1 Department of Biomedical Sciences, Creighton University, Omaha, Nebraska 68178, USA
2 Department of Internal Medicine, Section of Pulmonary, Critical Care and Sleep Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA
3 Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
Author for correspondence (e-mail: hallw{at}creighton.edu)
Accepted 17 June 2005
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Summary |
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Key words: Axonemal function, ß Tubulin isotype, Cilia, Ciliary beat frequency
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Introduction |
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There are seven known isotypes of ß tubulin in mammals. Their presence and composition are tissue-type specific, but no functional correlation has been made to date (Hallworth and Ludueña, 2000; Ludueña, 1993
). To investigate the functional significance of the C-termini of ß tubulin isotypes in mammals, we used a preparation of isolated, de-membranated ATP-activated beating bovine cilia (Hastie et al., 1986
; Wyatt et al., 2005
). We measured the effect on ciliary beat frequency (CBF) of isotype-specific monoclonal antibodies directed against the C-termini of tubulin and monoclonal antibodies directed against other epitopes of ß tubulin, as well as monoclonal antibodies against various epitopes of
tubulin, using a new, rapid digital video motility analysis method (Sisson et al., 2003
). In addition, we examined the effect on CBF of peptides containing (1) the C-terminal amino acid sequences of ß tubulin isotypes against which the antibodies were raised (C-terminal tail peptides, CTT peptides) and (2) the peptides containing the amino acid sequences of the isotype-specific axonemal motif or the closest equivalent. This is the first study to investigate the isotype-specific differences of tubulin isotypes in mammalian cilia in a functional, motility-based assay.
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Materials and Methods |
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Activation of cilia and measuring CBF
A thawed aliquot of frozen demembranated cilia was mixed with resuspension buffer and cAMP (in resuspension buffer, final concentration 1.0 mM) in a 1:2:1 dilution (by volume). 20 µl of the solution containing 0.25 mg/ml protein were then plated into wells of a 48 well culture plate (Corning Inc., Corning, NY). The plate was centrifuged at 400 g for 2 minutes at room temperature to attach the cilia to the bottom of each well. A first measurement was taken (time t=0) and 20 µl of ATP in resuspension buffer (Sigma, MO) (final concentration 1.25 mM) were added to activate ciliary beating, typically at a frequency of 8-10 Hz. Test solutions containing antibodies or peptides in resuspension buffer were added at time t=3 minutes, after three readings of activated cilia. Measurements of CBF were made at intervals of 1 minute for 15-20 minutes. ATP depletion occurred at approximately 25 minutes after ATP addition. Reactivation of cilia was possible with further ATP addition, but was performed for control reasons only. For CBF measurements, antibodies and peptides were diluted in resuspension buffer.
CBF was measured using the rapid automated digital analysis system described earlier (Sisson et al., 2003). Cilia were visualized using phase contrast microscopy (Olympus IMT-2 inverted phase-contrast microscope, Olympus America Inc., Melville, NY) directly connected to a digital camera (Kodak Megaplus ES 310 analog/digital video camera; Eastman Kodak Motion Analysis System Division, San Diego, CA) and a PC workstation (Dell Inc., Round Rock, TX). CBF was determined using Fourier analysis of the entire field of view from the digitized video.
Generation of antibodies against ß tubulin isotypes
Antibodies against the C-termini of ß tubulin isotypes were raised in mouse hybridoma cells and purified as previously described (Banerjee et al., 1990; Banerjee et al., 1992
; Banerjee et al., 1988
; Roach et al., 1998
). Each monoclonal antibody was prepared to an epitope unique to the C-terminus of that isotype. The C-termini of ßIVa and ßIVb are identical in amino acid sequence and therefore the antibody against ßIV tubulin was unable to discriminate between them. All antibodies used in this study were purified monoclonal IgG class 1 antibodies except for anti ßII and ßIII tubulin, which were IgG2b (Table 2). The antibody against the ßV tubulin C-terminus has not been previously reported (A.B. et al., unpublished). The monoclonal antibody SHM 12G11 specific for mouse ßV tubulin was generated by immunizing mice with the C-terminal peptide EEEINE. The peptide, coupled to keyhole limpet hematocyanin (KLH), was used to immunize mice. The antibody was purified from the hybridoma supernatant using a protein-G-sepharose column. Initial immunization was performed with peptide-KLH while the subsequent immunizations were performed with peptide coupled to BSA, according to Banerjee et al. (Banerjee et al., 1988
)
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Other antibodies were commercially obtained. The details are summarized in Table 2. Antibodies were used at a concentration of 20 µg/ml, which was within the range of concentrations used in several previous studies that employed antibodies to interfere with ciliary and flagellar beating (Bré et al., 1996; Audebert et al., 1999
; Cosson et al., 1996
; Gagnon et al., 1996
). Controls to assure specific antibody binding to axonemal tubulin were performed by immunohistochemistry and immunoblotting.
Visualization of antibody-labeled bovine cilia
Isolated cilia were plated onto concanavalin A-coated glass microscope slides (Sigma), allowed to settle for 1 hour and fixed using a solution of 1% paraformaldehyde in PBS. No permeabilizing step was necessary since the cilia are already demembraneted. Bound antibodies were visualized using goat anti-mouse secondary antibodies conjugated to Alexa 488 (Molecular Probes, Eugene, OR). Specimens were viewed using an Axioskop II microscope (Carl Zeiss, Jena, Germany) equipped with 40x and 100x objectives and captured using a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Images were prepared for presentation using Photoshop (Adobe Systems, San Jose, CA). Negative controls were performed by omitting the primary antibody. Positive controls for the isotype-specific ß tubulin antibodies were performed by staining organ of Corti tissue. The results seen were in concordance with previous observations (Hallworth and Ludueña, 2000; Hallworth et al., 2000
; Vent et al., 2004) (see supplementary material Fig. S1).
Immunoblotting
Immunoblots were performed under standard conditions with all antibodies used in the above experiments. A uniform amount of homogenized axonemal proteins (15 µg) was loaded onto each lane of a 10% polyacrylamide gel (Biorad, Hercules, CA). The protein content had been determined in a Bradford dye assay prior to aliquoting the extracted cilia using a microplate reader and protein (BSA) standards (MPM III 1.080, microplate reader, Biorad). The lanes were run at 100 V and 250 mA for 1 hour in sodium dodecyl sulfate electrophoresis running buffer. Proteins were then transferred from the gels to nitrocellulose sheets at 100 V and 100 mA for 1 hour in a transfer buffer containing Trizma base and glycine (Sigma). The nitrocellulose sheets containing the transferred protein lanes were then cut into strips and were exposed to one of the primary antibodies overnight at 4°C. After thorough rinsing with 1% milk in PBS, the secondary antibody (anti-mouse IgG linked to biotin, Cell Signaling Technology, Beverly, MA), was added and the strips were incubated on a rocker for 1 hour at room temperature. The protein strips were then rinsed three times in 1% milk in PBS for 10 minutes and twice in PBS for 10 minutes. They were then treated with SuperSignal West Pico Chemiluminiscent Substrate (Pierce Biotechnology, Inc., Rockford, IL) for 3 minutes and exposed to a CL-XPosure blue X-ray film (Pierce Biotechnology, Inc., Rockford, IL) for three seconds.
Generation of peptides
Axonemal motif peptides were synthesized using a previously published solid phase method (Taylor et al., 2005). Briefly, N-
-butyloxycarbonyl (Boc)-amino acid derivatives (Bachem Biosciences Inc., King of Prussia, PA and ChemImpex, Wood Dale, IL) were coupled to methylbenzhydrylamine resin (Bachem Biosciences Inc., King of Prussia, PA) using the coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in excess base. All coupling yields were >99% as determined by the quantitative ninhydrin method (Sarin et al., 1981
). Boc groups were removed in trifluoroacetic acid (TFA, Acros Organics, Pittsburgh, PA). Peptides were cleaved from the resin using a mixture of trifluoromethanesulphonic acid/TFA/ethanedithiol/thioanisole (1/9/0.5/1, v/v) and immediately desalted on a BioGel P6 column (90x2.5 cm) (Bio Gel P6, Bio Rad, Hercules, CA) in 5% aqueous acetic acid. The desalted material was loaded on to a Vydac C18 HPLC column (25x2.5 cm) (Vydac C18 column, The Sep/a/ra/tions Group, Hesperia, CA) previously equilibrated with a mixture of water and acetonitrile (9/1, v/v) (Fisher Scientific, Pittsburgh, PA) containing 0.1% TFA. The concentration of acetonitrile in the eluent was increased from 10 to 40% over 50 minutes to elute the peptides from the column. Fractions containing the desired peptides were pooled and lyophilized. Peptides were judged to be >95% pure by analytical RP-HPLC and possessed satisfactory amino acid compositions.
The peptides containing the sequences EGEAEEE, EGEGEEE, EDEDEGE and GEMYEDD were custom made peptides purchased from Bachem Biosciences Inc. (King of Prussia, PA). The C-terminal tail peptides were the peptides against which the ß tubulin isotype specific antibodies were raised (see Table 1 and Table 2 for sequences). Their synthesis is described elsewhere (Banerjee et al., 1990; Banerjee et al., 1992
; Banerjee et al., 1988
; Renthal et al., 1993
) (A.B. et al., unpublished). Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich Co. (St Louis, MO).
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Results |
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The effect of the antibodies on CBF is concentration dependent
We hypothesized that the inhibition of ciliary beating by antibodies would be specific and thus concentration dependent. To test this hypothesis, we examined the effect of antibodies at different concentrations. When the concentration of antibodies against ßI, ßIV and ßV tubulin was decreased in a logarithmic manner, the time taken for reduction of CBF to zero was increased. Fig. 1B shows the effect of the antibody against ßI tubulin on CBF at varying concentrations (20 µg/ml, 2 µg/ml and 0.2 µg/ml final concentrations, equivalent to 0.33 µM, 0.033 µM and 3.3 nM, respectively). Comparable results were obtained with antibodies against ßI and ßV tubulin (data not shown). Reduction of antibody concentration by at least two orders of magnitude from the initial concentration was required to reduce the effect of the antibodies to control levels.
Preincubation of axonemes with antibodies inhibited reactivation
As a further test of the specificity of the antibody effect, we pre-incubated cilia with a molar excess (10 µM) of antibody for 1 hour on ice. The high concentration was chosen to assure all epitopes would be bound by antibody. After equilibration to room temperature, ATP was added to activate ciliary beating. Normal activation to a CBF of 10 Hz was achieved in all samples except the ones containing antibodies against ßI, ßIV and ßV tubulin, which did not activate (see supplementary material Fig. S2).
Antibodies against other epitopes of ß tubulin, or against tubulin, have no effect on CBF
We hypothesized that the effects of the antibodies against the C-termini of ßI, ßIV and ßV tubulin on CBF were specific to the C-terminus of tubulin isotypes. To test this hypothesis, we applied monoclonal antibodies against other epitopes of ß and tubulin. Fig. 1C shows the effect on CBF of an antibody against a C-terminal, non-isotype-specific epitope of
tubulin (DM1A) and an antibody against a non-isotype-specific epitope (within position 281-446) of ß tubulin (TUB 2.1). The antibodies had no effect on CBF at the same concentration as the maximum concentration used in the above described studies. Even higher concentrations (200 µg/ml or 0.66 µM) did not affect CBF (data not shown). Fig. 1D shows the effect of two monoclonal non-isotype specific antibodies raised against the C- and N-termini of
tubulin, respectively. They had no effect on CBF at comparable concentrations. These experiments further support the inference that the effects of the antibodies against ßI, ßIV and ßV tubulin were specific to the binding sites of those antibodies, and that axonemal motility is dependent on the C-terminal epitope of ß tubulin isotypes or its direct proximity against which the antibodies are directed. Furthermore, the results indicate that there is no steric hindrance by ineffective antibodies, even against the C-terminus of
tubulin, to the dynein binding site. They also exclude any non-specific inhibitory effect of the antibodies.
Specific peptides reduce CBF
Nielsen et al. (Nielsen et al., 2001) hypothesized that a certain amino acid sequence, the axonemal motif, is required for ciliary function and assembly. Because of our antibody findings, we tested the axonemal motif hypothesis using synthesized heptapeptides containing the axonemal motif or amino acid sequences in corresponding positions of mammalian ßI, ßII, ßIII, ßIV and ßV tubulin, hypothesizing that the peptides would mimic the C-terminus of ß tubulin and thus competitively inhibit beating. We compared their effects to the effects of the C-terminal tail peptides against which the antibodies were raised.
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The results of these experiments are shown in Fig. 2A-E and are summarized in Table 3. In general, the axonemal motif peptides were effective in reducing CBF while the C-terminal tail peptides were much less so. For example, the ßIV and ßV tubulin axonemal motif peptides completely abolished ciliary beating in 15 minutes or less, while the corresponding C-terminal tail peptides had only weak effects on CBF (Fig. 2D,E). Both the axonemal motif and C-terminal tail peptide of ßIII tubulin were ineffective in reducing CBF (Fig. 2C), as expected since ßIII tubulin is not present in the bovine cilia preparation. However, the axonemal motif peptide of ßII tubulin, which is also not present in bovine tracheal cilia, was effective in abolishing ciliary beating, whereas the corresponding C-terminal tail peptide had only marginal effect (Fig. 2B). Further, both ßI tubulin peptides were effective in reducing CBF (Fig. 2A).
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The effect of the axonemal motif peptide is concentration dependent
We hypothesized that the effect of the ßIV tubulin axonemal motif peptide EGEFEEE is specific and therefore concentration dependent. To test this, the peptide was added to the activated cilia preparation at concentrations of 0.33 µM, 0.033 µM and 3.3 nM. The highest concentration of peptide reduced CBF the fastest, while the lowest concentration was essentially ineffective (Fig. 2F).
The central phenylalanine is important in the axonemal motif sequence
We observed that the axonemal motif peptides that reduced CBF contained, in addition to several acidic residues, a central F at position 436. Also, F436 is not present in the axonemal motif peptide of ßIII tubulin, which did not reduce CBF. To test the hypothesis that F436 is important, we replaced F436 in the axonemal motif sequence (EGEFEEE) with alanine, resulting in the sequence EGEAEEE. As shown in Fig. 3A, this peptide had no effect on CBF, demonstrating the importance of F436.
Axonemal motif-like sequences in the C-terminus of tubulin are not involved in ciliary beating
An E-rich sequence similar to the axonemal motif is present in the C-termini of several tubulin isotypes between positions 441 and 447, but these sequences lack the central F (Table 4). To test the hypothesis that the C-terminus of
tubulin is not involved in ciliary beating, we synthesized two peptides that represented the sequences of four
tubulin isotypes: EGEGEEE (
II,
III) and EDEDEGE (
I,
IV) (Ludueña and Banerjee, 2005a
; Ludueña and Banerjee, 2005b
; Ludueña and Banerjee, 2005c
; Ludueña and Woodward, 1975
). Neither peptide affected ciliary beating at comparable concentrations to previous experiments, supporting the antibody observations that the C-terminus of
tubulin may not be directly involved in ciliary beating (Fig. 1C,D and Fig. 3B).
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Antibodies are specific for axonemal tubulin
We hypothesized that the effects of antibodies against ßI, ßIV and ßV tubulin on ciliary beating were specific for binding at the ß tubulin isotype specific epitopes and are not due to unspecific inhibition of beating. To test this hypothesis, all antibodies were applied to fixed preparations of bovine cilia and processed for immunofluorescence as described in the Materials and Methods. Antibodies against ßI, ßIV and ßV tubulin, which blocked ciliary beating, labeled cilia (Fig. 4). The same was observed for the antibodies against the C- and N-termini of tubulin, as well as for antibodies against conserved, non-isotype-specific epitopes in the
and ß tubulin protein (DM1A and TUB 2.1). However, they did not inhibit ciliary beating, making steric hindrance as a reason for CBF inhibition by anti ßI, ßIV and ßV tubulin antibodies unlikely. As expected, antibodies against ß tubulin isotypes not present in tracheal cilia (anti ßII and anti ßIII tubulin) did not label isolated cilia in the described preparation (Fig. 4A,B).
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Immunoblots of purified de-membranated cilia with all of the above mentioned primary antibodies indicated that labeling is restricted to a single band of molecular mass close to 55 kDa (Fig. 4B), the expected molecular mass of ß tubulin (Ludueña, 1998). The amino acid sequence difference between ß tubulin isotypes is so small that the isotypes are indistinguishable by molecular mass alone. Strong labeling appeared with anti ßI, DM1A and Tub 2.1 (antibodies against conserved, non-isotype specific epitopes of
and ß tubulin) as well as the C- and N-terminus specific antibodies against
tubulin. As expected, antibodies against ßII and ßIII tubulin did not label the axonemal proteins at all, because ßII and ßIII tubulins are not synthesized in bovine tracheal cilia. A weak band occurred at 55 kDa with labeling by antibodies against ßIV and ßV tubulin. Controls for the well-established antibodies against ßII and ßIII tubulin were performed by staining the organ of Corti (supplementary material Fig. S1).
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Discussion |
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The axonemal motif sequence pivots around the central F
We found that, in general, C-terminal acidic residues (E or D) are required for ciliary beating. The higher the content of acidic amino acids in the peptide, the stronger was the inhibitory effect on beating. Further, we showed that this region must pivot around the central F436 found in some ß tubulin isotypes. However, the study referred to earlier by Okada and Hirokawa did not evaluate dynein and did not focus on the isotype-specific differences in the C-termini of ß tubulins (Okada and Hirokawa, 2000). In our study, the further requirement of a central F was established in addition to acidity as a key characteristic for axonemal function. In general, the C-terminal tail peptides, which did not contain F, were less effective than the axonemal motif peptides, with the exception of the peptides for ßI and ßV tubulin, which were equally effective. Our hypothesis is supported by the lack of effect of the two
tubulin peptides (Fig. 3) which, despite being highly acidic, lack the central F.
tubulin is not directly involved in ciliary beating
Our evidence that tubulin is not directly involved in ciliary beating is, on first inspection, surprising. Three different antibodies against
tubulin, two of which were directed against a conserved, non-isotype specific C-terminal amino acid sequence (419-435 and 426-450), had no effect on ciliary function. Furthermore, peptides of the
tubulin axonemal motif sequence did not affect CBF. Our results are consistent with recent literature that suggests no involvement of
tubulin in ciliary beating (Audebert et al., 1999
; Cosson et al., 1996
). Some earlier investigations favoured
tubulin being important for ciliary function (Gagnon et al., 1996
; Hirose et al., 1999
). Goldsmith et al. (Goldsmith et al., 1991
; Goldsmith et al., 1995
) suggest the involvement of both
and ß tubulin for dynein binding (Goldsmith et al., 1991
; Goldsmith et al., 1995
). The role of
tubulin might lie in other, yet undetermined functions. For example,
tubulin may function in binding of microtubule-associated proteins (MAPs) (Rodionov et al., 1990
) or interact with dynein for control of microtubule dynamics (Hunter and Wordeman, 2000
). However, those studies were conducted on cytoplasmic dynein. Hunter and Wordeman showed that the C-terminus of tubulin is necessary for MAP binding and further suggested a tubulin-binding site of dynein outside the C-terminus. Hoenger et al. describe that each kinesin dimer occupies two microtubule-binding sites (Hoenger et al., 2000
). That might be true for dynein also, but has not been investigated so far.
The periodicity of the ß tubulin heterodimer in the microtubule is 8 nm. In cytoplasmic dynein, the step size has been proposed to be dependent on cargo load and to have a minimum of 8 nm (Mallik et al., 2004
). If the step size of axonemal dynein is similar, dynein may skip
tubulin and interact only with the C-terminus of ß tubulin.
The sequences of the peptides EDEDEGE447 (in I and
IV tubulin) and EGEGEEE447 (in
II and
III tubulin) were derived from Ludueña and Banerjee (Ludueña and Banerjee, 2005a
). Due to the absence of isotype specific antibodies for
tubulins, previous information on the possible distribution of
tubulin isotypes was obtained by in situ hybridization. The presence of these
isotypes (I-IV) in the bovine tracheal cilia preparation is plausible but not established (Ludueña and Banerjee, 2005a
), thus there is a small possibility that the peptides have no effect due to the absence of these
tubulin isotypes in bovine cilia. However, antibodies against various non-isotype-specific epitopes of
tubulin, including the C-terminus, also did not affect CBF.
Post-translational modifications may alter the secondary structure of the C-terminus
Post-translational modifications (PTM) in tubulins occur mainly at the highly flexible C-terminus (Ludueña, 1998). The most recently discovered PTM, polyglycylation, has been shown to occur in axonemal tubulin from Paramecium to sea urchin and mammalian spermatozoa (Bré et al., 1996
). Bré et al. also suggested an involvement of polyglycylated tubulin in axoneme motility since AXO 49 and TAP 952, monoclonal antibodies against mono- and polyglyclyated C-terminal peptides from Paramecium axonemal tubulin, specifically inhibited the reactivated motility of sea urchin spermatozoa. Polyglycylation occurs at the C-terminus and is highly variable in its amount (up to 32 glycines on a side chain off the
-carboxyl chain of glutamic acids E435, E437 and E438). Polyglycylation has not yet been shown to be functionally required, but may affect the secondary structure of the highly flexible C-terminus in tubulin. Gagnon et al. reported that a different PTM, polyglutamylation, in the lateral chain of
tubulin plays a dynamic role in spermatozoan motility (Gagnon et al., 1996
). Possibly due to steric hindrance by the large antibody, Gagnon et al. could not determine the amino acid sequence required for ciliary beating.
Isotype specificity of ß tubulin in axonemes
This leads us back to the initial question of the existence of functional correlations of ß tubulin isotypes: why do some cells synthesize ß tubulin isotypes in one pattern and others synthesize different isotypes? Why, if the isotypes are so similar in amino acid sequence, is there still a requirement for the different isotypes? The answer may lie in the highly variable C-terminus.
It was previously found that ßI and ßIV tubulin are synthesized by all ciliated cells types tested in the gerbil (Jensen-Smith et al., 2003). It was inferred that both isotypes are required for axonemal assembly and/or function. However, those observations were based on immunohistochemistry alone. ßV tubulin was recently found to be in some but not all motile cilia (R. Hallworth and R. F. Ludueña, unpublished observations). Therefore, ßV tubulin may be capable of supporting ciliary beating, but may not be absolutely required for ciliary function. ßII tubulin has so far not been detected in cilia. However, its axonemal motif-like sequence differs only in the first amino acid from the axonemal motif sequence of ßIV tubulin (E433 to Q433). Further, the axonemal motif peptide of ßII tubulin was nearly as potent as that of ßIV tubulin in blocking ciliary beating. Is ßII tubulin incompatible with the correct axonemal assembly or function? A change in only position 433 from glutamate to glutamine is observed, resulting in a removal of one charge in ßII tubulin. This may indicate that the secondary structure of the synthetic peptide resembles the axonemal motif peptide more closely than does the corresponding sequence in ßII tubulin. It is worth noting that the unusual ß tubulin isotypes ßII and ßIII are in some circumstances capable of supporting axonal assembly, if not motility. In the globose basal cells of the nose (the olfactory stem cells) ßI, ßII and ßIII tubulin are synthesized and incorporated into microtubules. As soon as the cell matures and develops long, immotile sensory cilia, ßIV tubulin is also synthesized (Woo et al., 2002
). The isotypes do not compartmentalize, that is, all synthesized isotypes are incorporated into all microtubule structures in the cell, including the cilia. Thus ßII tubulin and ßIII tubulin are at least capable of assembling into immotile cilia. It remains to been seen if ßII tubulin and ßIII tubulin are capable of supporting ciliary motility and the assembly into a 9+2 structure, or if they disrupt these.
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Acknowledgments |
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Footnotes |
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* Present address: University Hospital of Cologne, Department of Otolaryngology, Head and Neck Surgery, Kerpener Straße 62, 50924 Cologne, Germany
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