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Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, 606-8501, and RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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Abstract |
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Introduction |
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Gene knockout experiments have revealed critical roles of p120 in epithelial morphogenesis (Davis & Reynolds 2006). However, the molecular functions of this protein are still controversial. A major function of the p120 associating with cadherins seems to be the regulation of cadherin turnover (Ireton et al. 2002; Davis et al. 2003; Xiao et al. 2003; Yanagisawa et al. 2004); without p120, cadherin molecules in the cell membranes are down-regulated. Another action of p120 is to inhibit Rho (Anastasiadis et al. 2000; Noren et al. 2000); this activity was recently shown to be elicited via the interaction of p120 with p190RhoGAP (Wildenberg et al. 2006). p120 also has been implicated in E-cadherin-mediated Rac signaling (Goodwin et al. 2003). Despite such an increase in information on this catenin, how these complex activities of p120 relate to each other remains largely unknown. One clear point is, however, that the armadillo repeat and N-terminal domains of p120 have distinct functions. The ability of p120 to stabilize cadherin turnover depends upon its binding to cadherin via the armadillo domain (Ireton et al. 2002), but many other activities of p120 require the N-terminus (Aono et al. 1999; Grosheva et al. 2001; Aho et al. 2002; Cozzolino et al. 2003).
In the present study, we asked whether p120 plays any role in the control of MT dynamics. To explore p120's intrinsic functions exclusively, we purposely used a cadherin-deficient cell line; this allowed us to avoid potential complexities in the interpretation of the results, which may arise from the cooperative actions of p120 and cadherin. Our results show that p120 interacted with tubulins or MTs via the N-terminal domain of the former and that its over-expression stabilized MTs. On the contrary, RNAi-mediated knockdown of p120 made MTs more sensitive to nocodazole, a MT-destabilizing reagent. Furthermore, p120 depletion caused an increase in the plus end dynamics of MTs. In addition, p120 knockdown altered cell motility in a characteristic way: it accelerated the motility of isolated cells, but suppressed wound healing. These findings support the idea that p120 has the ability to regulate MT dynamics independently of the cadherin system, and demonstrate that this protein is necessary to sustain ordered cell movement.
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Results |
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We immunostained Neuro-2a (N2a) cells, a cadherin-deficient neuroblastoma line (Matsunaga et al. 1988), for endogenous p120, and found that this protein was diffusely distributed throughout the cytoplasm. To examine the p120 localization with higher sensitivity, particularly in living cells, we transfected the cells with EGFP-tagged p120 (p120-EGFP; Fig. 1A). The exogenous p120 molecules were diffusely localized throughout the cytoplasm as the endogenous ones; notably, however, a certain fraction of them was concentrated on the centrosomes in 13%–14% of the transfected cells, not only in fixed cells (Fig. 1B,E) but also in live cells (data not shown). The centrosomal localization of p120 did not require MTs, because nocodazole treatment had no effects on it (Fig. 1E). The centrosomal p120 could be detected only as autofluorescence emanating from the attached EGFP tag, but not by immunostaining with antibodies against p120 or the EGFP-tag (Fig. 1B), suggesting that the centrosome-associated p120 was sterically hindered from being accessed by antibodies.
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N) and the N-terminal domain of p120 (N346) (Fig. 1A). The results showed that a fraction of N346-EGFP molecules was concentrated on the centrosomes but that the N-EGFP was not (Fig. 1B,E). Likewise, the N-EGFP was unable to associate with MTs, indicating that the binding of p120 to centrosomes or MTs required the N-terminal region of this protein. However, over-expressed N346-EGFP never co-localized with MTs nor induced MT bundling, suggesting that the mechanisms responsible for p120 to interact with these two structures are not completely identical.
The localization of p120 on the centrosomes suggested its interaction with centrosomal components. We indeed found that GST-fused peptides corresponding to amino acid 158–346 of the N-terminal domain of p120, but not those corresponding to amino acid 158–244 or 158–295, could pull-down the endogenous 
-tubulin from N2a cell lysates (Supplementary Fig. S1). We also confirmed that p120 could co-precipitate with taxol-stabilized MTs in cell lysates (data not shown), as found previously (Yanagisawa et al. 2004).
We next used nocodazole to examine whether p120 over-expression affected the polymerizing properties of MTs. When non-transfected N2a cells in low-density cultures had been treated with nocodazole (4 µM), the MTs became diffuse, leaving signals of MT polymers only around the centrosome. However, in p120-overexpressing cells, dense arrays of MTs remained throughout the cytoplasm even after the nocodazole treatment (Fig. 1D). N-EGFP, on the other hand, had no such effects. These results suggest that p120 was capable of stabilizing MTs in an N-terminal region-dependent manner.
p120 depletion causes destabilization of microtubules (MT)
Next, we depleted p120 in N2a cells by treating them with two independent constructs of siRNA; and other cells were treated with an unrelated siRNA as a control (Fig. 2A). The knockdown effect with either construct reached its maximum at day 3 after transfection, and persisted at least for 6 days. Immunostaining for p120 showed that 96.9% ± 2.8% (n = 3 experiments) of the cells exhibited only the background level of staining, although the rest of them retained the normal level of signals (Fig. 2B). In these p120-depleted cells, -tubulin showed the normal localization; and the radial networks of MTs looked normal (Fig. 2C). However, when we measured the nocodazole sensitivity of the MTs by using a tubulin fractionation assay, we noticed a difference in MT stability. We treated cells in confluent cultures with nocodazole (3.3–33 µM), subjected them to the tubulin fractionation assay, and found that the ratio of the soluble to the polymerized fraction of MTs was always higher in the p120-depleted cells than in the control in repeated experiments (n = 4, Fig. 3Aa,c). Consistent results were obtained when the cells treated with nocodazole under the same culture conditions as above were immunostained for MTs. Some of MT arrays remained polymerized in the control cells, whereas MTs were almost completely depolymerized in the p120-depleted cells after 10 µM nocodazole treatment (Fig. 3B); the differences in the nocodazole sensitivity of MTs seen between this experiment and that for Fig. 1D was due to that MTs were more resistant to this reagent in confluent cultures than in sparse ones, used for the respective experiments. These results suggest that the MTs in p120-depleted cells were more liable to become depolymerized by nocodazole than those in the control ones.
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p120 controls dynamics of the microtubule (MT) plus ends
To further explore the role of p120 in MT dynamics, we decided to observe MT behavior by live cell imaging. We firstly generated N2a cells stably expressing EGFP-
-tubulin, subsequently knocked down their p120 expression, and analyzed the tubulin dynamics in these cells. However, because of dense and bundled networks of MTs in N2a cells, we could hardly identify the ends of individual MTs, the tracking of which is necessary for quantitative analysis of MT dynamics.
To circumvent the above difficulty, we prepared cells transiently expressing GFP-fused EB1, an APC-binding protein. EB is known to preferentially localize on the growing plus-end of MTs (Mimori-Kiyosue et al. 2000). Time-lapse recording of EB1 signals enabled us to clearly track the behavior of the MT ends. In both, control and p120-knockdown cells, EB1-GFP exhibited comet-like signals with a typical radial distribution, emanating from the centrosomal areas (Fig. 4B). Time-lapse recording of the EB1 in living cells showed that, while EB1 ‘comets’ were moving in one direction, their signals often decreased or disappeared during the movement. Some of them, however, reappeared within several seconds at exactly the same positions as prior to their disappearance (8 s at most; Fig. 4A, brackets), whereas others never reappeared within the 2 min of recording (Fig. 4A, asterisk). The former cases were judged as pause events of MTs, and the latter were presumed to be catastrophe events. This judgment was made by comparing our results of the duration for EB1 disappearance with those obtained from the direct observation of fluorescent MTs (Shelden & Wadsworth 1993). Using this methodology, we could trace the ‘growth history’ of individual MTs, which consisted of several short growth and pause events and a single catastrophe event. When we traced the growth history of MTs, we noticed that, in p120-knockdown cells, MTs tended to have shorter growth durations until catastrophe (Fig. 4B). Dynamic instability parameters of these EB1-positive MTs, estimated in Table 1, showed that the MTs in p120-knockdown cells had higher catastrophe frequencies, as well as fewer pause events than those in the control cells. Although the growth rate of MTs was increased by treatment with one of the siRNA constructs, the other construct had no significant effect, suggesting that p120 depletion may have no effect on this particular parameter. The obtained values of the growth rates and catastrophe frequencies of MTs were comparable to those measured previously in other systems using fluorescence-labeled tubulin (Shelden & Wadsworth 1993). These results thus confirmed that MT dynamics was altered as a result of p120 depletion.
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Next, we asked whether p120 knockdown had any effect on cell behavior. We first noticed that p120-depleted N2a cells tended to clump more conspicuously than control cells (Fig. 5A). The p120 depletion, on the other hand, had no effect on cell growth (Fig. 5B). Time-lapse tracing of isolated cells in sparse cultures showed that the migration speed of individual cells slightly increased as a result of p120 depletion (Fig. 5C, D). Closer examination revealed that cell shape changed more rapidly in the p120-depeleted cells; for example, protrusions and retractions of the cell peripheries were more active in these cells than in the control ones (Fig. 5E, and Supplementary Movies 1 and 2).
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Discussion |
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p120-dependent MT dynamics
We obtained two lines of evidence for the role of p120 in MT dynamics: the forced increase or reduction in the p120 level influenced the nocodazole sensitivity of MTs and p120 knockdown affected the MT plus-end dynamics. The N-terminal domain of p120 was required for its interactions with MTs or -tubulin, as well as for its localization on the centrosome, suggesting that the interactions of p120 with tubulins or MTs are mediated by this domain, in contrast to the armadillo-domain dependency of cadherin stabilization.
Time-lapse observations of EB1-GFP indicated that the relatively long-lived and pausing population of EB1-positive MTs was reduced in p120-depleted cells. This finding suggests that more rapid turnover of MTs occurred in the absence of p120, consistent with the results showing that MTs had higher nocodazole sensitivity in these cells. The molecular mechanism governing how p120 regulates MT dynamics, however, remains to be elucidated; although we can speculate about several possibilities. We showed that EGFP-tagged p120 was localized on the centrosomes and also that p120 could interact with -tubulin, a key component of the MT-nucleating machinery in the centrosomes. These observations suggest the possibility that p120 may control MT stabilization via its interaction with -tubulin or the centrosome. It is, in fact, known that some molecules that interact with the centrosome, such as XMAP215, can regulate the plus-end stability of MTs (Kinoshita et al. 2002, 2005). Although we have no evidence that endogenous p120 really localizes at the centrosome in the present cellular system, p120 was reported to localize on the centrosome in a specific developmental stage of Drosophila embryos and in mitotic cells of some mammalian cell lines (Myster et al. 2003; Franz & Ridley 2004). A relative of p120, p0071, was also recently shown to associate with the centrosome (Wolf et al. 2006). On the other hand, the majority of endogenous p120 molecules were distributed diffusely in the cytoplasm. This finding suggests another possibility that p120 may interact not only with centrosomal -tubulin but also with a cytoplasmic pool of -tubulin. This latter interaction might affect MT stability, although the role of cytoplasmic -tubulin in MT dynamics is not well understood. As a third possible mechanism, p120 may stabilize MTs via their interaction with cytoplasmic factors other than -tubulin. In order to settle these questions, a search for other molecules that mediate or participate in the p120-MT interactions will be necessary.
It has been reported that cadherin also can stabilize MTs in centrosome-depleted CHO cells through some yet undefined signaling pathway (Chausovsky et al. 2000); that is, MT stability was enhanced by the formation of cadherin-mediated adhesion. This activity of cadherin was inhibited by the expression of soluble cadherin cytoplasmic domains, indicating that some cadherin-binding molecules were involved. Based on our findings, we propose p120 to be a candidate for such factors, and suggest that the MT-stabilizing activity of p120 might be enhanced through its interaction with cadherins.
Microtubules (MT) vs Rho small GTPases in the action of p120
Several reports indicated that p120 is a positive regulator of Rac1/Cdc42, and a negative regulator of RhoA (Anastasiadis et al. 2000; Noren et al. 2000). The view that p120 functions as a RhoA inhibitor was recently supported by conditional p120 knockout in epidermal keratinocytes (Perez-Moreno et al. 2006), as well as its knockdown in fibroblasts (Wildenberg et al. 2006), although no genetic interaction between Rho1 and p120 was found in Drosophila (Fox et al. 2005). In the present study, we observed only subtle changes in actin stress fibers after p120 depletion, but this may still support the idea of the functional link between p120 and RhoA.
The activities of small GTPases and MT dynamics are known to be interconnected (reviewed in Waterman-Storer & Salmon 1999). MT depolymerization by nocodazole activates Rho (Zhang et al. 1997), and MT regrowth after washout of nocodazole activates Rac1 (Waterman-Storer et al. 1999). These observations lead us to propose a possible scheme in which the primary role of p120 is to stabilize MTs, with the suppression of RhoA occurring as a secondary effect of this p120 action. On the contrary, activities of small GTPases were shown to influence the parameters of dynamic instability of MTs (Grigoriev et al. 2006): RhoA, activated by LPA, can stabilize MT through the pathway involving its effector mDia (Cook et al. 1998; Palazzo et al. 2001). This raises another possibility that p120 firstly suppresses RhoA activity, and in turn regulates MT stability. This simple pathway is, however, unlikely in our cellular system, because p120 did stabilize MTs, opposite to what this scheme would predict. We also experimentally tested whether LPA influenced p120-dependent MT stabilization, and obtained negative results. Thus, at least in the N2a cell system, p120-dependent stabilization of MTs appears to take place independently of RhoA, although leaving the possibility that p120-dependent MT stabilization is upstream of RhoA regulation.
The role of p120 in the control of cell motility
We demonstrated that p120 depletion affected cell motility. This finding conceptually accords with previous results showing that over-expression of p120 enhanced cell motility (Grosheva et al. 2001) and p120 depletion suppressed cellular invasiveness (Yanagisawa et al. 2004). Consistent with the latter observation, we also found that p120 knockdown suppressed the cell migration from the edges of wounded cell sheets. It should be noted that MT stability is known to be essential for directional movement of some cells (Kodama et al. 2003); and we confirmed that nocodazole treatment indeed suppressed cell migration from wound edges. These observations suggest that the p120-dependent MT stability is important for directed migration of cells necessary for wound healing.
Intriguingly, however, both p120-depletion and nocodazole treatment enhanced the motile activity of isolated cells, apparently opposite to the phenomenon observed during wound healing. Time-lapse observations of p120-depleted cells showed that the morphology of their cell peripheries changed more dynamically than that of control cells. Nocodazole-treated N2a cells also exhibited deformed outlines, indicating that MT depolymerization enhances cell motility, at least in the case of N2a cells. Appropriate levels of MT polymerization are presumably necessary for controlling the ordered motility of cell peripheries; and the aberrantly high motility of the cell peripheries, induced by p120 depletion, might have rather prevented the cells from organizing themselves to move in a polarized fashion during wound healing. After all, all of our observations are consistent with the hypothesis that p120 regulates cell migration by modulating MT polymerization.
In Xenopus embryos, the migration of cranial neural crest cells is disturbed by p120 knockdown (Ciesiolka et al. 2004). Also, Drosophila p120 mutants show slower dorsal closure (Fox et al. 2005). These observations are consistent with the concept that p120 controls cell movement. Since p120 normally binds cadherins, the motility-regulating activity of p120 might also operate at cell junctions in the cadherin-positive cells, although this potential mechanism has not been tested yet. Cell–cell adhesion and cell movement do not counteract each other; rather, cells often migrate while maintaining their contacts (reviewed in Solnica-Krezel 2005). Cadherins are even required for some processes of cell movement, for example, for epiboly movement, a tissue expansion that involves cell intercalations and directed cell migrations, in zebrafish embryos (Babb & Marrs 2004; Kane et al. 2005; Shimizu et al. 2005). The cadherin-p120 complex may play a role in coordination of cell–cell contact and cell movement, which is essential for such morphogenetic processes.
In conclusion, we have provided evidence that p120 has the ability to control MT dynamics, and that this ability may be important for cell migration control. To elucidate how the MT-regulating activity of p120 cooperates with the other functions of this protein, such as binding to cadherins and control of Rho GTPases, is an important future issue.
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Experimental procedures |
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The following primary antibodies were used: mouse or rat monoclonal antibodies against p120 (pp120, Transduction), -tubulin (DM1A, SIGMA), -tubulin (GTU88, SIGMA), ß-actin (AC15, SIGMA), paxillin (clone 349, Transduction) and GFP (GF090R, Nakalai), as well as rabbit polyclonal antibodies specific for -tubulin (SIGMA). Secondary antibodies were goat anti-mouse IgG, anti-rabbit IgG, each conjugated to Alexa Fluor (Molecular Probes). F-actin was detected with Alexa Fluor-labeled phalloidin (Molecular Probes).
Expression vectors
pCAsalEGFP and pCAsal6xHis were constructed such that a SalI site was settled immediately upstream of the coding sequence of EGFP (Clontech) or 6xHis, respectively, and that genes subcloned into the SalI site were expressed under the CAG promoter. A silent mutation of the SalI site of the mouse p120 isoform 1N sequence near its 5'-end was created, and the stop codon was replaced by a SalI site using PCR with 88aL4 (gift of Dr Reynolds) as a template to obtain the p120sal sequence. Sequences for N-terminal fragments of p120 were constructed by employing similar strategies. These sequences and an N-terminally truncated mutant (N346; Aono et al. 1999) were inserted into the SalI site of pCA-based vectors to express C-terminally tagged constructs. For the selection of stable transfectants, an internal ribosomal entry site (IRES, the PvuII-NcoI fragment from pCITE-4a(+) [Novagen]) combined with a drug-resistance gene was constructed. NcoI-neo and IRES sequences were inserted into pCAsal6xHis to obtain pCAN. The neo sequence was replaced by a mutant neo sequence (neo*) (Yenofsky et al. 1990) to obtain pCANw. For stably expressing EGFP--tubulin, an EGFP-fused human -tubulin fragment from pEGFP-Tub (Clontech) was inserted into pCANw to obtain pCANw-EGFPTub. For the expression of EB1 fused with C-terminal GFP under the cytomegalovirus (CMV) promoter, EB1/pGFP-NKB (gift of Dr Mimori-Kiyosue) was used.
Cell culture and transfection
For the N2a cells, we isolated a clone, N2a-6, with high affinity for its substratum, for use as the parental line for transfection. Cells were cultured in a 1 : 1 mixture of DMEM and Ham's F12 supplemented with 10% FCS at 37 °C in a 5% CO2 incubator. For transient expression analyses, cells were plated at 1.4 x 104 cells/cm2 on glass coverslips, transfected at day 2 with pCA-based plasmids by using Effectene (QIAGEN), and analyzed at day 1.5 after the transfection. For RNAi experiments, cells were transfected with 40 pmol of siRNA per well (containing 1 mL medium and about 3.5 x 105 cells) by using Lipofectamine 2000 (Invitrogen) and cultured for at least 3 days before starting the assays. The following siRNAs were obtained from Invitrogen: p120–1, 5'-AAACGAUGCAGGUCCACACUGCUUC-3', p120–2, 5'-UACAGAAGGUGGUUGUGAUCCUGGG-3' and Stealth RNAi Negative Control Medium GC (48% GC content) as a control (Stealth siRNA, Invitrogen). For live-cell imaging of cells in low-density cultures, the cells were treated with siRNA as above, reseeded on the following day at 1.7 x 103 cells/cm2, and examined at day 3 after the transfection. Prior to the imaging, the cell culture medium was replaced with Leibovitz L15 medium supplemented with 10% FCS. For visualization of MTs in living cells, we prepared a non-cloned population of N2a-6 cells stably expressing EGFP--tubulin; the cells were siRNA-treated and reseeded as above, and the medium was replaced 4 h before starting the recording. For the observation of EB1-GFP dynamics, siRNA-treated cells were further transfected with the EB1-GFP expression vector on the following day, reseeded at 6.9 x 103 cells/cm2 after the 4 h of incubation, and examined as in the case of EGFP--tubulin. For the wound healing assay, confluent cell layers at day 3 after siRNA transfection were scraped with pipette tips and rinsed with prewarmed medium (DH10, or L15 + 10% FCS for live-cell imaging).
Immunofluorescence techniques
The conditions for cell fixation were optimized for the preservation of MTs. Immediately after cells had been taken from the CO2 incubator, a 20% paraformaldehyde stock solution (pH 7.0), stored at –20 °C and prewarmed to 37 °C just before use, was added directly to the medium for a final concentration of 4%. The cells were left on a heat block at 37 °C or at room temperature for 20 min. After fixation, the samples were quenched and washed with 50 mM NH4Cl in PBS. The fixed cells were permeabilized with PBST (0.1% Triton X-100 in PBS) for 10 min, and then incubated in a blocking buffer (20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl, 2% BSA, 0.1% Triton X-100, 0.1% NaN3) for 15 min. The cells were next incubated with the desired primary antibody for 60 min, washed with PBS, and then incubated with a secondary antibody with or without Alexa Fluor-phalloidin for 30 min. After having been washed with PBS and rinsed in H2O, the coverslips were mounted on slides with FluorSave (Calbiochem).
Microscopy
Laser-scanning confocal microscopy was performed by using an LSM510 mounted on an Axiovert 100 M microscope equipped with a Plan-Apochromat 63x/1.40 objective (Carl Zeiss). Adobe Photoshop was used to linearly adjust the brightness of the digital images. In a room maintained at 37 °C, time-lapse differential interference contrast or fluorescent images were taken with a DeltaVision system (Applied Precision) mounted on an IX71 inverted microscope equipped with 10x/0.40 D Plan Apo UV and 100x/1.40 Plan Apo IX70 objectives (Olympus) and CoolSNAP HQ cooled CCD camera (Photometrics). Fluorescent time-lapse images were deconvolved by using Deltavision's decon3d with the default settings.
Image analysis
Cell motion was traced by MetaMorph (Molecular Devices), using its ‘Track Objects’ or ‘Track Points’ functionalities. The movement of EB1 comets was quantified from EB1-GFP image sequences obtained at 1-s intervals. Raw data were preprocessed with MetaMorph's ‘Flatten Background’ to subtract background signals, and the positions the comets were manually tracked using MetaMorph's ‘Track Points’ functionality. EB1-GFP comets that existed within 2/3-radius of the cells on the first frame were tracked. The data on positions and the elapsed times were further processed by Microsoft Excel and home-made software. Estimations of dynamic instability parameters of MTs from the EB1-GFP tracks were made as follows: The growth rate was calculated as the mean instantaneous speed, which was obtained by dividing the sum of displacements of a comet by its growth duration until catastrophe. Although the catastrophe frequency is normally calculated as the inverse number of the mean interval between two catastrophe events (Shelden & Wadsworth 1993), the obtained data were durations from the time 0 of the image sequences to the first catastrophe. As the time 0 was an arbitrary time point in each growth history, the real durations of the histories can be estimated as the values 2 times longer than the durations from the time 0 s to the end of the histories on average. Thus, the catastrophe frequency was calculated as the inverse number of 2x the mean growth duration until catastrophe. The pause events were counted when the distance of a comet position from its initial position did not change more than 1 pixel length (= 0.066 µm) in successive frames longer than 2 s. The pause frequency was calculated dividing the number of pause events of a comet by its growth duration until catastrophe. All the statistical analyses were done by using R (The R Foundation).
GST pull-down assay
For GST pull-down, GST-fused N-terminal fragments of p120 were expressed in E. coli strain DH5 by using the pGEX-6P-1 vector and purified with glutathione-Sepharose 4B beads according to the manufacturer's protocol (Amersham). N2a cells were lysed in pull-down lysis buffer (20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM MgCl2, 1 mM EGTA) and centrifuged at 20 000 g for 2 x 15 min at 4 °C. The supernatant was precleared with glutathione-Sepharose 4B beads, and aliquots containing 250-µg protein were incubated for 1 h at 4 °C with those containing 25 µg of the GST-fused protein immobilized on beads. The beads were washed 3 times with the pull-down lysis buffer and then analyzed by Western blotting. For preparation of whole-cell lysates, cells were lysed in a 20 µL/cm2 solution of whole-cell lysis buffer (10 mM Tris–HCl, pH 7.5, containing 2 mM EDTA, 1% SDS), shaken vigorously, and boiled for 15 min. The protein concentration of the lysate was determined by use of the BCA assay (PIERCE).
MT nocodazole-sensitivity assay
For tubulin fractionation assays, confluent cells at day 3 after siRNA transfection were treated with nocodazole for 2.5 h, and then fractioned essentially as previously described (Grindstaff et al. 1998). Cells (about 1.2 x 107) were incubated at 37 °C for 20 min in 360 µL of a MT-stabilizing buffer (0.1 M PIPES, pH 6.8, containing 2 M glycerol 1 mM MgSO4, 1 mM EDTA) supplemented with 0.1% Triton X-100. The cells were then freed with a cell scraper, and the free cells in the buffer were centrifuged at 20 000 g for 10 min at 30 °C. The supernatant was sampled, and the pellet was lysed in 360 µL of the cell-lysis buffer. An equivalent volume of each fraction was subjected to Western blotting. The band intensities of the blots were measured by Scion Image (Scion Corporation): The blots were digitalized by scanning the X-ray films, and the images were background-subtracted by using the 2D rolling ball algorithm; and the intensities of the bands were calculated by summing up the intensity values of pixels forming each band. For visual analysis of nocodazole-resistant MTs in the pellet, cells just before the centrifugation were fixed in 20% PFA, and immunostained with mAb DM1A. For the assay of LPA-stimulated cells, cells were incubated in serum-free L15 medium for 2 h, then incubated with the same L15 supplemented with 10 µM LPA for 30 min, and finally incubated in the presence of both 10 µM LPA and nocodazole for 2.5 h. Thereafter they were used for tubulin fractionation.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: takeichi{at}cdb.riken.jp
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References |
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Accepted: 22 March 2007
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