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1 Graduate School of Science and Technology, and 2 Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Cytoplasmic dynein, a multi subunit complex consisting of two heavy chains and several intermediate, light intermediate and light chains, is a minus end-directed microtubule motor protein involved in multiple cellular functions, including mitosis, maintenance of the Golgi apparatus and intracellular transport (Karki & Holzbaur 1999; Karcher et al. 2002). In membrane transport, cytoplasmic dynein is thought to act in transport from ER and endosomes to the minus ends of microtubules (King 2000). Dynactin is a large multi subunit complex including p150Glued, dynamitin, p24 and ARP1 subunits (Allan 1996; Holleran et al. 1998; Karki & Holzbaur 1999) that stimulate cytoplasmic dynein-mediated movement in vitro (Schroer & Sheetz 1991). Among them, p150Glued, the mammalian homologue of the product of the Drosophila gene Glued (Holzbaur et al. 1991), binds to both microtubules (Waterman-Storer et al. 1995) and cytoplasmic dynein (Gill et al. 1991). It was also reported that over-expression of dynamitin disrupts dynein–cargo interaction, thereby inducing fragmentation of the Golgi apparatus (Echeverri et al. 1996; Burkhardt et al. 1997). Thus, dynactin mediates the binding of dynein to membranous cargoes. However, detailed molecular mechanisms for interaction of dynactin and their cargo are poorly understood.
A giant anchoring protein centrosome and Golgi-localized PKN associated protein (CG-NAP) (Takahashi et al. 1999), also known as AKAP350 (Schmidt et al. 1999), AKAP450 (Witczak et al. 1999) or AKAP9, localizes to centrosomes throughout the cell cycle and the Golgi apparatus at interphase. CG-NAP has been found to anchor various signaling molecules such as protein kinase A (PKA), PKN, protein phosphatase 1, protein phosphatase 2 A, phosphodiesterase 4D, calmodulin, casein kinase 1
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, cdc42 interacting protein 4 (CIP4), Ran and Cyclin E/Cdk2 (Takahashi et al. 1999; Sillibourne et al. 2002; Takahashi et al. 2002; Keryer et al. 2003; Larocca et al. 2004; Nishimura et al. 2005). Thus, CG-NAP may coordinate the location and activity of these enzymes to regulate the phosphorylation states of specific substrates at the centrosome and the Golgi apparatus. We have found that CG-NAP is localized to the centrosome via the carboxyl-terminal region and serves as microtubule nucleation sites by anchoring the 
-tubulin ring complex (-TuRC) at the centrosome (Takahashi et al. 2002). Furthermore, suppression of CG-NAP expression by RNA interference alters the Golgi morphology (Larocca et al. 2004). However, the mechanism for this alteration as well as for the localization of CG-NAP to the Golgi remains to be determined.
Here we demonstrated that CG-NAP localized to the Golgi apparatus in a microtubule-dependent manner, and CG-NAP was well co-localized with dynein–dynactin subunits on the microtubules during recovery both from on-ice and acetate treatments. Endogenous CG-NAP interacted with p150Glued, and expression of p150Glued-binding region of CG-NAP fused with mitochondria-targeting sequence induced recruitment of mitochondria to pericentriolar area, suggesting that this region interacts with functional cytoplasmic dynein in vivo. Furthermore, over-expression of this region caused fragmentation of the Golgi, probably by sequestering the cytoplasmic dynein to cytosol. These results imply that CG-NAP is recruited to the Golgi apparatus by interacting with cytoplasmic dynein, and possibly involved in the organization and maintenance of the Golgi apparatus.
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Results |
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To investigate the Golgi-targeting mechanism of CG-NAP, we examined subcellular localization of CG-NAP after various treatments. Among them, when the cells were placed on ice for 30 min, we found that CG-NAP disappeared from the Golgi apparatus and only centrosome-localized CG-NAP was observed (Fig. 1Ac). Under this condition, microtubules were depolymerized (Fig. 1Ad), and the Golgi apparatus represented a relatively loose shape (Fig. 1Ah). When the cells were incubated again at 37 °C for 20 min, microtubules were repolymerized (Fig. 1Af) and CG-NAP was relocalized to the Golgi (Fig. 1Ae). CG-NAP dissociation from the Golgi by the on-ice treatment was suppressed in the presence of taxol, a microtubule stabilizing agent (Fig. 1Bi). Furthermore, nocodazole, a microtubule depolymerizing agent, suppressed CG-NAP relocalization to the Golgi apparatus after recovery from the on-ice treatment (Fig. 1Bk). These results indicate that CG-NAP localizes to the Golgi apparatus in a microtubule-dependent manner.
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To further characterize the Golgi-targeting mechanism of CG-NAP, we investigated the process during recovery from the on-ice treatment. To visualize cytoskeleton-associated proteins clearly, cells were briefly extracted with detergent prior to fixation. After 4 min from recovery, microtubules were nucleated from the centrosomes to form asters (Fig. 2Ab). At this time point, CG-NAP was detected along with the microtubule asters (Fig. 2Aa). After 7 min, the amount of microtubule-associated CG-NAP was increased (Fig. 2Ad) in conjunction with elongating microtubule asters (Fig. 2Ae). After 20 min, when the microtubule network became nearly normal (Fig. 2Ah), microtubule-associated CG-NAP was barely detectable and only pericentriolar staining was observed (Fig. 2Ag). Similar results were obtained with both HeLa cells and the cells recovered from nocodazole treatment (our unpublished results). These results suggest that CG-NAP is recruited to the microtubules until relocalizing to the Golgi apparatus.
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CG-NAP co-localizes with dynein–dynactin subunits during recovery from the on-ice treatment
At 4–7 min after recovery from the on-ice treatment, CG-NAP was not co-localized with entire length of microtubules (Fig. 2A). CG-NAP represented a continuous dot-like staining pattern along the microtubules, which is reminiscent of the distribution of motor proteins. Therefore, we compared the localization of CG-NAP with cytoplasmic dynein subunits at 4 min after recovery. As shown in Fig. 3, microtubule-associated CG-NAP was well co-localized with dynamitin, p150Glued and dynein intermediate chain (DIC). These co-localization patterns persisted until CG-NAP localized mostly to the Golgi apparatus (our unpublished results). These results suggest that CG-NAP is recruited to the microtubules by the dynein–dynactin complex.
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Brief acetate treatment, which leads to cytoplasmic acidification, has been known to induce redistribution of several organelles, including lysosomes (Heuser 1989), endosomes (Parton et al. 1991) and the Golgi apparatus (Vaughan et al. 1999). Though detailed mechanism has been elusive, it has been thought that dynactin–vesicle interaction is disrupted under this condition (Vaughan et al. 1999). After removal of acetate, dynein-mediated motility is initiated, and then those organelles are reformed. We examined CG-NAP behavior under this condition. In control cells, CG-NAP was mainly detected at the pericentriolar area and only a faint amount was co-localized with microtubules (Fig. 4A). After 15 min exposure to acetate, the Golgi apparatus exhibited slight diffusion, monitored by an anti-GM130 antibody (Fig. 4B). CG-NAP exhibited diffused localization surrounded by vesicle-like staining along microtubules. At 4 min after acetate wash-out, CG-NAP was partially co-localized with radial microtubules, which were well co-localized with dynamitin (Fig. 4C). CG-NAP was also well co-localized with DIC along microtubules (our unpublished results). Thirty minutes later, CG-NAP became relocalized to the Golgi apparatus in accordance with the decrease in microtubule-associated fractions (Fig. 4D), which coincided with the distribution of dynamitin. These results again suggest that CG-NAP is transported along microtubules by cytoplasmic dynein, and thereby localizes to the Golgi apparatus.
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To investigate whether CG-NAP associates with the cytoplasmic dynein complex, we performed a co-immunoprecipitation study of endogenous proteins. Among several subunits examined, p150Glued was found to be co-immunoprecipitated with CG-NAP by anti-CG-NAP antibody from HeLa cell extracts (Fig. 5A). Furthermore, the interaction was also observed between FLAG-CG-NAP and HA-p150Glued exogenously expressed in COS7 cells (Fig. 5B). To identify the domains of CG-NAP responsible for this interaction, we performed co-immunoprecipitation study using cell extracts co-expressing various FLAG-tagged deletion mutants of CG-NAP (see Fig. 2C) and HA-tagged p150Glued. HA-p150Glued was reciprocally co-immunoprecipitated with either FLAG-CG-NAP1229–1917 or CG-NAP2875–3899 (Fig. 5C). The interaction of HA-p150Glued with FLAG-CG-NAP1229–1917 was stronger than that with FLAG-CG-NAP2875–3899. These results suggest that CG-NAP can associate with p150Glued through amino acid residues 1229–1917 or 2875–3899.
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Next, we monitored subcellular localization of these deletion mutants. Since we have already shown that CG-NAP2875–3899 is localized to the centrosomes (Takahashi et al. 2002), we focused on the distribution of CG-NAP1229–1917. Unexpectedly, FLAG-CG-NAP1229–1917 was mainly localized in the cytoplasm and, in part, at pericentriolar region (Fig. 6A), suggesting that this region cannot be efficiently transported to the minus ends of microtubules by cytoplasmic dynein. When a longer fragment FLAG-CG-NAP1229–2444 was expressed, it was detected at the pericentriolar area, whereas the extended region FLAG-CG-NAP1917–2444 was distributed in the cytoplasm (Fig. 6A). To examine the localization of these deletions more clearly, we fused them with a mitochondria-targeting sequence (Mt) of the D-AKAP1 (see Experimental procedures). D-AKAP1 is an A-kinase anchoring protein that binds and targets PKA to the outer mitochondrial membranes (Ma & Taylor 2002), and thus we expected that expression of Mt-tagged deletions of CG-NAP alter mitochondrial distribution. As a control, we fused the Mt sequence to the amino terminus of GFP (Mt-GFP), which was co-localized with mitochondria throughout the cytoplasm (Fig. 6Ba), and had no effect on mitochondrial distribution (Fig. 6Bb). Mt-FLAG-CG-NAP1229–2444 exhibited pericentriolar localization (Fig. 6Bc). Furthermore, it caused drastic alteration of mitochondrial distribution from cytoplasm to the pericentriolar area (Fig. 6Bd), suggesting that this region can be efficiently loaded onto minus end-directed transport. On the other hand, neither Mt-FLAG-CG-NAP1229–1917 nor Mt-FLAG-CG-NAP1917–2444 could change the mitochondrial distributions (Fig. 6Bf,Bh). Since the fragment FLAG-CG-NAP1229–2444 was not co-localized with endogenous CG-NAP at the Golgi apparatus (our unpublished results), this region may mediate constitutive transport of CG-NAP to the minus ends of microtubules from cytosol before association with the Golgi apparatus that may need an additional region. Next we examined whether the fragment CG-NAP1229–2444 can associate with microtubules and p150Glued more tightly than CG-NAP1229–1917. FLAG-CG-NAP1229–2444 also interacted with both microtubules (Fig. 6C) and p150Glued (Fig. 6D), however, at almost similar levels to FLAG-CG-NAP1229–1917, implying that efficient loading of CG-NAP1229–2444 to the minus end-directed transport is not mediated by a stronger interaction with p150Glued and/or microtubules but by some additional mechanism(s). These results suggest that the region comprising amino acids 1229–2444 is responsible for the minus end-directed transport of CG-NAP by cytoplasmic dynein complex in vivo.
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Finally, we examined the effect of over-expression of FLAG-CG-NAP1229–2444 on the organization of the Golgi apparatus. Cells over-expressing FLAG-CG-NAP1229–2444 exhibited fragmented Golgi structures monitored by anti-GM130 antibody (Fig. 7A), which was similar to the effect of dynamitin over-expression (Fig. 7B). Dynamitin over-expression is known to disrupt cytoplasmic dynein complex, resulting in the fragmentation of the Golgi apparatus to form mini-stacks maintaining cis- and trans-organization (Fig. 7B). To examine whether the scattered Golgi structures within the cells over-expressing FLAG-CG-NAP1229–2444 also form such mini-stacks, we monitored the localization of GM130 and golgin 97, cis- and trans-Golgi proteins, respectively. These two proteins were detected at adjacent but distinct positions in the same Golgi fragments (Fig. 7A right, magnified images) similar to those in dynamitin over-expressing cells (Fig. 7B right, magnified images), suggesting that FLAG-CG-NAP1229–2444 over-expression inhibits the cytoplasmic dynein function. To investigate whether this was caused by sequestering p150Glued, we monitored the localization of p150Glued (Fig. 7C). As reported previously (Vaughan et al. 1999), microtubule-associated p150Glued was unaffected by dynamitin over-expression (Fig. 7C bottom, magnified image). On the other hand, in the cells over-expressing FLAG-CG-NAP1229–2444 p150Glued was displaced from the radial microtubules (Fig. 7C top, magnified images). These results suggest that over-expression of FLAG-CG-NAP1229–2444 causes fragmentation of the Golgi by sequestering p150Glued, and presumably intact cytoplasmic dynein complex, involved in the organization of the Golgi apparatus.
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Discussion |
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CG-NAP interacted with a dynactin subunit p150Glued through amino acid residues 1229–1917 (Fig. 5C); however, this region was not sufficient but an additional carboxyl-terminal region (amino acid residues 1918–2444) was required for loading onto the minus end-directed transport by cytoplasmic dynein (Fig. 6). Furthermore, over-expression of this longer fragment (amino acid residues 1229–2444) caused fragmentation of the Golgi. At the initial stage of cargo transport tethering of cargo to microtubule is mediated by dynactin complex through the microtubule binding domain of p150Glued, and then dynein complex is recruited to dynactin complex followed by switching the microtubule binding domain from p150Glued to dynein heavy chain to initiate transport. This switching was reported to be triggered by phosphorylation of p150Glued by PKA (Vaughan et al. 2002). CG-NAP is an A-kinase anchoring protein (AKAP) having two RII-binding domains (Takahashi et al. 1999), one of which is located in the p150Glued-binding region (amino acid residues 1229–1917). Nevertheless, this fragment was not sufficient to elicit minus end-directed transport. One possibility is that the latter region (amino acid residues 1918–2444 of CG-NAP) plays a role in recruiting dynein complex and/or facilitating the formation of dynein–dynactin complex, although the association of this region with dynein subunits has not been detected so far. Another possibility is that the latter region is required for maintaining the phosphorylation states of p150Glued for proper complex formation. In addition, it was reported that CG-NAP/AKAP350 associates with CIP4 at the Golgi apparatus thorough amino acid residues 1076–2143 of AKAP350 (Larocca et al. 2004), which corresponds to 1452–2519 of CG-NAP overlapping with the region (amino acid residues 1229–2444) elicited the minus end-directed transport. Larocca et al. also presented that over-expression of the CIP4 binding region of CG-NAP/AKAP350 or suppression of CG-NAP/AKAP350 by RNA interference alters the Golgi morphology. It is possible that this alteration was caused by inhibiting cytoplasmic dynein function but not by sequestering CIP4, or by the combined effect of sequestering both cytoplasmic dynein and CIP4. CIP4 might be involved in the formation of the complex mediating the minus ends-directed transport of CG-NAP along microtubules. Further studies will be needed to address these possibilities.
CG-NAP remains at centrosomes under various conditions such as on-ice treatment (Fig. 1) and nocodazole treatment (Takahashi et al. 1999), suggesting microtubule independency in localization to the centrosomes. However, centrosomal CG-NAP may also be recruited by cytoplasmic dynein before stable attachment to centrosomes through the centrosome-targeting domain (Gillingham & Munro 2000; Takahashi et al. 2002). Pericentrin, sharing homology with CG-NAP (Takahashi et al. 2002), was demonstrated to interact with dynein light intermediate chain 1 and be recruited to centrosomes in a cytoplasmic dynein-dependent manner (Tynan et al. 2000; Young et al. 2000), supporting this idea. Further, dynamitin over-expression caused the reduction of centrosomal CG-NAP (our unpublished results), suggesting that both centrosome and Golgi-localized CG-NAP are recruited to the minus ends of microtubules by cytoplasmic dynein followed by steady-state localization to these organelles. If this is the case, it will be of interest to elucidate the mechanism how these two destinations are determined during transport of CG-NAP as a cargo.
It has been clear that the Golgi apparatus is dependent on microtubules for its static maintenance as a single organelle near the centrosome as well as for its dynamic formation via vesicular transport. Several non-motor microtubule-binding proteins have been proposed as linkers between Golgi membranes and microtubules. A cis-Golgi microtubule binding protein GMAP210 was proposed to recruit -TuRC to the Golgi membranes to nucleate short microtubules and contribute to the assembly and maintenance of the Golgi around the centrosome (Rios et al. 2004); however, several contradictory evidences were presented (Barr & Egerer 2005). A microtubule-binding Hook3 was also proposed to participate in defining the architecture and localization of the Golgi (Walenta et al. 2001). Another candidate was a Golgi-localized binding partner for cytoplasmic dynein, Bicaudal-D (Hoogenraad et al. 2001), which was proven to function mainly in a microtubule-dependent recycling pathway from the trans-Golgi network to the ER (Young et al. 2005). Thus, molecules involved in the interaction between microtubules and the steady-state Golgi stacks still remain elusive. We have demonstrated that CG-NAP was dissociated from the Golgi by the on-ice treatment (Figs. 1 and 2), indicating the microtubule dependency in its localization to the steady-state Golgi. Under this condition localization of the Golgi matrix protein GM130 was also affected but far less than that of CG-NAP (Fig. 1). This sharp sensitivity of CG-NAP may reflect its involvement in the interface between microtubules and the steady-state Golgi stacks. The interaction of CG-NAP with microtubules might be mediated by cytoplasmic dynein as is the case during transport along microtubules. However, addition of the antibody m70.1, which recognizes DIC and blocks dynein-mediated binding to microtubules (Young et al. 2000), affected the binding of CG-NAP to microtubules only slightly (our unpublished results), suggesting that intact cytoplasmic dynein is not required for this interaction. We assume that interaction of CG-NAP with microtubules at the steady-state Golgi is mediated by p150Glued alone or dynactin complex, although we cannot exclude the possibility of its direct interaction with microtubules. In addition, it was reported that the Golgi is a microtubule-organizing organelle (Chabin-Brion et al. 2001), and we have previously shown that CG-NAP serves as microtubule nucleation sites by anchoring -TuRC at centrosomes (Takahashi et al. 2002). Therefore, another possibility is that CG-NAP functions as an interface by nucleating short microtubules near the Golgi that are connected to the preexisting microtubule arrays and the Golgi stacks via some microtubule-associated proteins, as was proposed for the function of GMAP-210 (Rios et al. 2004).
It was reported that treatment of cells at low temperature (5 °C) induces PKA dissociation from the Golgi to cytosol but not from the centrosomes (Martin et al. 1999), which is very similar to the behavior of CG-NAP, suggesting that CG-NAP is a responsible AKAP at the Golgi. Recently, it was also reported that PKA activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from a separated membrane stack (Bejarano et al. 2006). Taken together with the results of an RNA interference study (Larocca et al. 2004), these observations suggest that CG-NAP recruited to and maintained at the Golgi is involved in the formation of a Golgi ribbon as a major AKAP and/or as an interface between microtubules and the Golgi.
In summary, we have demonstrated that CG-NAP is recruited to the minus ends of microtubules by cytoplasmic dynein, is localized to the steady-state Golgi apparatus in a microtubule-dependent manner and, possibly, participates in the biogenesis and maintenance of the Golgi structural integrity.
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Experimental procedures |
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Mammalian expression plasmids for CG-NAP deletion mutants were constructed by inserting the corresponding cDNA fragments into pTB701-FLAG (Takahashi et al. 1999). cDNAs of p150Glued and p50 dynamitin were cloned by PCR using HeLa Marathon-Ready cDNA library (Clontech) with appropriate primers and subcloned into pTB701-HA and pcDNA3.1 Myc-His (Invitrogen). FLAG-tagged deletion mutants were fused with mitochondrial targeting sequence, amino acid residues 16–30 of D-AKAP1 at its amino-terminus (Ma & Taylor 2002), and subcloned into pcDNA3 (Invitrogen).
Cell culture, transfection and treatments
COS7, HEK293T and HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 50 units/mL penicillin and 50 µg/mL streptomycin at 37 °C in a humidified 5% CO2 atmosphere. COS7 cells were transfected with expression plasmids by electroporation using GENEPULSER II (Bio-Rad); HeLa and HEK293T cells were transfected using TransIT LT-1 Transfection Reagent (Mirus).
To depolymerize microtubules, cells were placed on ice for 30 min in the presence of 25 mM HEPES (pH 7.3). To repolmerize microtubules, the cells were incubated again at 37 °C for appropriate time points (we referred to this as the "recovery" process). For acetate treatment, COS7 cells were treated with Ringers acetate, pH 6.4 (80 mM NaCl, 70 mM sodium acetate, 10 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 2 mM NaPO4, 1 mM MgCl2) for 15 min in 37 °C as previously described (Vaughan et al. 1999). The cells were washed with PBS twice and incubate with prewarmed medium at 37 °C for appropriate periods.
Antibodies
Polyclonal antibody to CG-NAP (
EE) was previously described (Takahashi et al. 1999). Polyclonal antibodies to GRASP65 (Sutterlin et al. 2002) and golgin 97 (Yoshimura et al. 2004) were kindly provided by Dr Vivek Malhotra (University of California San Diego) and Dr Nobuhiro Nakamura (Kanazawa University), respectively. The following antibodies were purchased: anti--tubulin DM1A, rabbit anti-FLAG, mouse anti-FLAG clone M2 (Sigma); rat anti-hemagglutinin (HA) clone 3F10, mouse anti-HA clone 12CA5 (Roche Diagnostics); anti-GM 130, anti-p150Glued, p50 dynamitin (BD Biosciences); mouse anti-beta actin (Abcam); mouse anti-dynein intermediate chains, rhodamine or fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Chemicon International); and mouse anti-c-myc clone 9E10, horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).
Immunofluorescence microscopy
Cells grown on cover glasses were fixed with cold methanol at –20 °C for 5 min. To visualize microtubule associated proteins clearly, cells were briefly extracted with 0.1% Triton X-100 for 2 min in a buffer consisting of 50 mM piperazine-1, 4-bis (2-ethanesulfonic acid)/KOH at pH 6.9, 5 mM MgCl2 and 1 mM EDTA prior to fixation. Fixed cells were rehydrated with PBS, blocked with 5% (Cardone et al. 2002) normal donkey serum or 5% (w/v) BSA in Ca2+, Mg2+-free phosphate buffered saline (PBS) containing 0.1% Triton X-100, and incubated with the relevant antibody at room temperature for 2 h or at 4 °C overnight. Then the primary antibodies were visualized by incubation with appropriate secondary antibodies conjugated with either rhodamine or FITC at room temperature for 1 h. DNA was visualized by incubating with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI). Mitochondria were visualized using Mito Tracker Red CMXRos (Molecular probes) according to the manufacturer's instructions, then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. Fixed cells were permeabilized using 0.1% Triton X-100. The fluorescence of rhodamine and FITC was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan).
Immunoprecipitation and immunoblotting
Cells were lyzed in a lysis buffer consisting of 50 mM Tris–HCl at pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 µg/mL leupeptin, 1 mM PMSF and 1 mM dithiothreitol at 4 °C for 30 min. After centrifugation, the supernatants were incubated with appropriate antibodies at 4 °C for 2 h. Then Protein A-Sepharose beads (GE Healthcare) were added and incubated for further 30 min. The beads were washed with the same buffer three times, and the bound proteins were processed for immunoblotting.
Microtubule binding assays
Cells were lyzed in a buffer consisting of 100 mM PIPES at pH 6.9, 1 mM EGTA, 1 mM MgCl2 (PEM) and 1% Triton X-100 at 4 °C for 30 min and centrifuged at 100 000 g for 30 min. Microtubule binding experiments were performed according to Balczon et al. (1999) with slight modifications. In brief, cell lysates (100 µg) were mixed with 0.5 mM GTP with or without 20 µM taxol, and then incubated for 20 min at 37 °C. Lysates were overlaid on a cushion of PEM buffer containing 20% sucrose, 0.5 mM GTP with or without 10 µM taxol and followed by centrifugation at 30 000 g for 30 min at 25 °C. Microtubule pellets were washed and collected with sample buffer. Bound proteins were separated by SDS-PAGE and processed for immunoblotting.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: tmikiko{at}kobe-u.ac.jp
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References |
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Received: 23 October 2006
Accepted: 20 December 2006
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