with the indicated hook2 protein with α-tubulin or ninein showed that the indicated proteins accumulated at spindle poles or centrosomes. Arrows point to such ninein-positive structures. Scale bars in panels C and D represent 20 μm. E) The graph shows the percentage of cells with prominent hook-immunoreactive juxtanuclear accumulation in cells transfected with the indicated constructs; at least 400 cells were counted for each construct in at least two separate assays. For hook2 and hook2 truncations more than ten assays were counted. Bars show standard errors. MW, molecular weight.

As shown above (Figures 2 and 6), overexpressed hook2 accumulated in a single perinuclear location in about 50% (n > 5000) of cells in contrast to overexpressed hook3 that never showed such a distribution (Figure 6C,E). However, the C-terminal domain of hook3 partially rescued juxtanuclear localization of ΔC-hook2 because the chimeric protein 223 had a distribution pattern similar to that for hook2 and in 48% of cells accumulated at centrosomes (Figure 6C–E). At a lower frequency, chimeric protein 322 also localized to the centrosomal region (Figure 6C,D). Hook3 and constructs containing mostly hook3 sequences, such as 332, 233 and 323, distributed throughout the cells. The C- and N-terminal domains of hook3 at least partially restored the juxtanuclear localization of hook2, but 232, a chimera with the central domain of hook3 replacing the corresponding region in hook2, distributed diffusely. Therefore, it appears that the region between aa 161 and 439 in hook2 carries specific information that is necessary for the perinuclear localization of hook2 but is not sufficient as 323 also distributes diffusely (Figure 6E). Additional structural elements are needed from throughout the protein, but these are found in both hook2 and hook3. In short, the structural requirements for hook2 centrosomal localization are complex and not restricted to the C-terminal domain that interacts with centriolin/CEP110.

Expression of hook2 and hook2 mutants alters the distribution of centrosomal proteins and centrosomal function

Centrosomal markers and hook2 not only colocalized but also the intensity of staining for some centrosomal proteins increased in cells in which overexpressed hook2 prominently localized to centrosomes. Specifically, hook2 induced accumulation of endogenous ninein at the centrosome (Figure 7A). Interestingly, when two centrosomes were visible, overexpressed hook2 accumulated in only one of the two, and this correlated with ninein accumulation in the same centrosome (Figure 7A, inset). No change in the distribution of ninein and pericentriolar material-1 (PCM-1) was seen in cases when hook2 distributed diffusely (Figure 7B,F).

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Figure 7. Expression of hook2 truncation mutants leads to a redistribution of centrosomal proteins. Vero cells were transfected with the indicated hook2 construct and double stained for hook2 (red in merged images) and endogenous ninein (A–D), or GM130 (E) or PCM-1 (F–H). If overexpressed hook2 localized to a centrosome, accumulation of endogenous ninein was also observed (A); in the inset, an example is shown when overexpressed hook2 accumulated only at one of the two ninein-positive centrosomes and ninein accumulated at that same centrosome. For comparison, arrows in A–C point to ninein-positive centrosomes in untransfected cells. Overexpressed hook2 at the centrosome did not colocalize with the Golgi marker, GM130, and unlike hook3 (15), it did not lead to Golgi dispersal (E). Expression of ΔN-hook2 resulted in dispersal of ninein and PCM-1 (C and G). G shows at higher magnification, the colocalization between ΔN-hook2 and PCM-1 (asterisks). (H) ΔC-hook2 expression did not alter the staining pattern for either ninein or PCM-1. The inset in H shows the PCM-1-positive centrosome (green) at higher magnification. Scale bars represent 20 μm.

In addition, the N-terminal truncation mutant induced a mislocalization of centrosomal proteins and colocalized with scattered centrosome components. As shown above, N-terminally truncated hook2 that lacks the putative domain for microtubule interaction (Figure 6A) forms small insoluble aggregates (Figure 4D) that distribute throughout the cell (Figures 4D and 6C). Because ΔN-hook2 formed granules in a random pattern throughout the cells, proteins that colocalized with ΔN-hook2 are likely to be part of the same protein complex. Among several other proteins we looked at, we found that endogenous PCM-1, a component of the pericentriolar matrix (29), colocalized with ΔN-hook2 particles (Figure 7G); by contrast, ninein (and γ-tubulin; not shown) did not significantly colocalize with ΔN-hook2 (Figure 7C), but there was an apparent reduction in the intensity of ninein staining at the centrosome in cells that expressed ΔN-hook2 (Figure 7C).

Expression of the C-terminal truncation of hook2 did not have a visible effect on ninein or PCM-1 localization. Ninein (Figure 7D) and PCM-1 (Figure 7H) were seen as small juxtanuclear dots, similar in both localization and intensity to surrounding untransfected cells. Therefore, overexpressed hook2 and the N-terminally truncated hook2, but not the C-terminally truncated hook2 that lacks the centriolin/CEP110-binding site, induced a change in the distribution of other proteins that are enriched at the centrosome.

Next, we tested whether hook2 truncation mutants altered functions associated with the centrosome. One of the hallmarks of centrosomes is that they function as MTOC, and as such, they can nucleate microtubule growth and anchor microtubules to generate and maintain the radial organization of the microtubule cytoskeleton. Therefore, as a first measure of centrosomal function, we examined cells that expressed truncated hook2 constructs for changes in microtubule organization. The radial arrangement of microtubules appeared normal in cells in which hook2 accumulated at the centrosome (Figure 8A) but was mildly disturbed in cells expressing hook2 diffusedly throughout the cell (Figure 8B). In cell populations expressing hook2, 48% (n = 227) of the transfected cells did not show a radial array of microtubules while this was only observed in 24% (n = 287) of untransfected or green fluorescent protein-transfected cells. This effect was more pronounced after expression of hook2 truncation mutants. Microtubules were disorganized and showed poor radial organization in cells that expressed ΔN-hook2 (Figure 8C) and ΔC-hook2 (Figure 8D) compared with surrounding nontransfected cells or cells with hook2 accumulations at the centrosome (Figure 8A,B). The disruption of microtubule radial organization by truncation mutants of hook2 was observed in the majority of cells examined: 68% (n = 253) for ΔN-hook2 and 78% (n = 240) for ΔC-hook2 and was also reflected in the distribution of dynein (Figure 8E).

image

Figure 8. Interference with hook2 function disrupts the radial array of microtubules. Vero cells were transfected with the indicated hook2 proteins and double stained for hook2 (red in merged images) and endogenous α-tubulin (A–D, green in merged images) or dynein (E). Hook2 accumulation at centrosomes did not interfere with the radial microtubule array in interphase cells (A and B), but expression of the truncated hook2 proteins did (C–E). Scale bars represent 20 μm.

As a further measure of centrosomal function, we analyzed the ability of cells to regrow microtubules after depolymerization with nocodazole. In untransfected cells, 3 min after drug washout, single foci of α-tubulin-containing asters were obvious (Figure 9A), after 15 min, these had grown into short arrays of radially organized microtubules (Figure 9B) and after 45 min, regrowth of microtubules appeared complete (Figure 9C). At 15 min, 93% of wild-type cells had asters. By contrast, in cells transfected with hook2 (Figure 9D), microtubule asters were evident in a subset of cells at 15 min (50%; n = 50). This corresponds to the fraction of cells in which exogenous hook2 accumulates juxtanuclearly at the centrosome (Figure 6E). When hook2 foci were present, microtubule asters emanated from those foci. Furthermore, expression of ΔC-hook2 (Figure 9F) and, especially ΔN-hook2 (Figure 9E), also led to a decrease in the number of cells that had a microtubule aster: only 49% of cells that expressed ΔC-hook2 and only 15% of cells that expressed ΔN-hook2 had an aster 15 min after nocodazole washout (more than 100 cells were examined for each construct). After 45 min, microtubules did reappear in all cells. However, in cells that expressed truncation mutants, microtubules lacked radial organization, as had been observed in untreated cells that expressed truncation mutants of hook2 (Figure 8C,D). Furthermore, microtubule density appeared sparser after regrowth in nocodazole-treated cells that expressed hook2 truncation mutants (not shown) than in cells that expressed full-length hook2 or were untransfected. Taken together, these data indicate that interference with hook2 localization and function disrupted the MTOC activity of centrosomes.

image

Figure 9. Interference with hook2 function attenuates the regrowth of microtubule asters. In Vero cells transfected with the indicated hook2 constructs, microtubules were depolymerized by a 60-min incubation with 10 μm nocodazole. In untransfected cells (A–C), microtubule asters were evident after 3 min and microtubule elongation after 15 min. Microtubule regrowth appeared complete after 45 min (C). This course of cytoskeletal regrowth was not altered in cells expressing full-length hook2 at the centrosome (D). However, in cells that expressed truncated hook2 proteins, microtubule regrowth was attenuated, and fewer cells had microtubule asters at 15 min (E and F) than in control cells at the same time-point (B and D). Arrows point at growing microtubule asters. Scale bars represent 20 μm.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References

Cell lines

The following cell lines were used in this study, with tissue and species source for each line in parenthesis: HEK293 (human embryonic kidney), Vero (adult African green monkey kidney), SH-SY5Y (human brain neuroblastoma), C6 (brain glia from rat with glioma), RAW (B lymphocytes from mouse) and MEF (mouse embryo fibroblast); these cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and maintained as recommended by ATCC. The CAD cell line (mouse brain catecholaminergic neurons) was a gift from Dr D. Chikaraishi (Duke University, Durham, NC, USA).

Sources of antibodies

Polyclonal antibodies against hook2 (aa 427–719) and hook3 (aa 423–630) have been described (15); these were diluted 1:200–1:400 to detect endogenous hooks and 1:400–1:600 to detect overexpressed hooks by immunohistochemistry and 1:2000–1:10 000 for immunoblotting. Antibodies against α-tubulin (DM1A), dynein (74.1) and γ-tubulin (GTU-88) and anti-hsp 70 antibodies were from Sigma (St Louis, MO, USA; sigma-aldrich.com), GM130 from BD Transduction Labs (bdbiosciences.com), anti-ninein antibodies were a gift from Gordon Chan (Cross Cancer Institute, Edmonton, Alberta, Canada) and anti-PCM-1 antibodies (31) were a gift from Andreas Merdes (University of Edinburgh, UK). Chicken anti-Myc antibodies were from Aves Labs (aveslab.com). Secondary antibodies for immunoblots were horseradish peroxidase-labeled goat anti-mouse and anti-rabbit from Bio-Rad (Hercules, CA, USA), each used at a dilution of 1:10 000. Secondary antibodies for immunofluorescence were goat anti-mouse or goat anti-rabbit antibodies conjugated with Alexa-488, -564, or -594 from Molecular Probes (Eugene, OR, USA), each used at a dilution of 1:400.

Preparation of tissue homogenates

Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) and C57BL/6J mice were handled by protocols that conformed to National Institutes of Health guidelines approved by UT Southwestern Animal Research Committee. Animals were killed in CO2 chambers. Dissected tissues were frozen rapidly in liquid N2 and stored at −80°C, then thawed in ice-cold homogenization buffer [HB: 50 mm Tris, 150 mm NaCl, 0.2 % Triton-X-100 and protease inhibitor cocktail for mammalian cells (Roche, Indianapolis, IN, USA)]. Samples were homogenized on ice with a glass homogenizer. Protein concentration of homogenates was measured by BCA Protein Assay (Pierce Biotechnology, Rockford, IL, USA), and samples were brought to the same concentration in HB and electrophoresis buffers [EB: 0.35 m Tris–HCl pH 6.8, 10.3% SDS (Pierce), 36% glycerol, 5 mm DTT, 0.01% bromophenol blue].

Differential detergent extraction of live cells and preparation of whole cell lysates

Cells grown on 35- to 60-mm plates were placed on a slide warmer and rinsed several times with 37°C PHEM buffer (60 mm 1,4-Piperazinediethanesulfonic acid, 25 mm HEPES, 10 mm EGTA and 1 mm MgCl2, pH 7.4). Cells were sequentially extracted with increasing concentrations of detergents (0.01–1% Triton-X-100 and finally 1% SDS) in 100-300 μL of PHEM buffer containing protease inhibitors for mammalian cells (Roche). Each fraction was centrifuged at 20 000 × g, and all supernatants were mixed with EB, then frozen at −20°C. Whole cell lysates were obtained by scraping cells directly into EB after washes in serum-free medium at 37°C.

Complementary DNA constructs and transfections

Constructs 223, 322, 332, 233, 323 and 232 are chimeras of hook2 and hook3: 223 contains hook2 aa 1–567 and hook3 aa 573–719, 322 contains hook3 aa 1–213 and hook2 aa 210–719, 332 contains hook3 aa 1–572 and hook3 aa 567–719, 233 contains hook2 aa 1–210 and hook3 aa 215–719, 323 is hook3 with a hook2 insert from aa 213 to 572 and 232 is hook2 with a hook3 insert from aa 210 to 567. Chimeras were designed to approximate locations of the previously mapped N-terminal microtubule-binding region, the central coiled-coil homodimerization domain and the proposed organelle-binding C-terminal domain boundaries (15,28). Myc-ΔC-hook2, ΔN-hook2 and ΔCl-hook2 are deletion constructs of hook2: from ΔC-hook2, aa 533–719 were deleted; from ΔN-hook2, aa 1–161 and from ΔCl-hook2, aa 439–554. Myc-hook2401–719 expresses the C-terminal region of hook2. Each of the constructs was inserted into mammalian expression vector pcDNA3.1 that contains a cytomegalovirus promoter (Invitrogen, Carlsbad, CA, USA; invitrogen.com).

Vero and RAW cells were transfected with Fugene (Roche) using a ratio of 1 μg DNA per 3 μL Fugene for RAW cells and a 1:6 ratio for Vero cells.

Immunoblotting and immunoprecipitation

Equal amounts of tissue proteins, cell extracts or cell homogenates were separated by SDS-PAGE gels (Criterion Precast Gels; Bio-Rad) and transferred to nitrocellulose (Schleicher & Schuell, Keene, NH, USA). Prior to the application of antibodies, membranes were blocked in 3% nonfat dry milk in wash buffer (20 mm Tris–HCl pH 7.5, 150 μm NaCl, 1% Tween-20) for 1 h at room temperature or overnight at 4°C. Primary antibodies were applied in block for 1-2 hrs at room temperature. Secondary antibodies [goat anti-mouse or anti-rabbit immunoglobulin G–horseradish peroxidase conjugate (Bio-Rad)] were diluted 1:10 000 and applied for 1 h at room temperature. Blots were rinsed at least three times in wash buffer. Immunoreactive bands were visualized on Blue Biofilm (Denville Scientific, Metuchen, NJ, USA) after the application of SuperSignal West Pico Stable or West Femto Stable ECL substrate (Pierce). Finally, the efficiency of protein transfer to blots was assessed by staining membranes with the MemCode protein stain (Pierce). Immunoprecipitations were done as described using mouse anti-myc or rabbit anti-hook antibodies at 1:1000 (28).

Immunocytochemistry and microscopy

For immunohistochemistry, cells were grown on 4-well Lab-Tek II chamber slides (Nalgene-Nunc, Rochester, NY, USA) at a density of ∼5000 cells/cm2. Cells were rinsed several times with either serum-free medium or PHEM buffer at 37°C, then fixed in methanol at −20°C for 8 min. Fixed cells were rinsed in PBS, and non-specific binding was blocked with 2.5% gelatin/3% BSA/0.2 Triton-X-100 in PBS pH 7.4 for at least 45 min at 37°C. Antibodies were diluted in blocking solution; primary antibodies were applied overnight at 4°C and secondary antibodies for 1 h at room temperature. Washes were in 0.2% Tween-20 in PBS. Nuclei were stained with 0.5 μg/mL Hoechst (Aldrich Chemicals, St Louis, MO, USA). After rinses in 50 mm Tris (pH 8.0), sections were mounted with Gel/Mount (Biomedia, Foster City, CA, USA).

Fluorescent cell preparations were viewed through 20× (Numerical aperture (NA) 0.5), 40× (NA 1.0) or 100× (NA 1.3) PL/Fluotar Leica objectives on a Leica Leitz, DMR microscope (Thornwood, NY, USA). Images were captured with an AxioCam camera (Zeiss) controlled by Axiovision 3.0. All images used for determining the colocalization of proteins were captured in sequential mode with a Leica CS SP2 confocal scanner on a Leica DMIRE2 microscope through a 63× HCX PL APO (NA 1.32) objective. Images were assembled into panels in Adobe Photoshop (Adobe Systems, Mountain View, CA, USA) and adjusted for contrast and brightness.

Microtubule regrowth assay

Vero cells were transfected with various hook2 constructs and grown for 24 h. Cells were incubated in 10 μmm nocodazole (Sigma) for 1 h to depolymerize microtubules. Microtubule asters started to regrow after cells were washed three times with DMEM and incubated in fresh medium. Cells were fixed 3, 15 or 45 min after drug washout and stained for hook proteins and microtubules as described above.

Yeast two-hybrid assay

Two-hybrid analysis was performed using the Matchmaker 3 system (Clontech, Palo Alto, CA, USA). The plasmid pGBKT7-hHK2C expressing the C-terminal domain of human hook2 (aa 584–719) was used to isolate interacting candidates from a human adult brain complementary DNA (cDNA) library (Clontech) in the pACT vector. Candidate cDNAs were isolated based on His3 auxotrophy and retransformed in the AH109 strain together with pGBKT7-hHK2C or pGBKT7-p53 as negative control. Clones that specifically induced histidine and adenine auxotrophy and β-galactosidase expression in the presence of pGBKT7-hHK2C were further considered. Such interacting candidates were sequenced, and their identities determined using the Basic Local Alignment Search Tool (Blast) on the National Center for Biotechnology information database. For coimmunoprecipitation experiments, candidates were subcloned into a variant of pcDNA3.1 that adds an N-terminal Myc epitope, for centriolin/CEP110 that yielded in Myc-CEP1101985–2326.

Coimmunoprecipitation

Hook2-interacting candidates that scored positive after retesting were further evaluated by coimmunoprecipitation. Candidates were fused with a 2xMyc epitope in the DNA3.1 expression vector and cotransfected into Vero cells with hook expression vectors. After 24 h of expression, cells were lysed in 1% Triton-X-100, 142 mm KCl, 5 mm MgCl2, 10 mm HEPES pH 7.4, 1 mm DTT supplemented with a complete protease inhibitor cocktail (Roche). Candidates were precipitated using anti-Myc antibodies and protein A beads, washed three times and eluted by boiling for 2 min with SDS loading buffer. Controls for immunoprecipitation included cell extracts from untransfected cells and from cells transfected with only one of the candidate interacting pair. The amount of coprecipitated hook proteins was evaluated by Western blotting.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References

We thank Drs Andreas Merdes, Gordon Chan, Stephen Doxsey and Michel Bornens for providing antibodies. We thank Dr Adam Haberman for a critical reading of this manuscript. This work was supported by grants to H. K. from The Welch Foundation (I-1300) and the National Institutes of Health (EY10199 and NS43406).

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References