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Here we investigated whether the sensitivity of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression of katanin for short periods of time produced breaks preferentially in these regions. In fibroblasts with experimentally enhanced or diminished microtubule acetylation, the sensitivity of the microtubules to severing by katanin was increased or decreased respectively. In neurons, microtubules are notably more acetylated in axons than in dendrites. Experimental manipulation of microtubule acetylation in neurons yielded similar results on dendrites as observed on fibroblasts. However, under these experimental conditions, axonal microtubules were not appreciably altered with regard to their sensitivity to katanin. We hypothesized that this may be due to the effects of tau on the axonal microtubules, and this was validated by studies in which overexpression of tau caused microtubules in dendrites and fibroblasts to be more resistant to severing by katanin in a manner that was not dependent upon the acetylation state of the microtubules. Interestingly, none of these various findings apply to spastin, as the severing of microtubules by spastin does not appear to be strongly influenced either by the acetylation state of the microtubules or by tau. We conclude that sensitivity to microtubule-severing by katanin is regulated by a balance of factors including the acetylation state of the microtubules and the binding of tau to the microtubules. In the neuron, this contributes to regional differences in the microtubule arrays of axons and dendrites.
Microtubule-severing is a physiological process whereby severing proteins such as katanin and spastin break the lattice of the microtubule through ATP hydrolysis (Roll-Mecak and McNally, 2009). This results in higher numbers of microtubules and more free ends of microtubules to interact with various proteins and structures in the cell (Roll-Mecak and Vale, 2006). In addition, the shorter length of the microtubules renders them more readily transported and reorganized by molecular motor proteins (Baas et al., 2006). Cells generally express high amounts of the severing proteins, typically high enough to completely sever all cellular microtubules if the severing proteins had unfettered access to them (Solowska et al., 2008). This raises the question as to how the severing activities of katanin and spastin are regulated, and how they are targeted to particular microtubules or particular regions on microtubules.
Microtubule-severing is generally considered to impact more stable microtubules. An attractive possibility, in this regard, is that the severing proteins are targeted to microtubules that are rich in post-translational modifications that accompany stability. Indeed, there is a growing body of evidence suggesting that various microtubule-related proteins have differential affinities for microtubules rich or deficient in tubulin subunits that bear modifications such as acetylation, detyrosination, or polyglutamylation (Hammond et al., 2008). For example, recent studies suggest that acetylation may play an important role in regulating the degree to which kinesin-1 interacts with the microtubules (Reed et al., 2006).
Acetylation occurs on alpha-tubulin, and is unique among the various modifications in that it occurs on the luminal face of the microtubule (for review, see Hammond et al., 2008). It occurs on lysine 40, which is deeply embedded within the tubulin subunit. Thus, compared to the other modifications, it is less clear how acetylation could affect the properties of the microtubule or the manner by which it interacts with other proteins. In addition, it is unclear how the enzymes that add or remove the acetyl group access the relevant site. Two different enzymes have been identified which can remove the acetyl group, namely HDAC6 and Sirt2. Depletion of either enzyme results in markedly enhanced acetylation of microtubules, suggesting that the two enzymes may function coordinately. A variety of studies over the years indicate that acetylation is the result of stabilization and not the cause of it, although one somewhat controversial study suggests otherwise (Matsuyama et al., 2002).
In neurons, microtubule-severing is critically important for the development of axons and dendrites, and is particularly robust at sites of impending branch formation (Yu et al., 2005, 2008; Riano et al., 2009). It is known that microtubules in axons are generally richer in acetylated tubulin than are dendritic microtubules, that acetylated tubulin is unevenly distributed along the length of the microtubules, and that microtubules vary in their levels of acetylated tubulin in different regions of the axon (Cambray-Deakin and Burgoyne, 1987; Baas et al., 1991). For these reasons, acetylation makes particularly good sense in the neuron as a potential means to earmark certain microtubules for severing.
Constructs used for these studies were the following: pEGFP-C1 (control construct in experiments for neuron; BD Biosciences, Boston, MA); pEGFP-C1-p60 (C-terminally EGFP tagged rat p60-katanin) (Qiang et al., 2006); pEGFP-C1-M85 (C- terminally EGFP tagged mouse spastin translated from the second start codon); pcDNA-human HDAC6-flag (ID:13823) (addgene, Cambridge, MA); pRC/CMV-Flaghtau441wt (flag-tagged human four-repeat tau; provided by Dr. R. Brandt, University of Osnabrück, Barbarastrasse, Germany).
Trichostatin A (TSA) was purchased Upstate Biotechnology (Lake Placid, NY). Taxol was purchased from Sigma (St. Louis, MO). Tubacin was provided by Dr. Stuart L. Schreiber (Harvard University, Cambridge, MA).
RFL-6 cells: Rat RFL-6 fibroblasts were cultured as previously described (Qiang et al., 2006) and transfected with pEGFP-C1-P60, pEGFP-C1-M85, pHDAC6-flag, or pRC/CMV-Flaghtau441wt using a Nucleofector (Amaxa, Gaithersburg, MD) with the manufacturer’s program G-13. Fifteen micrograms of each plasmid and 106 cells were used for each transfection. The cells were plated at a density of 4000 cells per well on glass coverslips mounted in the bottom of 35-mm-diameterPetri dishes with holes drilled in the bottom. Taxol, tubacin, TSA were used at 5 μM, 10 μM, and 300 nM, respectively. Drugs were added to the culture immediately after transfection. Cells were cultured for 24 h (experiments in figure 1–3 and and6)6) or 12 h (experiments in figure 4) after transfection before being fixed. Hippocampal neurons: Rat hippocampal neurons were prepared as described previously (Yu and Baas, 1994), except that they were were cultured in the presence of conditioned media obtained from primary cultured astroglial cells derived from neonatal rat whole brain. The conditioned medium is a filtered supernatant of culture media of astroglial cells which had been cultured for two days under the hippocampal neuron plating medium (Neurobasal medium supplemented with 2% B27, 0.3% glucose, 1 mM glutamine, and 5% FBS). The cells were plated at a density of 3000 cells per well on glass coverslips mounted onto the bottom of 35-mm-diameterPetri dishes with holes drilled in the bottom. The transfections of pEGFP-C1-P60, pEGFP-C1-M85, pHDAC6-flag, or pRC/CMV-Flaghtau441wt were performed 8 d after the plating. For transfections of those constructs, we used Lipofectamine 2000 (Invitrogen, San Diego, CA). Two micrograms of DNA constructs and 5 μl of Lipofectamine 2000 were used per 35 mm dish. The cells were incubated in the DNA/Lipofectamine-containing medium for 5 h. Then neurons were transferred to fresh 37°C hippocampal neuron plating medium. In experiments using drugs, the drug was added to the medium at this time. Twenty four hours later, cells were fixed for immunostaining. Transfection efficiency was generally 5–10% for neuronal cultures and 15–30% for RFL-6 cell cultures.
Cy3 conjugated monoclonal anti-β-tubulin (1:150; for general tubulin staining), monoclonal anti-acetylated-tubulin (6-11B-1) (1:400), monoclonal anti-flag (M2) (1:500), and monoclonal anti-GFP (1:250; for the enhancement of GFP signals in experiments of neuron) antibodies were purchased from Sigma. Rabbit polyclonal anti-GFP antibody (1:250; for the enhancement of GFP signals in experiment of figure 4) was purchased (Abcam, Cambridge, UK). Rabbit polyclonal anti-human HDAC6 (H-300) (1:200) was purchased (Santa Cruz Biotechnology, Santa Cruz, CA). Monoclonal anti-polyglutamylated-tubulin antibody (GT335) (1:500) was provided by Dr. Carsten Janke (CNRS, Montpellier, France). Monoclonal anti-detyrosinated-tubulin antibody (4B8) (1:300) was provided by Dr. G.G. Gundersen (Columbia University, New York, NY). Monoclonal anti-tau antibody (Tau-1), used for dephosphorylated tau staining (1:500), was purchased from Sigma.
For immunofluorescence studies, cultures were briefly washed with 37°C phosphate buffered-saline (PBS) and then simultaneously fixed and extracted with 4% paraformaldehyde, 0.2% glutaraldehyde, and 0.1% Triton X-100 for 15 min. Cultures were washed with PBS three times for 5 min, quenched with 2 mg/ml sodium borohydride three times for 10 min, and then blocked with 10% normal goat serum and 10 mg/ml BSA in PBS for 1 h. For immunostaining of the expressed tau, HDAC6, and/or severing proteins, cultures were prepared as described previously (Karabay et al., 2004). In experiments using RFL-6 except for that of figure 4, we detected the GFP signals of tagged-severing proteins without further enhancement while in experiments using neurons or that of figure 4, we detected the GFP signals of tagged-severing proteins with enhancement by staining with monoclonal (for neurons) or polyclonal (for figure 4) anti-GFP antibody. We detected expressed HDAC6 with anti-human HDAC6 antibody which could reveal the presence of exogenous human HDAC6 in rat derived fibroblasts or neurons.
We detected expressed human tau protein by staining with Tau-1 antibody in both RFL-6 cells and neurons when they were co-stained with anti-acetylated-tubulin antibody. In RFL-6 cells we could clearly identify highly exogenous tau expressing cells since they do not express endogenous tau. Although neurons have endogenous tau, we could identify transfected cells with much higher expression levels of tau, especially in their dendrites. In exogenous tau expressing RFL-6 cells and neurons we identified morphological changes in general tubulin staining, namely microtubule bundle formation in RFL-6 cells and the generation of long straight microtubules crossing in all directions in the cell bodies of neurons. The tau expression by itself in neurons caused the reduction of microtubule levels in cell bodies. We identified tau expressing RFL-6 cells and neurons based on those morphological characteristics in the following experiments in which we tested the microtubule sensitivities to exogenous katanin.
To quantify microtubule mass, both fibroblasts and neurons were simultaneously fixed and extracted to remove free tubulin as described previously(Yu et al., 2005), then immunostained for HDAC6, tau (in the case of figure 6), and GFP (in the case of neurons and figure 4). Cultures were then treated with appropriate fluorescence-conjugated secondary antibodies, and subsequently with Cy3-conjugated anti-β-tubulin antibody as third antibody. So that samples could be compared against one another, microscope settings were adjusted to equilibrate the signal. In experiments on cultured neurons, we selected only neurons that were sufficiently separated from neighboring cells that we could distinguish axons from dendrites on the basis of morphology. Images were acquired with an AxioVert 200M microscope (Carl Zeiss, Oberkochen, Germany) coupled with an Orca-ER Digital CCD (Hamamatsu, Shizouka, Japan) and a 100x Plan-Neorofluar/1.3 numerical aperture objective. Except for the GFP signal quantification (see below), images to be compared were taken at identical settings of exposure time, brightness, and contrast and analyzed with Axiovision 4.0 software. Measurements were taken as total fluorescence intensity per cell in the case of RFL-6 cells, and total fluorescence intensity in 10 μm regions of axons or dendrites. In the latter case, we made sure to include the region of strongest fluorescence within any given axon or dendrite. Statistics were done using Student’s t test. In RFL-6 experiments, we classified GFP-fusion protein expressing cells according to their total GFP signal intensity per cell. We set exposure times 150 ms and 750 ms for GFP-katanin and GFP-spastin detection, respectively. Other settings including brightness and contrast are identical throughout the experiments. We defined cells possessing background-subtracted GFP fluorescence intensity from 0 to 300 and 0 to 400 arbitrary fluorescence units (AFU) as low expressers, from 300 to 800 AFU and from 400 to 700 as medium expressers, from 800 to 2000 and from 700 to 2000 as high expressers, for GFP-katanin and GFP-spastin expressing cells, respectively.
For the present studies, as with our earlier work on the microtubule-severing proteins (Qiang et al., 2006, 2010; Yu et al., 2008), we used RFL-6 rat fibroblasts and cultured rat hippocampal neurons. The fibroblasts are useful for high-resolution imaging of microtubules because they are so flat. In addition, they are useful for certain studies because they do not endogenously express microtubule-associated proteins such as tau that could impact the sensitivity of the microtubules to severing. The hippocampal neurons are useful because they generate bona fide and morphologically distinguishable axons and dendrites.
In an earlier study, we found that treatment of RFL-6 rat fibroblasts with taxol does not alter the diminution of microtubule mass observed with katanin overexpression, despite the fact that taxol stabilizes and bundles microtubules (Qiang et al., 2006). This was surprising because, even if taxol has no effect on sensitivity to katanin, we would have thought that the greater stability of the microtubules in the presence of taxol should reduce the ensuing depolymerization of microtubule mass that occurs as a result of the severing. On this basis, we became suspicious that taxol treatment might actually render the microtubules more sensitive to severing by katanin. Given that taxol causes a rapid accumulation of tubulin posttranslational modifications (Gundersen et al., 1987; Piperno et al., 1987), we hypothesized that it might be one or more of these modifications that renders the microtubules more sensitive to severing by katanin. To investigate this further, we used previously-characterized tools for the study of microtubule acetylation in living cells. HDAC6, a histone deacetylase that is specific for tubulin, has been reported to cause global deacetylation of microtubules when overexpressed in cells (Hubbert et al., 2002; Zhao et al., 2009). TSA is a membrane-permeable drug that inhibits a broad range of deacetylases, and hence causes microtubules in cells to become highly acetylated (Matsuyama et al., 2002; Chang et al., 2009; Zilberman et al., 2009). Tubacin, a more recently developed membrane-permeable drug, is preferable for these studies because it specifically inhibits HDAC6 and hence elevates tubulin acetylation without affecting other cellular proteins that can be acetylated (Haggarty et al., 2003; Chang et al., 2009; Zilberman et al., 2009).
In a first set of experiments, we confirmed the efficacy of these tools in RFL-6 fibroblasts. In some cases, cultures were transfected to overexpress HDAC6 (using a human HDAC6 construct with a flag tag; see Materials and Methods), and then fixed 24 hours later. Other cultures were treated with either 300 nM TSA, 10 μM tubacin, or 0.1% DMSO (vehicle alone, control) for 24 hours, and then fixed. The fixed cultures were then prepared for multi-channel immunofluorescence visualization of general tubulin and acetylated tubulin. In the case of the HDAC6 overexpression studies, cultures were also immuno-labeled for human HDAC6. Details on immunofluorescence procedures and antibodies are provided in the Materials and Methods section. Representative images are shown in figure 1. Figure 1 (panels a-c) show staining for general tubulin while lower panels (d-f) show staining for acetylated tubulin in the same cells. Control cells displayed a broad splayed array of microtubules revealed with the antibody for general tubulin, and a center-focused sub-array of curved microtubules revealed by the antibody for acetylated tubulin (figure 1a and d). This is the typical pattern for general and acetylated tubulin observed in most tissue culture fibroblasts (Piperno et al., 1987). In HDAC6-overexpressing cells, we observed notable reduction in staining for acetylated tubulin. The morphology of the cells appeared slightly more elongated than controls and the microtubules appeared slightly less splayed (figure 1b and e). It was necessary to select the most highly overexpressing cells to visualize the notable reduction in acetylation, so from this point forward for all studies reported here with HDAC6, we chose high expressers. In tubacin-treated cultures, all cells displayed uniformly and dramatically stronger staining for acetylated tubulin, but there were no recognizable differences in morphology or general tubulin staining relative to controls cells (figure 1c and f). TSA treatment yielded results similar to tubacin treatment (data not shown). The effects of tubacin, TSA, and HDAC6 were confirmed in three independent experiments for each condition. These results are consistent with those of previous reports (Hubbert et al., 2002, and Haggarty et al., 2003). Quantitative analysis (figure 3k) showed there are slight increases in total microtubule levels in tubacin and TSA treated cells, respectively but no such difference in HDAC6 expressing cells compared with controls.
Taxol treatment is known to cause microtubules in cells to become highly acetylated, detyrosinated, and polyglutamylated (Gundersen et al., 1987; Piperno et al., 1987). We wished to ascertain whether taxol might impact microtubule-severing by katanin differently if we specifically inhibit its capacity to enhance microtubule acetylation. For this, we introduced taxol into the cultures simultaneously with the HDAC6 transfection. As shown in figure 2A, this experimental regime was effective at severely diminishing the levels of microtubule acetylation with no diminution in the levels of the other two modifications. In addition, there was no apparent effect on the levels or bundling of the microtubules as a result of diminished acetylation during taxol treatment. These results are important because in some experimental regimes there seems to be a link among the modifications such that affecting one can affect the others (Redeker et al., 2005; Ikegami et al., 2007). Figure 2B (panels a-c) further confirms that taxol treatment results in dense microtubule bundles whether or not HDAC6 is overexpressed. The increase in total microtubule mass as a result of taxol treatment was 30% higher than control cells whereas the increase in total acetylated-microtubule mass was 616% higher than control cells (data not shown), indicating that the increased immunoreactivity for acetylated microtubule is indeed due to increased acetylation of microtubules, not just increased microtubule levels. High overexpression of katanin (rat GFP-p60-katanin; see Karabay et al., 2004; Yu et al., 2005, 2008; Qiang et al., 2006) resulted in 85% loss of microtubule mass in control cells, 83% loss in taxol-treated cells, and 75% loss in cells treated with taxol during HDAC6 overexpression (figure 2B, panel g). However, if we analyzed the medium-expressers, we observed only 45% loss of microtubule mass in control cells and 73% loss in taxol-treated cells, but no significant loss (p>0.05) of microtubule mass if HDAC6 overexpression was included with the taxol treatment (with significant difference between katanin alone and katanin+taxol (p<0.01) (n=3, 25 cells were analyzed for each condition) (figure 2B, panels d-g). These data support the hypothesis that the acetylation state of the microtubules is a critical factor in determining the sensitivity of the microtubules to being severed by katanin (although very high levels of katanin can apparently override this effect).
To further test the hypothesis, we conducted additional experiments without taxol but including the drugs that enhance microtubule acetylation. In addition, we included in these studies spastin, the other microtubule-severing protein that has been extensively studied in neuronal and non-neuronal cells (Solowska et al., 2008; Zhang et al., 2007). Again, cells were sorted with regard to levels of katanin or spastin expression, with medium expressers quantified separately from high expressers. Representative images are shown in figure 3a-j, and the quantitative data on microtubule levels are shown in figure 3k. In medium expressers of katanin (40% loss compared with control) but not spastin (57% loss), we recognized significant down-regulation or up-regulation of microtubule-severing in respective manipulations (figure 3c-e and h-j). As shown by the data quantification (figure 3k) (n=3, 25 cells were analyzed for each condition), the effects in both directions were statistically significant especially in middle level expressers. We observed 29 and 24% (p<0.01) significantly more severing in tubacin and TSA treated cells, respectively while 31% (p<0.01) significantly less severing in HDAC6 expressing cells. We should note that although HDAC6 and katanin co-expressers showed protection of total microtubules from severing, they also showed a tendency of loss of their convergence in microtubule array to cell center, suggesting potential involvement of previously reported HDAC6-mediated primary cilia absorption mechanisms in this phenomenon (Pugacheva et al., 2007). Taken together, these results support the conclusion from the taxol studies that microtubules rich in acetylated tubulin are favored for severing by katanin, but also indicate that the same is not true of spastin.
We next wished to pursue the conclusion from the above studies without the use of experimental manipulation of microtubule acetylation. If the conclusion from the above experiments is correct, we would surmise that katanin should preferentially cut the microtubules in regions normally rich in acetylated tubulin compared to regions deficient in acetylated tubulin, while spastin should have no such preference. When the severing proteins are overexpressed in cells, particularly for somewhat shorter periods of time, microtubules that had been in the process of being severed at the time of fixation can be readily visualized (see for example Yu et al., 2008). So, to pursue this, we fixed the cells after only 12 hours of katanin or spastin overexpression, and then stained them for general tubulin and acetylated tubulin with enhancement of GFP signals by rabbit polyclonal anti-GFP antibodies followed by FITC-conjugated anti-rabbit IgG as second antibody. For these studies, it was also helpful to focus on low expressers (in the presence of enhancement) so that we could more optimally discern microtubules in the process of being severed, and it was helpful to focus our analyses on the more central region of the cells where acetylated regions of microtubules are most abundant. Breaks were identified with the general tubulin staining, and then for each identified break, an image was obtained for acetylated tubulin. Each break was then scored as positive if a detectable signal for acetylated tubulin was observed within 1 μm of the break on both sides of the break, and scored as negative if this was not the case (see figure 4). Percentages were calculated per each of experiments, and then a mean percentage for each severing protein was calculated. Using this procedure, we found that 62% of the breaks observed with katanin were in acetylated regions of the microtubules (see figures 4a,b,d,e,g,h for two different examples), while only 24% of breaks observed with spastin were in acetylated regions (see figures 4c,f,i for a typical break, in a microtubule region not rich in acetylated tubulin). Three independent experiments were performed for each of conditions and more than 30 total breaks from more than 30 expressing cells were chosen for the analysis in each of experiments (katanin: 62 ± 4.3 %, spastin: 24 ± 5.3 %; there is a significant difference; p<0.01). To understand the meaning of these observations, we also calculated the percentage of randomly selected spots on microtubules that turned out to be flanked with acetylated regions. Microtubules were chosen in the same area of the control cells as in the case of the katanin or spastin overexpressing cells. The percentage of these spots that turned out to be flanked by acetylated regions was 20.0 ± 3.4 %, which is only slightly lower than the percentage of breaks that occurred in acetylated regions when spastin was overexpressed but markedly lower than the percentage of breaks that occurred in acetylated regions when katanin was overexpressed. These observations support the conclusion that katanin, but not spastin, has a strong preference for severing microtubule regions rich in acetylated tubulin.
The results on RFL-6 fibroblasts indicate that acetylation can be a critical factor in the sensitivity of cellular microtubules to severing by katanin, but the question remains as to whether this is a major regulatory mechanism in more complex cell types. Our next step was to test whether acetylation influences sensitivity of microtubules to katanin in neurons. Typical vertebrate neurons simultaneously maintain two different kinds of processes: a single axon and multiple dendrites. Microtubules in the axon are known to be much richer in acetylated tubulin than microtubules in dendrites (Cambray-Deakin and Burgoyne, 1987; Baas et al., 1991), so acetylation makes sense as a potential means to locally differentiate microtubule-severing in each compartment. We previously reported that 12 hours of katanin overexpression results in substantial loss of microtubule mass from the cell body and dendrites of cultured rat hippocampal neurons, but not from the axon (Yu et al., 2005). On the surface, this would appear to be the opposite of what would be expected on the basis of the results in fibroblasts described above, given that axonal microtubules are more highly acetylated. To investigate further, we used the same tools to manipulate acetylation as we used on the experiments with fibroblasts.
For these studies, we used rat hippocampal neurons that had been grown in culture for 9 days, by which time axonal arbors were extensive and bona fide dendrites had formed. Tubacin, TSA or DMSO alone was included in the media during the last 24 hours of culture. Transfection with HDAC6 was conducted (using lipofectamine for transfection, see Materials and Methods) during the final day of culture as well. Treatment with tubacin or TSA had no obvious effect on either the morphology of the neurons or amount of microtubule, and the same was true of HDAC6 overexpression (figure 5a-c). Dendritic microtubules showed similar responses to our manipulations (figure 5e, f, and k) as that of fibroblasts while axonal microtubules did not (figure 5e, f, and j). On the other hand, neurons overexpressing spastin displayed no difference in the sensitivity of the microtubules in axons compared to dendrites, as we previously reported (Yu et al., 2008). Similarly, no changes in response to the acetylation manipulations were found (figure 5g, h, i, j, and k). Quantitatively, the amount of axonal microtubule in respective conditions are; control (2790 ± 231), HDAC6 (2630 ± 379), tubacin (2843 ± 175), katanin (2509 ± 262), katanin+HDAC6 (2559 ± 238), katanin+tubacin (1932 ± 162), spastin (1769 ± 264), spastin+HDAC6 (1608 ± 239), and spastin+tubacin (1678 ± 305) as indicated by (mean microtubule mass (AFU) ± S.D.) formula (n=3, 25 cells were analyzed for each condition). Dendritic microtubules in respective conditions are; control (2387 ± 404), HDAC6 (2405 ± 416), tubacin (2268 ± 307), katanin (1574 ± 217), katanin+HDAC6 (2480 ± 390), katanin+tubacin (891 ± 298), spastin (1401 ± 320), spastin+HDAC6 (1333 ± 356), and spastin+tubacin (1513 ± 471). There are significant differences between katanin-expressing dendrites and katanin+HDAC6 dendrites or between katanin-expressing dendrites and katanin+tubacin dendrites (p<0.01) while we could not find consistent significant differences in axons in the same sets of comparisons at the same significance level (i.e.; p<0.01). Also, we could not find significant differences (p>0.01) in either axons or dendrites of spastin expressers in the same sets of comparisons as above.
Taken together, these results indicate a very similar response with the dendrites to what was observed with the fibroblasts; specifically that elevation and diminution of acetylation causes the microtubules to be more and less sensitive to severing by katanin (but not spastin) respectively. By contrast, this was not the case with the axon.
Tau is generally considered to be an axonal protein (at least the dephosphorylated variant recognized by the tau-1 antibody; see for example Bradke and Dotti, 2000) but is not completely absent from dendrites (see for example, Kosik and Finch, 1987). In a recent study, we demonstrated that the stronger resistance to katanin of axonal microtubules compared to dendritic microtubules owes to the presence of tau on the axonal microtubules (Qiang et al., 2006). In this study, when tau was experimentally depleted from the axon, the microtubules became equally as sensitive to katanin overexpression as microtubules in dendrites, immature processes, and the cell body. Based on this, we wondered if the binding of tau to the microtubules renders the status of their acetylation less influential in determining their sensitivity to katanin. In order to test this hypothesis, we overexpressed tau in RFL-6 cells (figure 6Aa-f), either alone or in conjunction with HDAC6 overexpression or treatment with tubacin. Cells expressing tau alone displayed dense bundles of microtubules (figure 6Aa and d), and this was also true of tau-overexpressing cells with the two different acetylation manipulations (figure 6Ab, c, e, and f). The HDAC6 was successful in lowering microtubule acetylation and the tubacin treatment was successful in elevating acetylation in the tau-overexpressing cells (figure 6Ae and f). This was important to confirm because some studies have suggested that tau can interact with HDAC6 (Ding et al., 2008) and inhibit its activity (Perez et al., 2009). We examined if there were any responses in microtubule sensitivity to altered acetylation in tau-overexpressing RFL-6 cells (figure 6B). Katanin overexpressers showed no reduction in microtubules when they co-expressed tau (no microtubule loss in tau and medium-katanin co-expressing cells compared with 39% loss in medium-katanin alone expressing cells), which was consistent with our previous report. The manipulation of acetylation no longer changed the sensitivity (figure 6Bf-i). Quantitatively, in medium katanin expressers with tau co-expression, only slight reduction or increase in microtubule mass (compared with tau alone) were observed when they were treated with tubacin or HDAC6, respectively, and these reductions proved to be statistically insignificant (p values of 0.56 and 0.88, respectively) (n=3, 25 cells were analyzed for each condition). These observations are consistent with the conclusion that the strong protection against katanin afforded by tau overrides the influence of microtubule acetylation observed in the absence of tau.
Finally, we tested whether the sensitivity of ectopically tau-expressing dendrites was changed in response to altered acetylation. For this purpose, we manipulated neurons under tau overexpression and immunostained them for general tubulin and acetylated-tubulin (figure 7). Consistent with the effects we observed in dendrites in figure 5, we indeed observed significant effects of our tools with regard to the acetylation levels in all three compartments (figure 7a-f). In tau alone overexpressing neurons, in which we confirmed efficient translocation of expressed tau into dendrites (data not shown), we observed significant changes in the organization of microtubules (figure 7g), including tighter microtubule bundling than that in controls. Tau overexpression induced more acetylation in both axons and dendrites compared with controls (figure 7d and j), but even so, the tools used to alter microtubule acetylation still had demonstrable effects (figure 7k and l).
Figure 8 shows quantitative experiments in which we experimentally manipulated microtubule acetylation in control neurons and neurons overexpressing tau. Compared with acetylation-dependent changes in sensitivity of dendrites in the absence of tau expression (figure 8a-d), we no longer detected any changes in sensitivities that depended on acetylation when tau was overexpressed (figure 8e-h, and j). Although we found slight increase in the amount of microtubules in tau expressers, there were also no changes in sensitivities in axons (figure 8e-i). Quantitatively, the amount of axonal microtubule in respective conditions are; control (2694 ± 275), HDAC6 (2610 ± 97), tubacin (2507 ± 179), tau (3283 ± 324), tau+HDAC6 (3447 ± 162), tau+tubacin (3424 ± 171), katanin (2483 ± 396), katanin+HDAC6 (2621 ± 267), katanin+tubacin (2249 ± 263), tau+katanin (3272 ± 416), tau+katanin+HDAC6 (3346 ± 223), and tau+katanin+tubacin (3467 ± 137) as indicated by (mean microtubule mass (AFU) ± S.D.) formula (n=3, 25 cells were analyzed for each condition). Dendritic microtubule in respective conditions are; control (2152 ± 185), HDAC6 (2142 ± 361), tubacin (2064 ± 268), tau (2702 ± 421), tau+HDAC6 (2851 ± 462), tau+tubacin (2982 ± 342), katanin (1308 ± 261), katanin+HDAC6 (2130 ± 349), katanin+tubacin (835 ± 105), tau+katanin (2860 ± 427), tau+katanin+HDAC6 (2822 ± 556), and tau+katanin+tubacin (2889 ± 281). In both axons and dendrites, when katanin+tau is compared with katanin+tau+HDAC6 or katanin+tau+tubacin, there are no significant differences (p>0.01) in their microtubule levels.
These data indicate that the sensitivity of microtubules in dendrites to katanin is no longer affected by alterations in acetylation status in the presence of tau.
Microtubule-severing is an important event that occurs across many cell types such as plant cells and epithelial cells, and occurs in many cellular microtubule structures such as the meiotic and mitotic spindles and cilia (Stoppin-Mellet et al., 2006; Keating et al., 1997; McNally et al., 2006; Zhang et al., 2007; Sharma et al., 2007). We are mainly interested in neurons. Over the years, we have reported that microtubules destined for axons and dendrites are nucleated at the centrosome within the cell body of the neuron, and then detached from the centrosome so that molecular motor proteins can convey them into axons and dendrites (Yu et al., 1993; Ahmad et al., 1994; Ahmad and Baas, 1995; Sharp et al., 1996). The ongoing severing of microtubules throughout the neuron is important to generate mobility within the microtubule array, because only very short microtubules are mobile (Wang and Brown, 2002). Microtubule-severing is particularly important for growth cone shape and motility (Dent et al., 1999) as well as for the formation of axonal branches (Yu et al., 1994, 2008; Dent et al., 1999; Riano et al., 2009; Qiang et al., 2010). The question arises as to how microtubules in these locales are targeted for a higher frequency of severing than occurs generally along the axonal shaft.
It is notable that the total levels of katanin and spastin in the neuron are very high, exceeding the concentration needed to fully sever purified microtubules into subunits in vitro (Solowska et al., 2008). Thus a major challenge for the neuron is not only to augment severing in certain locales, but also to protect microtubules in other locales from too much severing. We suspect that there are a number of factors that determine when and where the severing of microtubules is most active, including the levels and distribution of the severing proteins themselves (Yu et al., 2005; 2008), the presence of co-factors such as P80-katanin (Yu et al., 2005), and the composition of microtubule-associated proteins that decorate the microtubule (Baas and Qiang, 2005). Such proteins, especially tau and its family members, strongly suppress the severing of the microtubule by katanin, and to a lesser extent by spastin (Qiang et al., 2006; Yu et al., 2008). As such, one potential mechanism for targeting the severing of microtubules is to locally detach such protective proteins from the microtubule in the vicinity where severing is desirable.
The main purpose of microtubule-severing is probably not to depolymerize microtubules, but rather to transform smaller numbers of long microtubules into higher numbers of short microtubules. For this reason, it makes sense for cells to target the more stable microtubules for severing, so that the resulting short microtubules do not simply depolymerize. Severing of stable microtubules would increase the number of microtubules, the number of free microtubule ends, and also potentially render the newly cut microtubules short enough to be actively transported by molecular motor proteins (Baas et al., 2006; Roll-Mecak and Vale, 2006). In this regard, post-translational modifications of tubulin may be an attractive means by which severing events are targeted to the more stable microtubules, because these modifications are known to accumulate on the most stable microtubules within cells (Schulze et al., 1987; Bulinski and Gundersen, 1991; Baas et al., 1991). Already, it is known that modifications such as acetylation and detyrosination are important for binding of certain motor proteins (Reed et al., 2006; Konishi and Setou, 2009), and hence it would not be surprising if the same were true of severing proteins.
The idea that post-translational tubulin modifications might be a factor in regulating sensitivity to katanin was originally proposed by Sharma and colleagues (2007) on the basis of observations on Tetrahymena. In these studies, it was observed that katanin-null mutants displayed dramatic elevations in acetylated and polyglutamylated tubulin (Sharma et al., 2007). On this basis, it was proposed that microtubules rich in these modifications might be the favored target of katanin-induced severing. Additional support for this view was provided in more recent studies on katanin-inhibited cells in culture, which also displayed elevations in microtubule acetylation (Sudo and Maru, 2008). The results reported in our studies more directly demonstrate that acetylation is a significant factor in the sensitivity of a microtubule to katanin. If microtubule acetylation is experimentally elevated or diminished in fibroblasts, the microtubules become more or less sensitive to katanin, respectively. Moreover, observation of recent breaks in microtubules indicates a strong preference for these breaks to be made by katanin in richly acetylated regions of microtubules rather than poorly acetylated regions. Notably, the same does not appear to be true of spastin, as experimental manipulations of acetylation appear to have no effect on the efficiency of spastin-mediated microtubule-severing, nor does spastin appear to make breaks preferentially in regions of microtubules that are more acetylated (see also Sharma et al., 2007, wherein no accumulation of acetylated tubulin was observed with spastin nulls). Our studies do not preclude the possibility that other tubulin modifications may play important roles as well in regulating the sensitivity of microtubules to katanin and/or spastin, but our taxol/HDAC6 results (figure 2) point to a particularly central role for acetylation in the case of katanin.
This is not the first instance in which we have observed differences in the properties of the two severing proteins. In fact, based on these differences, we have proposed that axonal branches can be produced either through a katanin-based severing of microtubules or a spastin-based severing of microtubules (Yu et al., 2008). In the case of katanin, the severing of microtubules at the appropriate sites is activated by the dissociation of tau and other protective proteins from the microtubule lattice. In the case of spastin, it is simply the focal accumulation of spastin that elicits sufficient severing at the site of branch formation. In neurons, using immunoelectron microscopy, we have documented dramatic heterogeneity in the levels of acetylation along the length of individual microtubules, with the highest levels existing in discrete patches within the more stable domains of the polymer (Baas et al., 1991). Such patches, similar to those observed in the present studies on fibroblasts, may represent the most sensitive sites for potential severing by katanin.
In dendrites, the microtubules are less acetylated than in axons, but still display regions of acetylation (Baas et al., 1991). In addition, it appears that the normal complement of MAPs on dendritic microtubules provides far less protection against katanin than the normal MAP complement of axonal microtubules. The reasons for this remain murky as dendrites contain MAP2 as well as some tau, both of which can protect microtubules against katanin (Qiang et al, 2006). Perhaps the difference lies in the amounts of these proteins decorating the microtubules in dendrites compared to axons, because overexpression of tau affords protection of dendritic microtubules against katanin. In any event, potential regions of enhanced acetylation on dendritic microtubules may be quite important for targeting severing of microtubules during dendritic differentiation. In support of this, it has been reported that acetylation of microtubules in dendrites is key for them to undergo appropriate branching (Ohkawa et al., 2008). For microtubule acetylation to play an important role in axonal branching, mechanisms would presumably have to exist to coordinate the acetylation status of the microtubule with the binding of tau in distinct locales of the axon.
The most perplexing issue that arises from our studies is exactly how a modification of alpha tubulin that occurs on the luminal face of the polymer could alter the sensitivity of the microtubule to katanin. One possibility is that the acetylation status alters the lattice of the polymer in such a way as to influence the efficiency with which different proteins interact with it. If this is correct, it remains to be resolved whether the affinity of the lattice for katanin is directly affected or if the effect is indirect, through the association of other proteins which are sensitive to subtle alterations in the lattice of the microtubule (such as Hsp90; see Giustiniani et al., 2009). There is already evidence that so-called “lattice flaws” in the microtubule are especially targeted for severing by katanin (Odde et al., 1999). Another possibility relates to the manner by which the severing proteins break the microtubule. Specifically, a domain of the severing protein reaches into the microtubule, through the lattice, and yanks out a subunit, thus causing the microtubule to break (Roll-Mecak and McNally, 2009). In this sense, the fact that acetylation occurs on the luminal face of the microtubule may actually provide the ideal location to influence the efficacy of the severing event. Interestingly, tau is thought to bind to the inside wall of the microtubules (Kar et al., 2003), which is consistent with the possibility that the sensitivity of the microtubule to katanin is regulated mainly by events on the luminar face of the microtubule.
This work was funded by grants from the NIH, NSF, and Alzheimer’s Association to PWB. Tubacin was kindly provided by Dr. Stuart L. Schreiber of Harvard University. We thank Dr. Carsten Janke of CNRS for advice and helpful discussions.