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βIII-Tubulin is required for interphase microtubule dynamics in untransformed human mammary epithelial cells
European Journal of Cell Biology 90 (2011) 872–878
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Short communication
III-Tubulin is required for interphase microtubule dynamics in untransformed human mammary epithelial cells Benjamin Pierre Bouchet a,b,c,∗ , Alain Puisieux a,b,c,1 , Carlos María Galmarini d a
Université de Lyon, F-69000 Lyon, France Université Claude Bernard Lyon 1, F-69000 Lyon, France INSERM U590, Centre LEON BERARD, F-69008 Lyon, France d Cell Biology Department, PharmaMar, S.A., Colmenar Viejo, Madrid E-28770, Spain b c
a r t i c l e
i n f o
Article history: Received 4 February 2011 Received in revised form 28 April 2011 Accepted 3 May 2011 Keywords: Microtubule III-Tubulin Tubulin isotype Mammary epithelial cells Breast cancer
a b s t r a c t Numerous works have questioned the pertinence of using II- and/or III-tubulin expression as markers of prognosis and/or prediction of breast cancer response to chemotherapy containing microtubuletargeting agents. The rationale of such studies was essentially based on microtubule dynamics analysis using purified tubulin in vitro and cancer cell lines. Nonetheless, the significance of II- and III-tubulin expression in the control of microtubule dynamics in normal mammary epithelium has never been addressed. Here we investigate the expression and the consequences of II- and/or III-tubulin depletion in interphase microtubule dynamics in non-tumor human mammary epithelial cells. We find that both isoforms contribute to the tubulin isotype composition in primary and immortalized human mammary epithelial cells. Moreover, while II-tubulin depletion has limited effects on interphase microtubule behavior, III-tubulin depletion causes a strong exclusion of microtubules from lamella and a severe suppression of dynamic instability. These results demonstrate that, while II-tubulin is dispensable, IIItubulin is required for interphase microtubule dynamics in untransformed mammary epithelial cells. This strongly suggests that III-tubulin is an essential regulator of interphase microtubule functions in normal breast epithelium cells. © 2011 Elsevier GmbH. All rights reserved.
Introduction Microtubules are cytoskeletal filaments assembled from protofilament sheets resulting from the polymerization of ␣tubulin heterodimers (Desai and Mitchison, 1997). These hollow tubes are polarized fibers with the ␣-tubulin subunits exposed at the minus-end which is generally stable and anchored to the nucleation site such as the centrosome, and -tubulin subunits exposed at the plus-end that is highly dynamic. Heterodimer exchanges at the plus-ends characterize the phenomenon called dynamic instability and defined by rapid switches between periods of polymerization and depolymerization (Mitchison and Kirschner, 1984). Transition from growth or pause to shortening is called catastrophe and transition from shortening to growth or pause is called rescue (Jordan and Wilson, 2004). Dynamic microtubules are required for the proper dynamics of other crucial cell components including the mitotic chromosomes, the Golgi apparatus, and focal and
∗ Corresponding author at: Division of Cell Biology, Faculty of Science, Utrecht University, Utrecht, NL-3584 CH, The Netherlands. Tel.: +31 0 30 253 4585; fax: +31 0 30 253 2837. E-mail address: [email protected] (B.P. Bouchet). 1 Current address: INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69000 Lyon, France. 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.05.005
cell–cell adhesion structures (Akhmanova et al., 2009; Dumont and Mitchison, 2009; Miller et al., 2009). Several types of factors are involved in the regulation of microtubule dynamics. Besides microtubule-associated proteins and post-translational modifications of tubulins, tubulin isotype composition was also proposed as a regulatory mechanism for microtubule dynamics, though its role is not completely understood (Etienne-Manneville, 2010; Lyle et al., 2009a,b; Orr et al., 2003; Panda et al., 1994; Westermann and Weber, 2003). The amino acid differences in the C-terminal region of the protein serve to distinguish seven -tubulin isotypes: I, II, III, IVa, IVb, V and VI (Verdier-Pinard et al., 2005). Different works have showed that modulation of the expression of the different -tubulin isotypes can cause abnormalities in cell growth, axoneme assembly and platelet function (Bhattacharya and Cabral, 2004; Hoyle and Raff, 1990; Schwer et al., 2001). Early works using purified tubulin showed that -tubulin isotypes can differentially modulate microtubule dynamics in in vitro models (Banerjee et al., 1990, 1992; Panda et al., 1994). For example, the microtubule dynamics suppressor paclitaxel, frequently used in cancer chemotherapy, was shown to be less effective in microtubules assembled from purified II-, III- and IV-tubulin-containing dimers than in those assembled from unfractionated tubulin (Derry et al., 1997). Moreover, multiple
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studies revealed that III-tubulin overexpression is a feature of various human malignancies and notably epithelial tumors (Kavallaris, 2010; Leandro-Garcia et al., 2010; Seve and Dumontet, 2008). More specifically, III-tubulin overexpression was defined as a marker of poor prognosis and chemoresistance in tumor cells (Galmarini et al., 2008; Goncalves et al., 2001; Hasegawa et al., 2003; Kavallaris et al., 1997; Paradiso et al., 2005; Tommasi et al., 2007). This isotype was also described as a promoter of microtubule dynamics (Banerjee et al., 1992; Panda et al., 1994). Yet, III-tubulin overexpression, while inducing paclitaxel resistance, did not prove to be sufficient by itself to alter the dynamic instability parameters in untreated cells (Kamath et al., 2005). A recent study also showed that III-tubulin depletion sensitizes to microtubule dynamics suppression by low-dose tubulin-binding agents but does not affect microtubule dynamics in untreated human non-small cell lung cancer (NSCLC) cells H460 (Gan et al., 2010). In addition, though its role in normal tissues has been poorly investigated, III-tubulin functional inactivation due to genetic mutation in a congenital syndrome was reported to correlate with axon guidance defects and decreased overall microtubule dynamics (Tischfield et al., 2010). Finally, similarly to III-tubulin, II-tubulin overexpression was observed in several tumor types, including NSCLC and breast cancers, and associated with poor taxane response both in cancer tissues and cell lines (Bernard-Marty et al., 2002; Cucchiarelli et al., 2008; Galmarini et al., 2008; Gan and Kavallaris, 2008; Haber et al., 1995). It is striking to notice that, although altered expression of II- and III-tubulin was shown to be associated with mammary tumorigenesis, the significance of the expression of these tubulin isotypes in normal breast epithelium is still unknown. Nonetheless, various immunostaining-based studies have clearly shown that II- and III-tubulin are expressed in non-neoplastic mammary epithelium (Dozier et al., 2003; Galmarini et al., 2008; Leandro-Garcia et al., 2010). Hence, to gain insight into the role of II- and III-tubulin in microtubule dynamics regulation in non-tumor epithelial cells, we have first investigated here their expression in both primary and immortalized non-tumor mammary epithelial cells. We have next analyzed the consequences of their individual and combined depletion in microtubule dynamics in interphase cells. Our results demonstrate that III-, but not II-tubulin, is required for interphase microtubule dynamics in non-tumor human mammary epithelial cells. Materials and methods Plasmids and siRNA The PAcGFP1-Tubulin vector coding for GFP-␣-tubulin was purchased from Clontech. A pool of four independent siRNAs (siGENOME SMARTpool, Dharmacon) targeting TUBB2A (M008260-03-0010), TUBB2B (M-017790-00-0010) and TUBB3 mRNA (M-020099-03-0010) was used to transitorily inhibit respectively II- (siII) and/or III-tubulin (siIII) expression. Individual siRNA sequences were verified for their identity (nucleotide blast, NCBI); two targeting sequences from TUBB2A SMARTpool were found to target TUBB2B and no common sequences were found by comparing TUBB2A/B and TUBB3 SMARTpools. A pool of four independent nontargeting siRNAs (siCONTROL Non-Targeting siRNA Pool #2, siCT, Dharmacon) was used as control in siRNA-mediated knockdown experiments. Cell culture and transfection Primary human mammary epithelial cells (HMECs, used at population doubling 7–14) were obtained from Lonza. Low passage
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immortalized human mammary epithelial cells (hTMECs, hTERTtransduced HMECs) were provided by R.A. Weinberg (Whitehead Institute, Cambridge, USA). Primary HMECs and hTMEC-derived cell lines were cultured in MEBM basal medium (Lonza) supplemented with 10 ng/ml human EGF, 5 g/ml insulin, 0.5 g/ml hydrocortisone, 0.4% bovine pituitary extract, 50 g/ml gentamicin and 50 g/ml amphotericin-B. Plasmid transfection in hTMECs was performed using FuGENE HD (Roche). Stably pAcGFP1-Tubulin-transfected hTMECs (GThMECs) were selected under 100 g/ml geneticin. Subclones were selected according to their optimal fluorescently tagged protein expression and localization, normal 2D mammary epithelial cell morphology and growth. Transfection of 10–30 nM siRNAs was performed in hTMECderived cell lines using Lipofectamine RNAiMAX (Invitrogen). In these conditions, transfectivity was estimated to be 100% by using fluorescent control siRNA (siGLO RISC-Free Control siRNA, Dharmacon; DY-547-conjugated, absorbance: 557, emission max: 570 nm) in all hTMEC-derived cell lines used in transient siRNA-mediated knockdown experiments (Supplementary Fig. S1). Antibodies and reagents Mouse monoclonal anti-II-tubulin (7B9) antibody was purchased from Novus and Sigma–Aldrich. Mouse monoclonal antibodies against I- (SAP.4G5), III- (SDL.3D10) and IV-tubulin (ONS.1A6), total -tubulin (TUB 2.1), total ␣-tubulin (B-5-1-2) and acetylated tubulin (6-11B-1) were purchased from Sigma–Aldrich. Rat monoclonal anti-␣-tubulin (YL1/2) and mouse monoclonal anti-III-tubulin (TUJ1) antibodies used in immunofluorescence experiments were respectively provided by Abcam and Covance Inc. Secondary Alexa Fluor 488 goat anti-rat IgG (H+L) and Alexa Fluor 594 F(ab )2 fragment of goat anti-mouse IgG (H+L) antibodies were supplied by Invitrogen. Vectashield mounting medium was purchased from Vector Laboratories. Trypsin neutralizing solution was obtained from Lonza. Oxyrase was purchased from Oxyrase Inc. Immunofluorescent staining, microscopy Double immunostaining of total ␣-tubulin and II or IIItubulin was performed on cells grown on a glass coverslip, fixed in 100% methanol at −20 ◦ C, permeabilized in 0.15% Triton X-100 1× PBS and blocked in 1% BSA/0.05% Tween 20 1× PBS (blocking buffer). Cells were successively incubated at room temperature with 1:200 rat anti-␣-tubulin, 1:300 Alexa Fluor 488 goat anti-rat, 1:250 mouse anti-II- or III-tubulin and 1:300 Alexa Fluor 594 F(ab )2 fragment of goat anti-mouse in blocking buffer. Ethanol dehydrated coverslips were mounted in Vectashield and sealed with nail polish. Fluorescence microscopy imaging was performed with a 100×/1.3 NA PL Fluotar objective on a DMRBE microscope (Leica) coupled to a C4880 CCD camera (Hamamatsu) automated in Wasabi! software (Hamamatsu). Live cell imaging and analysis Live cells were visualized on a 37 ◦ C heated Zeiss Axiovert 100M microscope automated by MetaMorph 7.5.6.0 software and equipped with a cooled CCD camera (CoolSNAP HQ monochrome; Photometrics). Experiments were performed on cells plated in glass-bottom dishes (MatTek) maintained on a 37 ◦ C heated stage and in a 5% CO2 and humidified atmosphere. Fluorescent microtubule imaging was performed with a 100×/1.4 NA Plan-Apochromat or a 63×/1.4 NA PlanApochromat objective on GT-hMECs cultured in phenol-free
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MEGM containing 5–10 l/ml oxyrase. Images were acquired from optimal focal planes in lamella of interphase cells, at 3.5-s intervals, with a 350-ms exposure and during a minimum of 2.5 min. Quantification of the shortest distance between the microtubule tip and the cell edge in distal lamella was manually performed on randomly chosen microtubule ends in a zone comprised between the cell edge and an internal limit approximately located at 8 m from the edge. Using this method, 14 microtubule tips were tracked in each analyzed cell. Quantification of the microtubule density in distal lamella was performed using iterative measurement of the microtubule lattice contained in a region of interest corresponding to a 5 m-diameter circle (∼19.6 m2 ) adjacent to the cell edge. Each measurement consisted in the calculation of the mean microtubule track (m) per m2 of distal lamella. A minimum of 5 lamella measurements per cell was used. For statistical analysis, measurements for each condition were pooled. Microtubule dynamic instability parameters were extracted by manually tracking microtubule plus ends in GFP time-lapse sequences using ImageJ 1.42q software (http://rsb.info.nih.gov/ij/) and MTrackJ plugin (Erik Meijering, Erasmus Medical Center Rotterdam, NLD; http://www.imagescience.org/meijering/software/mtrackj/). For analysis and presentation, image sequences were inverted, processed as stacks and submitted to bleach correction, FFT bandpass filter and contrast enhancing. The distance between a plus-end and a fixed origin on the microtubule body was used to approximate microtubule length changes and velocity between each time point. Only microtubule length changes ≥0.4 m between two consecutive time points were considered as growth or shortening events, while changes <0.4 m were considered as the pause event (based on the measurement of the mean error in 3 independent manual trackings of the same 90-frame sequence; superior to optical resolution for GFP imaging with this setup, 0.24 m/frame). Growth, shortening and pause phases were respectively identified as unique or consecutive growth, shortening and pause events. Mean growth and shortening rates were determined using a custom-written Excel macro (Microsoft) averaging microtubule velocity in each corresponding phase. The total microtubule length change for each phase was calculated as the product of mean growth or shortening rate by total phase duration. Catastrophes (transitions from growth or pause to shortening) and rescues (transitions from shortening to pause or growth) were counted by a custom-written Excel macro. Catastrophe frequencies were calculated by dividing the number of catastrophes by the sum of the time spent in growth and pause. Similarly, rescue frequencies were calculated by dividing the number of rescues by the time spent in shortening. Microtubule dynamicity was measured as the total microtubule length grown and shortened relative to the microtubule life span. Mean dynamicity was calculated for each condition as the average of microtubule dynamicities. For each condition, we analyzed a minimum of 70 microtubules in a minimum of 14 cells.
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Results and discussion ˇII- and ˇIII-tubulin are expressed in primary and hTERT-immortalized human mammary epithelial cells In order to address the eventual role of II- and III-tubulin in microtubule regulation in non-tumor breast epithelium, we have first analyzed the expression of both isotypes in primary and hTERT-immortalized untransformed human mammary epithelial cells (HMECs and hTMECs; Fig. 1A). These cells predominantly retain an euploid karyotype and a stable genetic background similar to that observed in normal human cells and therefore provide a relevant model to study mammary epithelium properties and functions (Romanov et al., 2001; Troester et al., 2004; Wang et al., 2000). We have also investigated the expression of I- and total IV-tubulin in the same cells. I-tubulin was described as a major constitutive isotype (Cleveland, 1987; Leandro-Garcia et al., 2010). IV-tubulin exists under two isoforms: IVa-tubulin, initially described as a brain-specific form and IVb-tubulin, constitutively expressed in human tissues (Cleveland, 1987; Leandro-Garcia et al., 2010). Since the anti-IV-tubulin antibody that we used here does not discriminate between both isoforms, we assume that we detected the IVb form in mammary epithelial cells; however we cannot exclude that the IVa form could be expressed in these cells as well. Western blot analysis on total cell lysates showed that II- and III-tubulin are expressed in both HMECs and hTMECs, indicating that these -tubulin isotypes significantly contribute to the tubulin isotype composition in non-tumor mammary epithelial cells (Fig. 1A). This result was confirmed by immunofluorescent staining of II- and III-tubulin in fixed hTMECs (Fig. 1B). This is consistent with previous data showing that II- and III-tubulin are expressed in normal mammary epithelial tissues (Dozier et al., 2003; Galmarini et al., 2008; Leandro-Garcia et al., 2010). Additionally, we found that I- and IV-tubulin also significantly contribute to the tubulin isotype composition in mammary epithelial cells (Fig. 1A). Besides, it is interesting to note that this composition is almost identical in HMECs and hTMECs, which strengthens the relevance of hTERT-immortalized cells as a model for primary mammary epithelial cells (Fig. 1A). Lastly, acetylation of ␣-tubulin, which is a post-translational modification associated with stable microtubules, was found in similar levels in HMECs and hTMECs (Fig. 1A; Westermann and Weber, 2003). This data suggests that both lines harbor equivalent stable microtubule population and reinforces the relevance of hTMECs to model microtubule dynamics in normal mammary epithelium. ˇIII- but not ˇII-tubulin inactivation causes suppression of interphase microtubule dynamics in untransformed human mammary epithelial cells In order to specify the involvement of II- and III-tubulin in microtubule regulation in normal breast epithelium, we have next
Fig. 1. Expression of II- and III-tubulin and consequences of their depletion in lamella microtubule organization in non-tumor human mammary epithelial cells. (A) Western blot analysis of I–IV tubulin isotypes, total -tubulin (-tot), acetylated (Ac-tub) and total ␣-tubulin (␣-tot) in whole lysates from HMEC and hTMEC cells. (B) Immunofluorescent labeling of total ␣-tubulin and II- or III-tubulin in hTMEC cells. Gray frame, zoomed region shown on the right panel. Main panel, bar, 10 m; zoom panel, bar, 3 m. (C) Western blot analysis of II, III, I, and IV tubulin isotypes, total - (-tot) and acetylated tubulin (Ac-tub) in whole lysates from hTMEC cells after a 72 h transfection with control siRNA pool (siCT), II (siII), III (siIII) or combined II and III tubulin-targeting siRNA pool (see Materials and methods). (D) GFP imaging in live GT-hMECs treated as in C; gray frame, zoomed region shown on the right panel; yellow dotted line, cell edge; bar, 5 m. (E) Quantification of the shortest distance between the microtubule (MT) tip and the cell edge (m) in live GT-hMECs treated as in C (see Materials and methods). Box plots indicate the 25th percentile (bottom boundary), median (middle line), mean (red open circle), 75th percentile (top boundary), nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and outliers (black cross). For each condition, n = 196 measures in 14 cells. Kruskal–Wallis analysis, P < 0.001; pairwise multiple comparison by Dunn’s method. Gray dotted line figures mean in siCT condition. (F) Quantification of the microtubule (MT) density in distal lamella in live GT-hMECs treated as in C (see Materials and methods). Box plots built as in (E); siCT, n = 53, 5 cells; siII, n = 51, 4 cells; siIII, n = 53, 5 cells, siII + III, n = 54, 6 cells. Kruskal–Wallis analysis, P < 0.001; pairwise multiple comparison by Dunn’s method. Gray dotted line figures mean in siCT condition. Molecular mass (kDa). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Effects of II and/or III tubulin depletion on microtubule dynamic instability in interphase non-tumor human mammary epithelial cells. (A–F) Dynamic instability parameters measured in GT-hMECs, after 72 h transfection with control siRNA pool (siCT, 70 microtubules, 14 cells), II- (siII, 70 microtubules, 20 cells), III- (siIII, 70 microtubules, 18 cells) or combined II- and III- tubulin-targeting siRNA pool (70 microtubules, 14 cells). Box plots built as in Fig. 1E. Kruskal–Wallis analysis, P < 0.001; pairwise multiple comparison by Dunn’s method. Gray dotted line in all panels, except in (E), figures mean in siCT condition. Error bar in time percentage graph, 95% confidence interval.
investigated the consequences of II- and/or III-tubulin depletion in microtubule dynamics in non-tumor mammary epithelial cells. We only used here hTERT-immortalized cells, because HMECs in culture have a limited proliferative potential due to replicative senescence that prevents them from bypassing 20 population doublings without acquiring further genetic alterations (Foster and Galloway, 1996; Kiyono et al., 1998; Romanov et al., 2001). At least two hTMEC-derived clones stably expressing GFP-␣-tubulin (GThMECs) and allowing live imaging of microtubule dynamics were thus transiently knocked down for II- and/or III-tubulin, after transfection of respectively TUBB2A/B- and/or TUBB3-targeting siRNAs (Fig. 1B; Rusan et al., 2001). These treatments allowed almost complete suppression of II- and/or III-tubulin expression (Fig. 1C). Of note, I-, IV- and total -tubulin levels seemed to be unaltered by II- and/or III-tubulin silencing (Fig. 1C). Similarly, II- and III-tubulin expressions appeared to be unmodified by the silencing of each other (Fig. 1C). This is in agreement with studies also showing unaltered expression of -tubulin isotypes and total -tubulin when II- or III-tubulin genes are silenced (Gan et al., 2007; Gan and Kavallaris, 2008). Though we noticed an apparent reduced proliferation in III-tubulin-depleted cells, we did not observe any significant mitotic arrest in II- and/or III-tubulin-depleted cells. Moreover, our previous works have indicated that the mitotic checkpoint in hTMECs is very sensitive to modified microtubule dynamics since those cells undergo mitotic arrest when treated with a low dose of the microtubule stabilizer paclitaxel (Bouchet et al., 2007). This suggests that IIand/or III-tubulin silencing could only have a limited effect on mitotic microtubule dynamics. However, the specific mitotic role of II- and III-tubulin need to be further investigated. Live fluorescence observation in GT-hMECs showed that microtubules in control interphase cells organize into a sparse network and stably invade lamella (Fig. 1D, supplementary Movie 1). Furthermore, microtubules grow toward the cell edge and frequently undergo bending and reorientation when they reach the
most distal part of the lamella so that they grow parallel to the edge (Fig. 1D, supplementary Movie 1). This phenomenon was previously described in epithelial cells and shown to involve the actinomyosin-based retrograde flow (Waterman-Storer and Salmon, 1997). Microtubules in II-tubulin-depleted cells showed similar behavior than in control cells (Fig. 1D and supplementary Movie 1). In contrast, microtubules in III-tubulin-depleted cells exhibited an aberrant behavior consisting in a tight network organization within proximal lamella and a strong exclusion from distal lamella (Fig. 1D, supplementary Movie 1). This behavior was characterized by the significant increase of the distance between microtubule tips and the cell edge in both III- and II-/IIItubulin-depleted cells (Fig. 1E, P < 0.05, Dunn’s post hoc test). IIIand II-/III-tubulin-depleted cells also showed a strong reduction of the microtubule density in distal lamella (Fig. 1F, P < 0.05, Dunn’s post hoc test). Of note, the mean microtubule tip-cell edge distance and density in distal lamella was not significantly affected by the depletion of II-tubulin alone (Fig. 1E and F). Moreover, IItubulin depletion did not significantly modify these features when combined with III-tubulin depletion (Fig. 1E and F). Next, microtubule dynamics was analyzed in detail (Fig. 2A–F). Based on our preliminary data indicating that transfectivity is 100% in our siRNA experiments (see Materials and methods), we chose a minimum of 14-cell/70-microtubule sampling to extract microtubule dynamic instability parameters. Of note, microtubule dynamics showed similar features to what we observed in a previous study (Bouchet et al., 2011). As suggested by the results described above (Fig. 1D–F), interphase microtubule dynamics showed only limited changes in II-tubulin-depleted cells when compared to control cells, including a reduced shortening rate (−8.1%, P < 0.05, Dunn’s post hoc test) with no significant impact on mean dynamicity (Fig. 1B and F). In contrast, interphase microtubule dynamics in III-tubulin-depleted cells was highly modified when compared with control cells (Fig. 2A–F). Alterations comprised a decreased growth rate (−9.0%, P < 0.05, Dunn’s post hoc
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test), a decreased shortening rate (−31.5%, P < 0.05, Dunn’s post hoc test), a decreased catastrophe frequency (−37.6%, P < 0.05, Dunn’s post hoc test) and an increased rescue frequency (+35.5%, P < 0.05, Dunn’s post hoc test). Moreover, in III-tubulin-depleted cells, the time spent by microtubules in pause was strongly increased when compared with control cells (+33.3%, P < 0.05, Dunn’s post hoc test). Finally, the mean microtubule dynamicity was dramatically decreased in III-tubulin-depleted cells compared to control cells (−70.8%, P < 0.05, Dunn’s post hoc test). Noticeably, in cells depleted of both II- and III-tubulin, none of the dynamic instability parameters were found to be significantly different from those observed in cells harboring only III-tubulin depletion (Fig. 2A–F, Dunn’s post hoc test). This result confirms our previous observation of the microtubule behavior in double knocked down cells (Fig. 1D–F). This also demonstrates that II-tubulin silencing neither potentiates nor rescues the suppression of microtubule dynamics caused by III-tubulin loss. Together with individual knockdown experiment results, these data indicate that II-tubulin has no major influence on interphase microtubule dynamics modulation in normal mammary epithelial cells. It is remarkable that, despite the decrease of overall microtubule dynamics, the ␣-tubulin acetylation, which is generally associated with microtubule stability, was not increased in IIItubulin-depleted cells (Fig. 1B; Westermann and Weber, 2003). This could indicate that III-tubulin inactivation affects only the population of dynamic microtubules without modifying the proportion of stable acetylated microtubules (Bulinski and Gundersen, 1991). As well, microtubule populations could differ in their tubulin isotype composition and stable microtubules assembly may involve less III-tubulin than dynamic microtubules, which would explain their unaltered state when III-tubulin is depleted. Alternatively, it is possible that microtubules could undergo plus-end dynamics alterations that do not affect acetylation in their stable lattice part. The link between tubulin isotype composition and accumulation of post-translational modifications is still poorly understood and should thus be specified in future studies. We found here that III-tubulin inactivation causes interphase microtubule dynamics suppression in untransformed human mammary epithelial cells. This result is in accordance with earlier studies showing that III-tubulin promotes microtubule dynamics (Banerjee et al., 1992; Panda et al., 1994). Moreover, our data are consistent with the recent description of a congenital syndrome that associates III-tubulin functional inactivation with severe defects of axon guidance, oculomotor nerve hypoplasia and suppression of microtubule dynamics (Tischfield et al., 2010). In contrast, our study does not confirm the observations previously made in the lung adenocarcinoma cell line H460 and describing an unaffected interphase microtubule dynamics consecutive to IIItubulin silencing (Gan et al., 2010). We assume that this discrepancy arises from the radical difference between cell models used in both studies. This actually implies that tissue-specificity and genetic background must be taken into account when determining microtubule dynamics properties in different non-tumor and tumor cell line models. Of note, the functional cause of microtubule dynamics suppression due to III-tubulin inactivation in non-tumor mammary epithelial cells is still unclear. Tischfield et al. (2010) recently showed that III-tubulin mutations causing microtubule dynamics suppression also inhibit microtubule plus-end binding of depolymerizing kinesin-8 family members such as Kip3p in yeast. It is thus possible that inactivation of the binding of depolymerizing kinesins and/or other microtubule plus-end tracking proteins could contribute to the suppression of microtubule dynamics in III-tubulin-depleted mammary epithelial cells. In the same study, III-tubulin mutation was also shown to correlate with suppressed microtubule binding of KIF21A which belongs to the family of
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plus-end-directed motor proteins (Marszalek et al., 1999; Tischfield et al., 2010). This suggests that III-tubulin inactivation could also affect kinesin-based transport toward the cell periphery. In the present study, we show that the cell periphery exhibits a severe loss of microtubules when III-tubulin is inactivated (Fig. 1C and supplementary Movie 1). Thus, the dynamics of key components of lamella such as actin and adhesion structures could be modified by defective plus-end-directed transport. Because these structures exhibit various mechanisms of cross-regulation with microtubules, their alteration could in turn contribute to the suppression of lamella microtubule dynamics in III-tubulin-depleted cells. Interestingly, numerous works have shown that dynamic microtubules regulate cell motility, notably through their ability to tune Rho GTPases activity, actin dynamics and focal adhesion turnover (Broussard et al., 2008; Palazzo and Gundersen, 2002; Rodriguez et al., 2003). Because III-tubulin depletion strongly suppresses lamella microtubule dynamics and according to preliminary observations (data not shown), we strongly suspect that III-tubulin is required for motility in untransformed human mammary epithelial cells. For these reasons and because the link between tubulin isotype composition and cell motility is poorly understood, the eventual role of III-tubulin in regulating cell motility should be addressed in future works. In conclusion, we show here that, while II-tubulin is dispensable, III-tubulin is required for interphase microtubule dynamics in human mammary epithelial cells. These results reveal that III-tubulin is a new candidate for physiological regulation of interphase microtubule dynamics in mammary epithelium. This could have major implications in understanding how III-tubulin specific functions participate in tissue homeostasis and how these functions are eventually altered during epithelial tumorigenesis, notably in human breast. Moreover, our data could also contribute to refine the investigation and the analysis of -tubulin isotype expression for breast cancer management. Acknowledgements We thank Prof. Anna Akhmanova (Division of Cell Biology, Faculty of Science, Utrecht University, Utrecht, NDL) for critical comments and support for final experiments. We also thank PLATIM (PLAteau Technique Imagerie/Microscopie, Ecole Normale Supérieure, Lyon, FRA) for microscopy support. This work was supported by FRM (Fondation pour la Recherche Médicale, FRA) and PharmaMar, S.A. (Colmenar Viejo (Madrid), ESP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejcb.2011.05.005. References Akhmanova, A., Stehbens, S.J., Yap, A.S., 2009. Touch, grasp, deliver and control: functional cross-talk between microtubules and cell adhesions. Traffic 10, 268–274. Banerjee, A., Roach, M.C., Trcka, P., Luduena, R.F., 1990. Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin. J. Biol. Chem. 265, 1794–1799. Banerjee, A., Roach, M.C., Trcka, P., Luduena, R.F., 1992. Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain. J. Biol. Chem. 267, 5625–5630. Bernard-Marty, C., Treilleux, I., Dumontet, C., Cardoso, F., Fellous, A., Gancberg, D., Bissery, M.C., Paesmans, M., Larsimont, D., Piccart, M.J., Di, L.A., 2002. Microtubule-associated parameters as predictive markers of docetaxel activity in advanced breast cancer patients: results of a pilot study. Clin. Breast Cancer 3, 341–345. Bhattacharya, R., Cabral, F., 2004. A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation. Mol. Biol. Cell 15, 3123–3131. Bouchet, B.P., Bertholon, J., Falette, N., Audoynaud, C., Lamblot, C., Puisieux, A., Galmarini, C.M., 2007. Paclitaxel resistance in untransformed human mammary
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epithelial cells is associated with an aneuploidy-prone phenotype. Br. J. Cancer 97, 1218–1224. Bouchet, B.P., Fauvet, F., Grelier, G., Galmarini, C.M., Puisieux, A., 2011. p21Cip1 regulates cell-substrate adhesion and interphase microtubule dynamics in untransformed human mammary epithelial cells. Eur. J. Cell Biol. 90, 631–641. Broussard, J.A., Webb, D.J., Kaverina, I., 2008. Asymmetric focal adhesion disassembly in motile cells. Curr. Opin. Cell Biol. 20, 85–90. Bulinski, J.C., Gundersen, G.G., 1991. Stabilization of post-translational modification of microtubules during cellular morphogenesis. Bioessays 13, 285–293. Cleveland, D.W., 1987. The multitubulin hypothesis revisited: what have we learned? J. Cell Biol. 104, 381–383. Cucchiarelli, V., Hiser, L., Smith, H., Frankfurter, A., Spano, A., Correia, J.J., Lobert, S., 2008. Beta-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas. Cell Motil. Cytoskeleton 65, 675–685. Derry, W.B., Wilson, L., Khan, I.A., Luduena, R.F., Jordan, M.A., 1997. Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes. Biochemistry 36, 3554–3562. Desai, A., Mitchison, T.J., 1997. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83–117. Dozier, J.H., Hiser, L., Davis, J.A., Thomas, N.S., Tucci, M.A., Benghuzzi, H.A., Frankfurter, A., Correia, J.J., Lobert, S., 2003. Beta class II tubulin predominates in normal and tumor breast tissues. Breast Cancer Res. 5, R157–R169. Dumont, S., Mitchison, T.J., 2009. Force and length in the mitotic spindle. Curr. Biol. 19, R749–R761. Etienne-Manneville, S., 2010. From signaling pathways to microtubule dynamics: the key players. Curr. Opin. Cell Biol. 22, 104–111. Foster, S.A., Galloway, D.A., 1996. Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. Oncogene 12, 1773–1779. Galmarini, C.M., Treilleux, I., Cardoso, F., Bernard-Marty, C., Durbecq, V., Gancberg, D., Bissery, M.C., Paesmans, M., Larsimont, D., Piccart, M.J., Di, L.A., Dumontet, C., 2008. Class III beta-tubulin isotype predicts response in advanced breast cancer patients randomly treated either with single-agent doxorubicin or docetaxel. Clin. Cancer Res. 14, 4511–4516. Gan, P.P., Kavallaris, M., 2008. Tubulin-targeted drug action: functional significance of class II and class IVb beta-tubulin in vinca alkaloid sensitivity. Cancer Res. 68, 9817–9824. Gan, P.P., McCarroll, J.A., Po’uha, S.T., Kamath, K., Jordan, M.A., Kavallaris, M., 2010. Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of betaIII-tubulin. Mol. Cancer Ther. 9, 1339–1348. Gan, P.P., Pasquier, E., Kavallaris, M., 2007. Class III beta-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res. 67, 9356–9363. Goncalves, A., Braguer, D., Kamath, K., Martello, L., Briand, C., Horwitz, S., Wilson, L., Jordan, M.A., 2001. Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics. Proc. Natl. Acad. Sci. U.S.A 98, 11737–11742. Haber, M., Burkhart, C.A., Regl, D.L., Madafiglio, J., Norris, M.D., Horwitz, S.B., 1995. Altered expression of M beta 2, the class II beta-tubulin isotype, in a murine J774.2 cell line with a high level of taxol resistance. J. Biol. Chem. 270, 31269–31275. Hasegawa, S., Miyoshi, Y., Egawa, C., Ishitobi, M., Taguchi, T., Tamaki, Y., Monden, M., Noguchi, S., 2003. Prediction of response to docetaxel by quantitative analysis of class I and III beta-tubulin isotype mRNA expression in human breast cancers. Clin. Cancer Res. 9, 2992–2997. Hoyle, H.D., Raff, E.C., 1990. Two Drosophila beta tubulin isoforms are not functionally equivalent. J. Cell Biol. 111, 1009–1026. Jordan, M.A., Wilson, L., 2004. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 4, 253–265. Kamath, K., Wilson, L., Cabral, F., Jordan, M.A., 2005. BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability. J. Biol. Chem. 280, 12902–12907. Kavallaris, M., 2010. Microtubules and resistance to tubulin-binding agents. Nat. Rev. Cancer 10, 194–204. Kavallaris, M., Kuo, D.Y., Burkhart, C.A., Regl, D.L., Norris, M.D., Haber, M., Horwitz, S.B., 1997. Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes. J. Clin. Invest. 100, 1282–1293. Kiyono, T., Foster, S.A., Koop, J.I., McDougall, J.K., Galloway, D.A., Klingelhutz, A.J., 1998. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396, 84–88.
Leandro-Garcia, L.J., Leskela, S., Landa, I., Montero-Conde, C., Lopez-Jimenez, E., Leton, R., Cascon, A., Robledo, M., Rodriguez-Antona, C., 2010. Tumoral and tissue-specific expression of the major human beta-tubulin isotypes. Cytoskeleton (Hoboken.) 67, 214–223. Lyle, K., Kumar, P., Wittmann, T., 2009a. Snapshot: microtubule regulators I. Cell 136, 380. Lyle, K., Kumar, P., Wittmann, T., 2009b. SnapShot: Microtubule regulators II. Cell 136, 566. Marszalek, J.R., Weiner, J.A., Farlow, S.J., Chun, J., Goldstein, L.S., 1999. Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B. J. Cell Biol. 145, 469–479. Miller, P.M., Folkmann, A.W., Maia, A.R., Efimova, N., Efimov, A., Kaverina, I., 2009. Golgi-derived CLASP-dependent microtubules control Golgi organization and polarized trafficking in motile cells. Nat. Cell Biol. 11, 1069–1080. Mitchison, T., Kirschner, M., 1984. Dynamic instability of microtubule growth. Nature 312, 237–242. Orr, G.A., Verdier-Pinard, P., McDaid, H., Horwitz, S.B., 2003. Mechanisms of Taxol resistance related to microtubules. Oncogene 22, 7280–7295. Palazzo, A.F., Gundersen, G.G., 2002. Microtubule-actin cross-talk at focal adhesions. Sci. STKE 2002, e31. Panda, D., Miller, H.P., Banerjee, A., Luduena, R.F., Wilson, L., 1994. Microtubule dynamics in vitro are regulated by the tubulin isotype composition. Proc. Natl. Acad. Sci. U.S.A. 91, 11358–11362. Paradiso, A., Mangia, A., Chiriatti, A., Tommasi, S., Zito, A., Latorre, A., Schittulli, F., Lorusso, V., 2005. Biomarkers predictive for clinical efficacy of taxol-based chemotherapy in advanced breast cancer. Ann. Oncol. 16 (Suppl. 4), iv14–iv19. Rodriguez, O.C., Schaefer, A.W., Mandato, C.A., Forscher, P., Bement, W.M., Waterman-Storer, C.M., 2003. Conserved microtubule-actin interactions in cell movement and morphogenesis. Nat. Cell Biol. 5, 599–609. Romanov, S.R., Kozakiewicz, B.K., Holst, C.R., Stampfer, M.R., Haupt, L.M., Tlsty, T.D., 2001. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409, 633–637. Rusan, N.M., Fagerstrom, C.J., Yvon, A.M., Wadsworth, P., 2001. Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin. Mol. Biol. Cell 12, 971–980. Schwer, H.D., Lecine, P., Tiwari, S., Italiano Jr., J.E., Hartwig, J.H., Shivdasani, R.A., 2001. A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets. Curr. Biol. 11, 579–586. Seve, P., Dumontet, C., 2008. Is class III beta-tubulin a predictive factor in patients receiving tubulin-binding agents? Lancet Oncol. 9, 168–175. Tischfield, M.A., Baris, H.N., Wu, C., Rudolph, G., Van, M.L., He, W., Chan, W.M., Andrews, C., Demer, J.L., Robertson, R.L., Mackey, D.A., Ruddle, J.B., Bird, T.D., Gottlob, I., Pieh, C., Traboulsi, E.I., Pomeroy, S.L., Hunter, D.G., Soul, J.S., Newlin, A., Sabol, L.J., Doherty, E.J., de Uzcategui, C.E., de, U.N., Collins, M.L., Sener, E.C., Wabbels, B., Hellebrand, H., Meitinger, T., de, B.T., Magli, A., Schiavi, C., Pastore-Trossello, M., Koc, F., Wong, A.M., Levin, A.V., Geraghty, M.T., Descartes, M., Flaherty, M., Jamieson, R.V., Moller, H.U., Meuthen, I., Callen, D.F., Kerwin, J., Lindsay, S., Meindl, A., Gupta Jr., M.L., Pellman, D., Engle, E.C., 2010. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance. Cell 140, 74–87. Tommasi, S., Mangia, A., Lacalamita, R., Bellizzi, A., Fedele, V., Chiriatti, A., Thomssen, C., Kendzierski, N., Latorre, A., Lorusso, V., Schittulli, F., Zito, F., Kavallaris, M., Paradiso, A., 2007. Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of beta-tubulins. Int. J. Cancer 120, 2078–2085. Troester, M.A., Hoadley, K.A., Sorlie, T., Herbert, B.S., Borresen-Dale, A.L., Lonning, P.E., Shay, J.W., Kaufmann, W.K., Perou, C.M., 2004. Cell-type-specific responses to chemotherapeutics in breast cancer. Cancer Res. 64, 4218–4226. Verdier-Pinard, P., Shahabi, S., Wang, F., Burd, B., Xiao, H., Goldberg, G.L., Orr, G.A., Horwitz, S.B., 2005. Detection of human betaV-tubulin expression in epithelial cancer cell lines by tubulin proteomics. Biochemistry 44, 15858– 15870. Wang, J., Hannon, G.J., Beach, D.H., 2000. Risky immortalization by telomerase. Nature 405, 755–756. Waterman-Storer, C.M., Salmon, E.D., 1997. Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling. J. Cell Biol. 139, 417–434. Westermann, S., Weber, K., 2003. Post-translational modifications regulate microtubule function. Nat. Rev. Mol. Cell Biol. 4, 938–947.
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Short communication
III-Tubulin is required for interphase microtubule dynamics in untransformed human mammary epithelial cells Benjamin Pierre Bouchet a,b,c,∗ , Alain Puisieux a,b,c,1 , Carlos María Galmarini d a
Université de Lyon, F-69000 Lyon, France Université Claude Bernard Lyon 1, F-69000 Lyon, France INSERM U590, Centre LEON BERARD, F-69008 Lyon, France d Cell Biology Department, PharmaMar, S.A., Colmenar Viejo, Madrid E-28770, Spain b c
a r t i c l e
i n f o
Article history: Received 4 February 2011 Received in revised form 28 April 2011 Accepted 3 May 2011 Keywords: Microtubule III-Tubulin Tubulin isotype Mammary epithelial cells Breast cancer
a b s t r a c t Numerous works have questioned the pertinence of using II- and/or III-tubulin expression as markers of prognosis and/or prediction of breast cancer response to chemotherapy containing microtubuletargeting agents. The rationale of such studies was essentially based on microtubule dynamics analysis using purified tubulin in vitro and cancer cell lines. Nonetheless, the significance of II- and III-tubulin expression in the control of microtubule dynamics in normal mammary epithelium has never been addressed. Here we investigate the expression and the consequences of II- and/or III-tubulin depletion in interphase microtubule dynamics in non-tumor human mammary epithelial cells. We find that both isoforms contribute to the tubulin isotype composition in primary and immortalized human mammary epithelial cells. Moreover, while II-tubulin depletion has limited effects on interphase microtubule behavior, III-tubulin depletion causes a strong exclusion of microtubules from lamella and a severe suppression of dynamic instability. These results demonstrate that, while II-tubulin is dispensable, IIItubulin is required for interphase microtubule dynamics in untransformed mammary epithelial cells. This strongly suggests that III-tubulin is an essential regulator of interphase microtubule functions in normal breast epithelium cells. © 2011 Elsevier GmbH. All rights reserved.
Introduction Microtubules are cytoskeletal filaments assembled from protofilament sheets resulting from the polymerization of ␣tubulin heterodimers (Desai and Mitchison, 1997). These hollow tubes are polarized fibers with the ␣-tubulin subunits exposed at the minus-end which is generally stable and anchored to the nucleation site such as the centrosome, and -tubulin subunits exposed at the plus-end that is highly dynamic. Heterodimer exchanges at the plus-ends characterize the phenomenon called dynamic instability and defined by rapid switches between periods of polymerization and depolymerization (Mitchison and Kirschner, 1984). Transition from growth or pause to shortening is called catastrophe and transition from shortening to growth or pause is called rescue (Jordan and Wilson, 2004). Dynamic microtubules are required for the proper dynamics of other crucial cell components including the mitotic chromosomes, the Golgi apparatus, and focal and
∗ Corresponding author at: Division of Cell Biology, Faculty of Science, Utrecht University, Utrecht, NL-3584 CH, The Netherlands. Tel.: +31 0 30 253 4585; fax: +31 0 30 253 2837. E-mail address: [email protected] (B.P. Bouchet). 1 Current address: INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69000 Lyon, France. 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.05.005
cell–cell adhesion structures (Akhmanova et al., 2009; Dumont and Mitchison, 2009; Miller et al., 2009). Several types of factors are involved in the regulation of microtubule dynamics. Besides microtubule-associated proteins and post-translational modifications of tubulins, tubulin isotype composition was also proposed as a regulatory mechanism for microtubule dynamics, though its role is not completely understood (Etienne-Manneville, 2010; Lyle et al., 2009a,b; Orr et al., 2003; Panda et al., 1994; Westermann and Weber, 2003). The amino acid differences in the C-terminal region of the protein serve to distinguish seven -tubulin isotypes: I, II, III, IVa, IVb, V and VI (Verdier-Pinard et al., 2005). Different works have showed that modulation of the expression of the different -tubulin isotypes can cause abnormalities in cell growth, axoneme assembly and platelet function (Bhattacharya and Cabral, 2004; Hoyle and Raff, 1990; Schwer et al., 2001). Early works using purified tubulin showed that -tubulin isotypes can differentially modulate microtubule dynamics in in vitro models (Banerjee et al., 1990, 1992; Panda et al., 1994). For example, the microtubule dynamics suppressor paclitaxel, frequently used in cancer chemotherapy, was shown to be less effective in microtubules assembled from purified II-, III- and IV-tubulin-containing dimers than in those assembled from unfractionated tubulin (Derry et al., 1997). Moreover, multiple
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studies revealed that III-tubulin overexpression is a feature of various human malignancies and notably epithelial tumors (Kavallaris, 2010; Leandro-Garcia et al., 2010; Seve and Dumontet, 2008). More specifically, III-tubulin overexpression was defined as a marker of poor prognosis and chemoresistance in tumor cells (Galmarini et al., 2008; Goncalves et al., 2001; Hasegawa et al., 2003; Kavallaris et al., 1997; Paradiso et al., 2005; Tommasi et al., 2007). This isotype was also described as a promoter of microtubule dynamics (Banerjee et al., 1992; Panda et al., 1994). Yet, III-tubulin overexpression, while inducing paclitaxel resistance, did not prove to be sufficient by itself to alter the dynamic instability parameters in untreated cells (Kamath et al., 2005). A recent study also showed that III-tubulin depletion sensitizes to microtubule dynamics suppression by low-dose tubulin-binding agents but does not affect microtubule dynamics in untreated human non-small cell lung cancer (NSCLC) cells H460 (Gan et al., 2010). In addition, though its role in normal tissues has been poorly investigated, III-tubulin functional inactivation due to genetic mutation in a congenital syndrome was reported to correlate with axon guidance defects and decreased overall microtubule dynamics (Tischfield et al., 2010). Finally, similarly to III-tubulin, II-tubulin overexpression was observed in several tumor types, including NSCLC and breast cancers, and associated with poor taxane response both in cancer tissues and cell lines (Bernard-Marty et al., 2002; Cucchiarelli et al., 2008; Galmarini et al., 2008; Gan and Kavallaris, 2008; Haber et al., 1995). It is striking to notice that, although altered expression of II- and III-tubulin was shown to be associated with mammary tumorigenesis, the significance of the expression of these tubulin isotypes in normal breast epithelium is still unknown. Nonetheless, various immunostaining-based studies have clearly shown that II- and III-tubulin are expressed in non-neoplastic mammary epithelium (Dozier et al., 2003; Galmarini et al., 2008; Leandro-Garcia et al., 2010). Hence, to gain insight into the role of II- and III-tubulin in microtubule dynamics regulation in non-tumor epithelial cells, we have first investigated here their expression in both primary and immortalized non-tumor mammary epithelial cells. We have next analyzed the consequences of their individual and combined depletion in microtubule dynamics in interphase cells. Our results demonstrate that III-, but not II-tubulin, is required for interphase microtubule dynamics in non-tumor human mammary epithelial cells. Materials and methods Plasmids and siRNA The PAcGFP1-Tubulin vector coding for GFP-␣-tubulin was purchased from Clontech. A pool of four independent siRNAs (siGENOME SMARTpool, Dharmacon) targeting TUBB2A (M008260-03-0010), TUBB2B (M-017790-00-0010) and TUBB3 mRNA (M-020099-03-0010) was used to transitorily inhibit respectively II- (siII) and/or III-tubulin (siIII) expression. Individual siRNA sequences were verified for their identity (nucleotide blast, NCBI); two targeting sequences from TUBB2A SMARTpool were found to target TUBB2B and no common sequences were found by comparing TUBB2A/B and TUBB3 SMARTpools. A pool of four independent nontargeting siRNAs (siCONTROL Non-Targeting siRNA Pool #2, siCT, Dharmacon) was used as control in siRNA-mediated knockdown experiments. Cell culture and transfection Primary human mammary epithelial cells (HMECs, used at population doubling 7–14) were obtained from Lonza. Low passage
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immortalized human mammary epithelial cells (hTMECs, hTERTtransduced HMECs) were provided by R.A. Weinberg (Whitehead Institute, Cambridge, USA). Primary HMECs and hTMEC-derived cell lines were cultured in MEBM basal medium (Lonza) supplemented with 10 ng/ml human EGF, 5 g/ml insulin, 0.5 g/ml hydrocortisone, 0.4% bovine pituitary extract, 50 g/ml gentamicin and 50 g/ml amphotericin-B. Plasmid transfection in hTMECs was performed using FuGENE HD (Roche). Stably pAcGFP1-Tubulin-transfected hTMECs (GThMECs) were selected under 100 g/ml geneticin. Subclones were selected according to their optimal fluorescently tagged protein expression and localization, normal 2D mammary epithelial cell morphology and growth. Transfection of 10–30 nM siRNAs was performed in hTMECderived cell lines using Lipofectamine RNAiMAX (Invitrogen). In these conditions, transfectivity was estimated to be 100% by using fluorescent control siRNA (siGLO RISC-Free Control siRNA, Dharmacon; DY-547-conjugated, absorbance: 557, emission max: 570 nm) in all hTMEC-derived cell lines used in transient siRNA-mediated knockdown experiments (Supplementary Fig. S1). Antibodies and reagents Mouse monoclonal anti-II-tubulin (7B9) antibody was purchased from Novus and Sigma–Aldrich. Mouse monoclonal antibodies against I- (SAP.4G5), III- (SDL.3D10) and IV-tubulin (ONS.1A6), total -tubulin (TUB 2.1), total ␣-tubulin (B-5-1-2) and acetylated tubulin (6-11B-1) were purchased from Sigma–Aldrich. Rat monoclonal anti-␣-tubulin (YL1/2) and mouse monoclonal anti-III-tubulin (TUJ1) antibodies used in immunofluorescence experiments were respectively provided by Abcam and Covance Inc. Secondary Alexa Fluor 488 goat anti-rat IgG (H+L) and Alexa Fluor 594 F(ab )2 fragment of goat anti-mouse IgG (H+L) antibodies were supplied by Invitrogen. Vectashield mounting medium was purchased from Vector Laboratories. Trypsin neutralizing solution was obtained from Lonza. Oxyrase was purchased from Oxyrase Inc. Immunofluorescent staining, microscopy Double immunostaining of total ␣-tubulin and II or IIItubulin was performed on cells grown on a glass coverslip, fixed in 100% methanol at −20 ◦ C, permeabilized in 0.15% Triton X-100 1× PBS and blocked in 1% BSA/0.05% Tween 20 1× PBS (blocking buffer). Cells were successively incubated at room temperature with 1:200 rat anti-␣-tubulin, 1:300 Alexa Fluor 488 goat anti-rat, 1:250 mouse anti-II- or III-tubulin and 1:300 Alexa Fluor 594 F(ab )2 fragment of goat anti-mouse in blocking buffer. Ethanol dehydrated coverslips were mounted in Vectashield and sealed with nail polish. Fluorescence microscopy imaging was performed with a 100×/1.3 NA PL Fluotar objective on a DMRBE microscope (Leica) coupled to a C4880 CCD camera (Hamamatsu) automated in Wasabi! software (Hamamatsu). Live cell imaging and analysis Live cells were visualized on a 37 ◦ C heated Zeiss Axiovert 100M microscope automated by MetaMorph 7.5.6.0 software and equipped with a cooled CCD camera (CoolSNAP HQ monochrome; Photometrics). Experiments were performed on cells plated in glass-bottom dishes (MatTek) maintained on a 37 ◦ C heated stage and in a 5% CO2 and humidified atmosphere. Fluorescent microtubule imaging was performed with a 100×/1.4 NA Plan-Apochromat or a 63×/1.4 NA PlanApochromat objective on GT-hMECs cultured in phenol-free
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MEGM containing 5–10 l/ml oxyrase. Images were acquired from optimal focal planes in lamella of interphase cells, at 3.5-s intervals, with a 350-ms exposure and during a minimum of 2.5 min. Quantification of the shortest distance between the microtubule tip and the cell edge in distal lamella was manually performed on randomly chosen microtubule ends in a zone comprised between the cell edge and an internal limit approximately located at 8 m from the edge. Using this method, 14 microtubule tips were tracked in each analyzed cell. Quantification of the microtubule density in distal lamella was performed using iterative measurement of the microtubule lattice contained in a region of interest corresponding to a 5 m-diameter circle (∼19.6 m2 ) adjacent to the cell edge. Each measurement consisted in the calculation of the mean microtubule track (m) per m2 of distal lamella. A minimum of 5 lamella measurements per cell was used. For statistical analysis, measurements for each condition were pooled. Microtubule dynamic instability parameters were extracted by manually tracking microtubule plus ends in GFP time-lapse sequences using ImageJ 1.42q software (http://rsb.info.nih.gov/ij/) and MTrackJ plugin (Erik Meijering, Erasmus Medical Center Rotterdam, NLD; http://www.imagescience.org/meijering/software/mtrackj/). For analysis and presentation, image sequences were inverted, processed as stacks and submitted to bleach correction, FFT bandpass filter and contrast enhancing. The distance between a plus-end and a fixed origin on the microtubule body was used to approximate microtubule length changes and velocity between each time point. Only microtubule length changes ≥0.4 m between two consecutive time points were considered as growth or shortening events, while changes <0.4 m were considered as the pause event (based on the measurement of the mean error in 3 independent manual trackings of the same 90-frame sequence; superior to optical resolution for GFP imaging with this setup, 0.24 m/frame). Growth, shortening and pause phases were respectively identified as unique or consecutive growth, shortening and pause events. Mean growth and shortening rates were determined using a custom-written Excel macro (Microsoft) averaging microtubule velocity in each corresponding phase. The total microtubule length change for each phase was calculated as the product of mean growth or shortening rate by total phase duration. Catastrophes (transitions from growth or pause to shortening) and rescues (transitions from shortening to pause or growth) were counted by a custom-written Excel macro. Catastrophe frequencies were calculated by dividing the number of catastrophes by the sum of the time spent in growth and pause. Similarly, rescue frequencies were calculated by dividing the number of rescues by the time spent in shortening. Microtubule dynamicity was measured as the total microtubule length grown and shortened relative to the microtubule life span. Mean dynamicity was calculated for each condition as the average of microtubule dynamicities. For each condition, we analyzed a minimum of 70 microtubules in a minimum of 14 cells.
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Results and discussion ˇII- and ˇIII-tubulin are expressed in primary and hTERT-immortalized human mammary epithelial cells In order to address the eventual role of II- and III-tubulin in microtubule regulation in non-tumor breast epithelium, we have first analyzed the expression of both isotypes in primary and hTERT-immortalized untransformed human mammary epithelial cells (HMECs and hTMECs; Fig. 1A). These cells predominantly retain an euploid karyotype and a stable genetic background similar to that observed in normal human cells and therefore provide a relevant model to study mammary epithelium properties and functions (Romanov et al., 2001; Troester et al., 2004; Wang et al., 2000). We have also investigated the expression of I- and total IV-tubulin in the same cells. I-tubulin was described as a major constitutive isotype (Cleveland, 1987; Leandro-Garcia et al., 2010). IV-tubulin exists under two isoforms: IVa-tubulin, initially described as a brain-specific form and IVb-tubulin, constitutively expressed in human tissues (Cleveland, 1987; Leandro-Garcia et al., 2010). Since the anti-IV-tubulin antibody that we used here does not discriminate between both isoforms, we assume that we detected the IVb form in mammary epithelial cells; however we cannot exclude that the IVa form could be expressed in these cells as well. Western blot analysis on total cell lysates showed that II- and III-tubulin are expressed in both HMECs and hTMECs, indicating that these -tubulin isotypes significantly contribute to the tubulin isotype composition in non-tumor mammary epithelial cells (Fig. 1A). This result was confirmed by immunofluorescent staining of II- and III-tubulin in fixed hTMECs (Fig. 1B). This is consistent with previous data showing that II- and III-tubulin are expressed in normal mammary epithelial tissues (Dozier et al., 2003; Galmarini et al., 2008; Leandro-Garcia et al., 2010). Additionally, we found that I- and IV-tubulin also significantly contribute to the tubulin isotype composition in mammary epithelial cells (Fig. 1A). Besides, it is interesting to note that this composition is almost identical in HMECs and hTMECs, which strengthens the relevance of hTERT-immortalized cells as a model for primary mammary epithelial cells (Fig. 1A). Lastly, acetylation of ␣-tubulin, which is a post-translational modification associated with stable microtubules, was found in similar levels in HMECs and hTMECs (Fig. 1A; Westermann and Weber, 2003). This data suggests that both lines harbor equivalent stable microtubule population and reinforces the relevance of hTMECs to model microtubule dynamics in normal mammary epithelium. ˇIII- but not ˇII-tubulin inactivation causes suppression of interphase microtubule dynamics in untransformed human mammary epithelial cells In order to specify the involvement of II- and III-tubulin in microtubule regulation in normal breast epithelium, we have next
Fig. 1. Expression of II- and III-tubulin and consequences of their depletion in lamella microtubule organization in non-tumor human mammary epithelial cells. (A) Western blot analysis of I–IV tubulin isotypes, total -tubulin (-tot), acetylated (Ac-tub) and total ␣-tubulin (␣-tot) in whole lysates from HMEC and hTMEC cells. (B) Immunofluorescent labeling of total ␣-tubulin and II- or III-tubulin in hTMEC cells. Gray frame, zoomed region shown on the right panel. Main panel, bar, 10 m; zoom panel, bar, 3 m. (C) Western blot analysis of II, III, I, and IV tubulin isotypes, total - (-tot) and acetylated tubulin (Ac-tub) in whole lysates from hTMEC cells after a 72 h transfection with control siRNA pool (siCT), II (siII), III (siIII) or combined II and III tubulin-targeting siRNA pool (see Materials and methods). (D) GFP imaging in live GT-hMECs treated as in C; gray frame, zoomed region shown on the right panel; yellow dotted line, cell edge; bar, 5 m. (E) Quantification of the shortest distance between the microtubule (MT) tip and the cell edge (m) in live GT-hMECs treated as in C (see Materials and methods). Box plots indicate the 25th percentile (bottom boundary), median (middle line), mean (red open circle), 75th percentile (top boundary), nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and outliers (black cross). For each condition, n = 196 measures in 14 cells. Kruskal–Wallis analysis, P < 0.001; pairwise multiple comparison by Dunn’s method. Gray dotted line figures mean in siCT condition. (F) Quantification of the microtubule (MT) density in distal lamella in live GT-hMECs treated as in C (see Materials and methods). Box plots built as in (E); siCT, n = 53, 5 cells; siII, n = 51, 4 cells; siIII, n = 53, 5 cells, siII + III, n = 54, 6 cells. Kruskal–Wallis analysis, P < 0.001; pairwise multiple comparison by Dunn’s method. Gray dotted line figures mean in siCT condition. Molecular mass (kDa). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Effects of II and/or III tubulin depletion on microtubule dynamic instability in interphase non-tumor human mammary epithelial cells. (A–F) Dynamic instability parameters measured in GT-hMECs, after 72 h transfection with control siRNA pool (siCT, 70 microtubules, 14 cells), II- (siII, 70 microtubules, 20 cells), III- (siIII, 70 microtubules, 18 cells) or combined II- and III- tubulin-targeting siRNA pool (70 microtubules, 14 cells). Box plots built as in Fig. 1E. Kruskal–Wallis analysis, P < 0.001; pairwise multiple comparison by Dunn’s method. Gray dotted line in all panels, except in (E), figures mean in siCT condition. Error bar in time percentage graph, 95% confidence interval.
investigated the consequences of II- and/or III-tubulin depletion in microtubule dynamics in non-tumor mammary epithelial cells. We only used here hTERT-immortalized cells, because HMECs in culture have a limited proliferative potential due to replicative senescence that prevents them from bypassing 20 population doublings without acquiring further genetic alterations (Foster and Galloway, 1996; Kiyono et al., 1998; Romanov et al., 2001). At least two hTMEC-derived clones stably expressing GFP-␣-tubulin (GThMECs) and allowing live imaging of microtubule dynamics were thus transiently knocked down for II- and/or III-tubulin, after transfection of respectively TUBB2A/B- and/or TUBB3-targeting siRNAs (Fig. 1B; Rusan et al., 2001). These treatments allowed almost complete suppression of II- and/or III-tubulin expression (Fig. 1C). Of note, I-, IV- and total -tubulin levels seemed to be unaltered by II- and/or III-tubulin silencing (Fig. 1C). Similarly, II- and III-tubulin expressions appeared to be unmodified by the silencing of each other (Fig. 1C). This is in agreement with studies also showing unaltered expression of -tubulin isotypes and total -tubulin when II- or III-tubulin genes are silenced (Gan et al., 2007; Gan and Kavallaris, 2008). Though we noticed an apparent reduced proliferation in III-tubulin-depleted cells, we did not observe any significant mitotic arrest in II- and/or III-tubulin-depleted cells. Moreover, our previous works have indicated that the mitotic checkpoint in hTMECs is very sensitive to modified microtubule dynamics since those cells undergo mitotic arrest when treated with a low dose of the microtubule stabilizer paclitaxel (Bouchet et al., 2007). This suggests that IIand/or III-tubulin silencing could only have a limited effect on mitotic microtubule dynamics. However, the specific mitotic role of II- and III-tubulin need to be further investigated. Live fluorescence observation in GT-hMECs showed that microtubules in control interphase cells organize into a sparse network and stably invade lamella (Fig. 1D, supplementary Movie 1). Furthermore, microtubules grow toward the cell edge and frequently undergo bending and reorientation when they reach the
most distal part of the lamella so that they grow parallel to the edge (Fig. 1D, supplementary Movie 1). This phenomenon was previously described in epithelial cells and shown to involve the actinomyosin-based retrograde flow (Waterman-Storer and Salmon, 1997). Microtubules in II-tubulin-depleted cells showed similar behavior than in control cells (Fig. 1D and supplementary Movie 1). In contrast, microtubules in III-tubulin-depleted cells exhibited an aberrant behavior consisting in a tight network organization within proximal lamella and a strong exclusion from distal lamella (Fig. 1D, supplementary Movie 1). This behavior was characterized by the significant increase of the distance between microtubule tips and the cell edge in both III- and II-/IIItubulin-depleted cells (Fig. 1E, P < 0.05, Dunn’s post hoc test). IIIand II-/III-tubulin-depleted cells also showed a strong reduction of the microtubule density in distal lamella (Fig. 1F, P < 0.05, Dunn’s post hoc test). Of note, the mean microtubule tip-cell edge distance and density in distal lamella was not significantly affected by the depletion of II-tubulin alone (Fig. 1E and F). Moreover, IItubulin depletion did not significantly modify these features when combined with III-tubulin depletion (Fig. 1E and F). Next, microtubule dynamics was analyzed in detail (Fig. 2A–F). Based on our preliminary data indicating that transfectivity is 100% in our siRNA experiments (see Materials and methods), we chose a minimum of 14-cell/70-microtubule sampling to extract microtubule dynamic instability parameters. Of note, microtubule dynamics showed similar features to what we observed in a previous study (Bouchet et al., 2011). As suggested by the results described above (Fig. 1D–F), interphase microtubule dynamics showed only limited changes in II-tubulin-depleted cells when compared to control cells, including a reduced shortening rate (−8.1%, P < 0.05, Dunn’s post hoc test) with no significant impact on mean dynamicity (Fig. 1B and F). In contrast, interphase microtubule dynamics in III-tubulin-depleted cells was highly modified when compared with control cells (Fig. 2A–F). Alterations comprised a decreased growth rate (−9.0%, P < 0.05, Dunn’s post hoc
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test), a decreased shortening rate (−31.5%, P < 0.05, Dunn’s post hoc test), a decreased catastrophe frequency (−37.6%, P < 0.05, Dunn’s post hoc test) and an increased rescue frequency (+35.5%, P < 0.05, Dunn’s post hoc test). Moreover, in III-tubulin-depleted cells, the time spent by microtubules in pause was strongly increased when compared with control cells (+33.3%, P < 0.05, Dunn’s post hoc test). Finally, the mean microtubule dynamicity was dramatically decreased in III-tubulin-depleted cells compared to control cells (−70.8%, P < 0.05, Dunn’s post hoc test). Noticeably, in cells depleted of both II- and III-tubulin, none of the dynamic instability parameters were found to be significantly different from those observed in cells harboring only III-tubulin depletion (Fig. 2A–F, Dunn’s post hoc test). This result confirms our previous observation of the microtubule behavior in double knocked down cells (Fig. 1D–F). This also demonstrates that II-tubulin silencing neither potentiates nor rescues the suppression of microtubule dynamics caused by III-tubulin loss. Together with individual knockdown experiment results, these data indicate that II-tubulin has no major influence on interphase microtubule dynamics modulation in normal mammary epithelial cells. It is remarkable that, despite the decrease of overall microtubule dynamics, the ␣-tubulin acetylation, which is generally associated with microtubule stability, was not increased in IIItubulin-depleted cells (Fig. 1B; Westermann and Weber, 2003). This could indicate that III-tubulin inactivation affects only the population of dynamic microtubules without modifying the proportion of stable acetylated microtubules (Bulinski and Gundersen, 1991). As well, microtubule populations could differ in their tubulin isotype composition and stable microtubules assembly may involve less III-tubulin than dynamic microtubules, which would explain their unaltered state when III-tubulin is depleted. Alternatively, it is possible that microtubules could undergo plus-end dynamics alterations that do not affect acetylation in their stable lattice part. The link between tubulin isotype composition and accumulation of post-translational modifications is still poorly understood and should thus be specified in future studies. We found here that III-tubulin inactivation causes interphase microtubule dynamics suppression in untransformed human mammary epithelial cells. This result is in accordance with earlier studies showing that III-tubulin promotes microtubule dynamics (Banerjee et al., 1992; Panda et al., 1994). Moreover, our data are consistent with the recent description of a congenital syndrome that associates III-tubulin functional inactivation with severe defects of axon guidance, oculomotor nerve hypoplasia and suppression of microtubule dynamics (Tischfield et al., 2010). In contrast, our study does not confirm the observations previously made in the lung adenocarcinoma cell line H460 and describing an unaffected interphase microtubule dynamics consecutive to IIItubulin silencing (Gan et al., 2010). We assume that this discrepancy arises from the radical difference between cell models used in both studies. This actually implies that tissue-specificity and genetic background must be taken into account when determining microtubule dynamics properties in different non-tumor and tumor cell line models. Of note, the functional cause of microtubule dynamics suppression due to III-tubulin inactivation in non-tumor mammary epithelial cells is still unclear. Tischfield et al. (2010) recently showed that III-tubulin mutations causing microtubule dynamics suppression also inhibit microtubule plus-end binding of depolymerizing kinesin-8 family members such as Kip3p in yeast. It is thus possible that inactivation of the binding of depolymerizing kinesins and/or other microtubule plus-end tracking proteins could contribute to the suppression of microtubule dynamics in III-tubulin-depleted mammary epithelial cells. In the same study, III-tubulin mutation was also shown to correlate with suppressed microtubule binding of KIF21A which belongs to the family of
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plus-end-directed motor proteins (Marszalek et al., 1999; Tischfield et al., 2010). This suggests that III-tubulin inactivation could also affect kinesin-based transport toward the cell periphery. In the present study, we show that the cell periphery exhibits a severe loss of microtubules when III-tubulin is inactivated (Fig. 1C and supplementary Movie 1). Thus, the dynamics of key components of lamella such as actin and adhesion structures could be modified by defective plus-end-directed transport. Because these structures exhibit various mechanisms of cross-regulation with microtubules, their alteration could in turn contribute to the suppression of lamella microtubule dynamics in III-tubulin-depleted cells. Interestingly, numerous works have shown that dynamic microtubules regulate cell motility, notably through their ability to tune Rho GTPases activity, actin dynamics and focal adhesion turnover (Broussard et al., 2008; Palazzo and Gundersen, 2002; Rodriguez et al., 2003). Because III-tubulin depletion strongly suppresses lamella microtubule dynamics and according to preliminary observations (data not shown), we strongly suspect that III-tubulin is required for motility in untransformed human mammary epithelial cells. For these reasons and because the link between tubulin isotype composition and cell motility is poorly understood, the eventual role of III-tubulin in regulating cell motility should be addressed in future works. In conclusion, we show here that, while II-tubulin is dispensable, III-tubulin is required for interphase microtubule dynamics in human mammary epithelial cells. These results reveal that III-tubulin is a new candidate for physiological regulation of interphase microtubule dynamics in mammary epithelium. This could have major implications in understanding how III-tubulin specific functions participate in tissue homeostasis and how these functions are eventually altered during epithelial tumorigenesis, notably in human breast. Moreover, our data could also contribute to refine the investigation and the analysis of -tubulin isotype expression for breast cancer management. Acknowledgements We thank Prof. Anna Akhmanova (Division of Cell Biology, Faculty of Science, Utrecht University, Utrecht, NDL) for critical comments and support for final experiments. We also thank PLATIM (PLAteau Technique Imagerie/Microscopie, Ecole Normale Supérieure, Lyon, FRA) for microscopy support. This work was supported by FRM (Fondation pour la Recherche Médicale, FRA) and PharmaMar, S.A. (Colmenar Viejo (Madrid), ESP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejcb.2011.05.005. References Akhmanova, A., Stehbens, S.J., Yap, A.S., 2009. Touch, grasp, deliver and control: functional cross-talk between microtubules and cell adhesions. Traffic 10, 268–274. Banerjee, A., Roach, M.C., Trcka, P., Luduena, R.F., 1990. Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin. J. Biol. Chem. 265, 1794–1799. Banerjee, A., Roach, M.C., Trcka, P., Luduena, R.F., 1992. Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain. J. Biol. Chem. 267, 5625–5630. Bernard-Marty, C., Treilleux, I., Dumontet, C., Cardoso, F., Fellous, A., Gancberg, D., Bissery, M.C., Paesmans, M., Larsimont, D., Piccart, M.J., Di, L.A., 2002. Microtubule-associated parameters as predictive markers of docetaxel activity in advanced breast cancer patients: results of a pilot study. Clin. Breast Cancer 3, 341–345. Bhattacharya, R., Cabral, F., 2004. A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation. Mol. Biol. Cell 15, 3123–3131. Bouchet, B.P., Bertholon, J., Falette, N., Audoynaud, C., Lamblot, C., Puisieux, A., Galmarini, C.M., 2007. Paclitaxel resistance in untransformed human mammary
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epithelial cells is associated with an aneuploidy-prone phenotype. Br. J. Cancer 97, 1218–1224. Bouchet, B.P., Fauvet, F., Grelier, G., Galmarini, C.M., Puisieux, A., 2011. p21Cip1 regulates cell-substrate adhesion and interphase microtubule dynamics in untransformed human mammary epithelial cells. Eur. J. Cell Biol. 90, 631–641. Broussard, J.A., Webb, D.J., Kaverina, I., 2008. Asymmetric focal adhesion disassembly in motile cells. Curr. Opin. Cell Biol. 20, 85–90. Bulinski, J.C., Gundersen, G.G., 1991. Stabilization of post-translational modification of microtubules during cellular morphogenesis. Bioessays 13, 285–293. Cleveland, D.W., 1987. The multitubulin hypothesis revisited: what have we learned? J. Cell Biol. 104, 381–383. Cucchiarelli, V., Hiser, L., Smith, H., Frankfurter, A., Spano, A., Correia, J.J., Lobert, S., 2008. Beta-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas. Cell Motil. Cytoskeleton 65, 675–685. Derry, W.B., Wilson, L., Khan, I.A., Luduena, R.F., Jordan, M.A., 1997. Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes. Biochemistry 36, 3554–3562. Desai, A., Mitchison, T.J., 1997. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83–117. Dozier, J.H., Hiser, L., Davis, J.A., Thomas, N.S., Tucci, M.A., Benghuzzi, H.A., Frankfurter, A., Correia, J.J., Lobert, S., 2003. Beta class II tubulin predominates in normal and tumor breast tissues. Breast Cancer Res. 5, R157–R169. Dumont, S., Mitchison, T.J., 2009. Force and length in the mitotic spindle. Curr. Biol. 19, R749–R761. Etienne-Manneville, S., 2010. From signaling pathways to microtubule dynamics: the key players. Curr. Opin. Cell Biol. 22, 104–111. Foster, S.A., Galloway, D.A., 1996. Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. Oncogene 12, 1773–1779. Galmarini, C.M., Treilleux, I., Cardoso, F., Bernard-Marty, C., Durbecq, V., Gancberg, D., Bissery, M.C., Paesmans, M., Larsimont, D., Piccart, M.J., Di, L.A., Dumontet, C., 2008. Class III beta-tubulin isotype predicts response in advanced breast cancer patients randomly treated either with single-agent doxorubicin or docetaxel. Clin. Cancer Res. 14, 4511–4516. Gan, P.P., Kavallaris, M., 2008. Tubulin-targeted drug action: functional significance of class II and class IVb beta-tubulin in vinca alkaloid sensitivity. Cancer Res. 68, 9817–9824. Gan, P.P., McCarroll, J.A., Po’uha, S.T., Kamath, K., Jordan, M.A., Kavallaris, M., 2010. Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of betaIII-tubulin. Mol. Cancer Ther. 9, 1339–1348. Gan, P.P., Pasquier, E., Kavallaris, M., 2007. Class III beta-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res. 67, 9356–9363. Goncalves, A., Braguer, D., Kamath, K., Martello, L., Briand, C., Horwitz, S., Wilson, L., Jordan, M.A., 2001. Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics. Proc. Natl. Acad. Sci. U.S.A 98, 11737–11742. Haber, M., Burkhart, C.A., Regl, D.L., Madafiglio, J., Norris, M.D., Horwitz, S.B., 1995. Altered expression of M beta 2, the class II beta-tubulin isotype, in a murine J774.2 cell line with a high level of taxol resistance. J. Biol. Chem. 270, 31269–31275. Hasegawa, S., Miyoshi, Y., Egawa, C., Ishitobi, M., Taguchi, T., Tamaki, Y., Monden, M., Noguchi, S., 2003. Prediction of response to docetaxel by quantitative analysis of class I and III beta-tubulin isotype mRNA expression in human breast cancers. Clin. Cancer Res. 9, 2992–2997. Hoyle, H.D., Raff, E.C., 1990. Two Drosophila beta tubulin isoforms are not functionally equivalent. J. Cell Biol. 111, 1009–1026. Jordan, M.A., Wilson, L., 2004. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 4, 253–265. Kamath, K., Wilson, L., Cabral, F., Jordan, M.A., 2005. BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability. J. Biol. Chem. 280, 12902–12907. Kavallaris, M., 2010. Microtubules and resistance to tubulin-binding agents. Nat. Rev. Cancer 10, 194–204. Kavallaris, M., Kuo, D.Y., Burkhart, C.A., Regl, D.L., Norris, M.D., Haber, M., Horwitz, S.B., 1997. Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes. J. Clin. Invest. 100, 1282–1293. Kiyono, T., Foster, S.A., Koop, J.I., McDougall, J.K., Galloway, D.A., Klingelhutz, A.J., 1998. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396, 84–88.
Leandro-Garcia, L.J., Leskela, S., Landa, I., Montero-Conde, C., Lopez-Jimenez, E., Leton, R., Cascon, A., Robledo, M., Rodriguez-Antona, C., 2010. Tumoral and tissue-specific expression of the major human beta-tubulin isotypes. Cytoskeleton (Hoboken.) 67, 214–223. Lyle, K., Kumar, P., Wittmann, T., 2009a. Snapshot: microtubule regulators I. Cell 136, 380. Lyle, K., Kumar, P., Wittmann, T., 2009b. SnapShot: Microtubule regulators II. Cell 136, 566. Marszalek, J.R., Weiner, J.A., Farlow, S.J., Chun, J., Goldstein, L.S., 1999. Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B. J. Cell Biol. 145, 469–479. Miller, P.M., Folkmann, A.W., Maia, A.R., Efimova, N., Efimov, A., Kaverina, I., 2009. Golgi-derived CLASP-dependent microtubules control Golgi organization and polarized trafficking in motile cells. Nat. Cell Biol. 11, 1069–1080. Mitchison, T., Kirschner, M., 1984. Dynamic instability of microtubule growth. Nature 312, 237–242. Orr, G.A., Verdier-Pinard, P., McDaid, H., Horwitz, S.B., 2003. Mechanisms of Taxol resistance related to microtubules. Oncogene 22, 7280–7295. Palazzo, A.F., Gundersen, G.G., 2002. Microtubule-actin cross-talk at focal adhesions. Sci. STKE 2002, e31. Panda, D., Miller, H.P., Banerjee, A., Luduena, R.F., Wilson, L., 1994. Microtubule dynamics in vitro are regulated by the tubulin isotype composition. Proc. Natl. Acad. Sci. U.S.A. 91, 11358–11362. Paradiso, A., Mangia, A., Chiriatti, A., Tommasi, S., Zito, A., Latorre, A., Schittulli, F., Lorusso, V., 2005. Biomarkers predictive for clinical efficacy of taxol-based chemotherapy in advanced breast cancer. Ann. Oncol. 16 (Suppl. 4), iv14–iv19. Rodriguez, O.C., Schaefer, A.W., Mandato, C.A., Forscher, P., Bement, W.M., Waterman-Storer, C.M., 2003. Conserved microtubule-actin interactions in cell movement and morphogenesis. Nat. Cell Biol. 5, 599–609. Romanov, S.R., Kozakiewicz, B.K., Holst, C.R., Stampfer, M.R., Haupt, L.M., Tlsty, T.D., 2001. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409, 633–637. Rusan, N.M., Fagerstrom, C.J., Yvon, A.M., Wadsworth, P., 2001. Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin. Mol. Biol. Cell 12, 971–980. Schwer, H.D., Lecine, P., Tiwari, S., Italiano Jr., J.E., Hartwig, J.H., Shivdasani, R.A., 2001. A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets. Curr. Biol. 11, 579–586. Seve, P., Dumontet, C., 2008. Is class III beta-tubulin a predictive factor in patients receiving tubulin-binding agents? Lancet Oncol. 9, 168–175. Tischfield, M.A., Baris, H.N., Wu, C., Rudolph, G., Van, M.L., He, W., Chan, W.M., Andrews, C., Demer, J.L., Robertson, R.L., Mackey, D.A., Ruddle, J.B., Bird, T.D., Gottlob, I., Pieh, C., Traboulsi, E.I., Pomeroy, S.L., Hunter, D.G., Soul, J.S., Newlin, A., Sabol, L.J., Doherty, E.J., de Uzcategui, C.E., de, U.N., Collins, M.L., Sener, E.C., Wabbels, B., Hellebrand, H., Meitinger, T., de, B.T., Magli, A., Schiavi, C., Pastore-Trossello, M., Koc, F., Wong, A.M., Levin, A.V., Geraghty, M.T., Descartes, M., Flaherty, M., Jamieson, R.V., Moller, H.U., Meuthen, I., Callen, D.F., Kerwin, J., Lindsay, S., Meindl, A., Gupta Jr., M.L., Pellman, D., Engle, E.C., 2010. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance. Cell 140, 74–87. Tommasi, S., Mangia, A., Lacalamita, R., Bellizzi, A., Fedele, V., Chiriatti, A., Thomssen, C., Kendzierski, N., Latorre, A., Lorusso, V., Schittulli, F., Zito, F., Kavallaris, M., Paradiso, A., 2007. Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of beta-tubulins. Int. J. Cancer 120, 2078–2085. Troester, M.A., Hoadley, K.A., Sorlie, T., Herbert, B.S., Borresen-Dale, A.L., Lonning, P.E., Shay, J.W., Kaufmann, W.K., Perou, C.M., 2004. Cell-type-specific responses to chemotherapeutics in breast cancer. Cancer Res. 64, 4218–4226. Verdier-Pinard, P., Shahabi, S., Wang, F., Burd, B., Xiao, H., Goldberg, G.L., Orr, G.A., Horwitz, S.B., 2005. Detection of human betaV-tubulin expression in epithelial cancer cell lines by tubulin proteomics. Biochemistry 44, 15858– 15870. Wang, J., Hannon, G.J., Beach, D.H., 2000. Risky immortalization by telomerase. Nature 405, 755–756. Waterman-Storer, C.M., Salmon, E.D., 1997. Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling. J. Cell Biol. 139, 417–434. Westermann, S., Weber, K., 2003. Post-translational modifications regulate microtubule function. Nat. Rev. Mol. Cell Biol. 4, 938–947.