Microtubule PTMs and Active Kinesin-1 Motors Are Both Enriched in the Developing Axon of Stage 3 Cells and the Same Subset of Neurites of Stage 2 Cells
Shortly after plating, dissociated hippocampal neurons put out several short, apparently identical processes, referred to as minor neurites (developmental stage 2; Dotti et al., 1988
). Neurons become morphologically polarized when one neurite undergoes an extended period of growth and becomes the axon (developmental stage 3). The remaining neurites subsequently acquire the properties of dendrites (developmental stage 4) and develop an extensive arborization with numerous synapses (stage 5). We focused on cells at stages 2 and 3, before the appearance of a population of minus-end out microtubules in dendrites (Baas et al., 1989
), when constitutively active Kinesin-1 can translocate only toward the neurite tips.
We first investigated whether axons are enriched in microtubule PTMs compared with minor neurites (immature dendrites) in polarized stage 3 hippocampal neurons. Immunofluorescence was performed with antibodies specific to α-tubulin and either acetylated α-tubulin, detyrosinated α-tubulin, or glutamylated α- and β-tubulin. To account for differences in tubulin levels in different neurites, we measured the ratio of modified tubulin to total tubulin in each neurite. The ratios of acetylation and detyrosination were significantly higher in the developing axon than in the minor neurites (, A and B), whereas the ratio of glutamylated tubulin was not appreciably different between these subcellular regions (, A and B). It is important to note, however, that glutamylation is a highly variable modification in that it occurs on both α- and β-tubulin subunits and involves the chain addition of one to six glutamate residues (Verhey and Gaertig, 2007
; Hammond et al., 2008
). Because the GT335 antibody recognizes mono- and polyglutamylation of both α- and β-tubulin (Wolff et al., 1992
; van Dijk et al., 2007
), we cannot rule out the possibility that specific glutamylation signals (α- vs. β-tubulin or chain length variations) could be enriched in axons or minor neurites.
Figure 1. Presence of higher levels of tubulin PTMs correlates with the polarized localization of CA-Kinesin-1 motors. (A) Immunofluorescence of polarized stage 3 hippocampal neurons stained with antibodies to total α-tubulin and either acetylated (top), (more ...)
When examined on a cell-by-cell basis, axonal microtubules were preferentially acetylated in 39 of 40 cells, preferentially detyrosinated in 30 of 40 cells, and preferentially glutamylated in only 19 of 40 cells (C). Truncated CA Kinesin-1 motors selectively accumulate in the axon in ~ 90% of transfected cells (Jacobson et al., 2006
). Thus, the enrichment of certain microtubule PTMs in axons correlates with the selective accumulation of CA-Kinesin-1 motors to this subcellular destination. This indicates that microtubule PTMs could provide a biochemical cue that directs the polarized transport of Kinesin-1 to axons.
We then tested whether microtubule PTMs are enriched in a subset of neurites in unpolarized stage 2 neurons and if so, whether Kinesin-1 preferentially traffics to these neurites. Previous results indicate that the localization of expressed truncated kinesins is a useful measure of where these motors translocate in neurons (Nakata and Hirokawa, 2003
; Jacobson et al., 2006
). The CA-Kinesin-1 motors [KIF5C(1-560) or KIF5C(1-509)] that were used contain the motor and stalk domains required for microtubule-based motility of a dimeric motor, but lack the C-terminal tail domains that contribute to autoinhibition and cargo binding. When tagged with fluorescent proteins and expressed in neuronal cells, such truncated motors accumulate at the tips of some neurites, providing a direct readout of Kinesin-1 activity regulated primarily by the microtubule/motor interface (Nakata and Hirokawa, 2003
; Jacobson et al., 2006
). Unpolarized stage 2 hippocampal neurons expressing CA-Kinesin-1-GFP motors were fixed and stained with antibodies against total α-tubulin and specific tubulin PTMs. We found that the ratio of acetylated tubulin to total tubulin was higher in one or two neurites than the others and that CA-Kinesin-1 motors accumulated more frequently in these neurites with higher levels of acetylation (, D and E). In contrast, detyrosination and glutamylation were not significantly enriched in one neurite over the others (E; data not shown) and were not significantly different between neurites that contained or lacked CA-Kinesin-1 motors (E). Based on its enrichment in the axon of stage 3 cells and the subset of stage 2 neurites preferred by Kinesin-1, microtubule acetylation is a likely candidate to direct the polarized transport of Kinesin-1.
The Enrichment of Tubulin PTMs Is Not a Result of Increased Microtubule Stability in the Axon
PTMs occur preferentially on stable (i.e., long-lived) microtubules. In mature sympathetic neurons, axonal microtubules are preferentially resistant to nocodazole-induced depolymerization (Baas et al., 1991
), a measure thought to correlate with microtubule stability. Similar results were obtained in stage 3 hippocampal neurons (Witte et al., 2008
). Thus, we wondered whether the differential distribution of tubulin PTMs in axons versus dendrites could be explained by a difference in the stability of microtubules in these domains.
To analyze microtubule stability in cultured hippocampal neurons, we used an α-tubulin construct tagged with dendra2, a photoswitchable variant of GFP whose fluorescence emission changes from green to red after illumination with 405 nm light (Gurskaya et al., 2006
). In polarized stage 3 hippocampal neurons, expressed dendra2-α-tubulin exhibited a diffuse distribution and a filamentous pattern (, A and B), indicating that it had been incorporated into the microtubule network. Before photoconversion, dendra2-α-tubulin fluorescence was detectable only in the green channel (, A and B). After photoconversion of a small segment in the proximal region of the axon or minor neurite, a red dendra2-α-tubulin signal was detected only in the illuminated region. In both axons and minor neurites, the red dendra2-α-tubulin fluorescence decayed initially rapidly and then more slowly so that after one hour, a significant fraction of the red fluorescence remained in both axons and minor neurites (, A and C), suggesting that each contains a significant population of stable microtubules. As a test of the sensitivity of this method, we also examined the decline in red dendra2-α-tubulin fluorescence in growth cones that contain predominantly dynamic, and hence unstable, microtubules. When dendra2-α-tubulin was photoconverted in the growth cone region, the red fluorescence signal disappeared within minutes (, B and C), confirming that microtubules in the growth cone turn over more rapidly than those in neurite shafts.
Figure 2. Rate of tubulin turnover is similar in axons and dendrites. (A) To estimate the temporal stability of axonal and dendritic microtubules in polarized stage 3 neurons, dendra2-α-tubulin was subjected to photoconversion in a segment of the axon (box (more ...)
The decay in dendra2-α-tubulin fluorescence in the axons and minor neurites could be mathematically fit by a two-component model. The rapid component decayed with a half-time of ~1.5 min, whereas the slow component decayed with a half-time of ~1 h. The rapidly decaying component probably represents the diffusion of soluble tubulin and the turnover of dynamic microtubules, including the loss of subunits due to depolymerization. The slowly decaying component probably represents tubulin subunits that have been incorporated into “stable” microtubules. The rate of decline of both the fast and slow phases was similar in axons and minor neurites (C). Although the half-time of stable microtubules was slightly longer in axons (64 vs. 42 min, respectively), this difference was not statistically significant. Furthermore, the distribution of dendra2-α-tubulin between the two phases is similar in axons and minor neurites (C). In contrast to these results, the dendra2-α-tubulin fluorescence decay in growth cones could be fit with a one-phase decay model, which yielded an average half life of 0.9 min, indicating that there were no slow-decaying components in this region. These results demonstrate that the increased levels of tubulin PTMs observed in the axon of stage 3 neurons cannot be accounted for by a higher proportion of stable microtubules in this domain. Instead, the activities of the enzymes responsible for tubulin modification must differ between the axon and minor neurites.
Increased Acetylation Is Not Sufficient to Alter the Selective Localization of Kinesin-1 Motors and Cargoes
Acetylated tubulin is present in higher levels in the developing axon in polarized stage 3 hippocampal neurons and in a subset of the neurites in unpolarized stage 2 neurons (). Microtubule acetylation can influence Kinesin-1 trafficking events in unpolarized (stage 2) neurons in that hyperacetylation causes the Kinesin-1 cargo JIP1 to accumulate in nearly all neurite tips rather than in only a subset of neurite tips as seen in control cells (Reed et al., 2006
). We thus tested whether microtubule acetylation is sufficient to direct Kinesin-1 to axons as opposed to minor neurites in polarized (stage 3) neurons.
Microtubule acetylation is a reversible modification that involves the addition of an acetyl group to lysine 40 of α-tubulin. In fibroblasts, microtubule acetylation can be greatly enhanced by TSA and tubacin, inhibitors of a known α-tubulin deacetylase histone deacetylase (HDAC)6 (Hubbert et al., 2002
; Matsuyama et al., 2002
; Haggarty et al., 2003
). We verified that TSA and tubacin treatments also cause a global increase in microtubule acetylation in polarized stage 3 neuronal cells by Western blotting and immunofluorescence. Treatment with TSA or tubacin caused a significant increase in the overall levels of microtubule acetylation compared with control dimethyl sulfoxide (DMSO) treatment (A). The increase in acetylated tubulin levels occurred in both the axon and the minor neurites (C) and resulted in nearly equivalent levels of acetylation between these two compartments (B). Thus, HDAC6 inhibition efficiently abolished the selective enrichment of acetylated microtubules in the developing axon. These results also suggest that tubulin acetylation occurs at about the same rate in minor neurites and axons but that a higher deacetylase activity in the minor neurites reduces the level of acetylated tubulin in this compartment.
Figure 3. Treatment with deacetylase inhibitors results in increased microtubule acetylation in both the axon and minor neurites. (A) Polarized stage 3 cortical cells were treated for 3 h with DMSO (control), 125 nM TSA, or 10 μM tubacin and then lysed (more ...)
We next tested whether hyperacetylation of microtubules throughout both axons and minor neurites was sufficient to misdirect CA-Kinesin-1 into minor neurites. Polarized stage 3 hippocampal neurons were treated for 3–4 h with DMSO, TSA, or tubacin. The cells were then transfected with CA-Kinesin-1-mCherry along with yellow fluorescent protein (YFP) as a soluble marker of the transfected cells and allowed to express the exogenous proteins under additional drug treatment for 4–5 h. Treatment with deacetylase inhibitors did not cause CA-Kinesin-1-mCherry motors to accumulate in the minor neurites. Rather, CA-Kinesin-1-mCherry motors localized specifically to the developing axon in both control and treated cells (, A and B). Similar results were obtained in mature (stage 5) neurons where treatment with deacetylase inhibitors did not alter the selective accumulation of CA-Kinesin-1 motors in the axon (B).
Figure 4. Increased microtubule acetylation does not alter the polarized sorting of CA-Kinesin-1 motors or JIP1 cargoes in polarized stage 3 neurons. (A) stage 3 hippocampal neurons were treated for 2–4 h with DMSO, 125 nM TSA, or 10 μM tubacin. (more ...)
We then tested whether hyperacetylation of microtubules could misdirect the localization of the Kinesin-1 cargo protein JIP1 from the developing axon in polarized stage 3 cells. In cells treated with DMSO, TSA, or tubacin for 3 h and then fixed and stained with antibodies to JIP1 and acetylated α-tubulin, JIP1 was still delivered exclusively to axons (, C and D). Together, the results on CA-Kinesin-1 motor and JIP1 cargo localization suggest that α-tubulin acetylation is not sufficient to provide the biochemical cue that drives the selective axonal translocation of Kinesin-1 motors in polarized neurons.
Taxol Treatment Results in Increases in Microtubule Posttranslational Modifications and Misdirection of Kinesin-1 Trafficking
What biochemical cues other than microtubule acetylation could account for the preferential accumulation of the Kinesin-1 motor domain in axons? One clue comes from a study of mature neurons (stage 5), which showed that treatment with low doses of Taxol resulted in accumulation of CA-Kinesin-1 motors in both axons and dendrites (Nakata and Hirokawa, 2003
). We thus asked whether the loss of selective Kinesin-1 accumulation after Taxol treatment could be due to changes in multiple microtubule PTMs. To explore this possibility, we tested whether Taxol treatment alters the levels of microtubule PTMs in neuronal cells. At submicromolar concentrations, Taxol suppresses microtubule dynamics without an increase in polymer mass (Jordan and Wilson, 1998
). Consistent with this, low dose Taxol treatment of polarized stage 3 neurons caused a significant increase in the fraction of stable microtubules in both axons and minor neurites (Supplemental Figure 3). Importantly, low-dose Taxol treatment of polarized stage 3 neurons also resulted in a significant increase in overall levels of tubulin acetylation, detyrosination, and glutamylation (A).
Figure 5. Taxol treatment increases all microtubule PTMs and disrupts the polarized localization of CA-Kinesin-1 motors and JIP-1 cargoes in polarized stage 3 and unpolarized stage 2 neurons. (A–E) Effects of Taxol in polarized stage 3 cells. (A) stage (more ...)
We verified that Taxol treatment results in misrouting of Kinesin-1 to dendrites in mature stage 5 neurons as described by Nakata and Hirokawa (2003)
; Supplemental Figure 2). Analysis of Kinesin-1 translocation at this stage of development is complicated by the presence of minus-end out microtubules in the dendrites. Thus, we examined the effects of Taxol treatment on Kinesin-1 accumulation at earlier stages of development, when the microtubules in all neurites are oriented plus end-out (Baas et al., 1989
). Treatment of polarized stage 3 neurons with Taxol markedly reduced the selectivity of Kinesin-1 accumulation (B). Specifically, Taxol treatment resulted in the accumulation of CA-Kinesin-1-mCherry in the minor neurites in >70% of the cells as compared with only 13% of DMSO-treated cells (, B and C). Similar effects were found on the localization of the endogenous JIP1 cargo protein; Taxol treatment caused JIP1 to accumulate in the minor neurites in 57% (100 nM Taxol) or 70% (10 nM Taxol) of cells compared with only 16% of DMSO treated cells (, D and E). In unpolarized stage 2 cells, Taxol treatment also caused a mislocalization of both active Kinesin-1 motors and JIP1 cargoes to a majority rather than a subset of neurite tips (, F–H). Together these results demonstrate that Taxol-induced changes in microtubule stability regulate the selective accumulation of Kinesin-1 and its cargoes in both unpolarized and polarized neurons. This change in Kinesin-1 sorting correlates with increased levels of multiple tubulin PTMs.
We next determined whether the Taxol-induced changes in microtubule PTMs and Kinesin-1 accumulation occur on the same time scale. Western blot analysis of unpolarized stage 2 neurons treated with 100 nM Taxol for 0–60 min revealed that the increase in microtubule PTMs (specifically acetylated, detyrosinated, and polyglutamylated tubulin) occurs rapidly, within 7.5 min of Taxol treatment (A). To investigate the reaction time of CA-Kinesin-1 to Taxol treatment, we used time-lapse microscopy to observe unpolarized (stage 2) or polarized (stage 3) neuronal cells that had been transfected with CA-Kinesin-1-monomeric citrine (mCit) plasmid at the time of plating. In unpolarized stage 2 cells where Kinesin-1 was evenly distributed throughout the cell before Taxol treatment, the motor began concentrating in nearly all neurite tips within minutes after Taxol treatment but not after DMSO treatment (, B and C), and accumulation was nearly complete by 10–15 min after treatment. Thus, in the same time scale, Taxol-induced changes in microtubule structure, stability, and PTMs resulted in loss of the preferential accumulation of CA-Kinesin-1. Taxol treatment also limited the dynamic nature of CA-Kinesin-1 accumulation in different neurite tips over time. With Taxol treatment, CA-Kinesin-1 remained in neurite tips for the duration of the recording (, B and C) and no longer underwent transient accumulations, as reported for untreated cells (Jacobson et al., 2006
Figure 6. Taxol-induced alterations in CA-Kinesin-1 trafficking and tubulin PTMs occur on the same time scale. (A) A mix of stage 2/3 hippocampal neurons were treated with 100 nM Taxol for the indicated times. Cell lysates were analyzed by western blotting with (more ...)
In both unpolarized stage 2 cells (data not shown) and polarized stage 3 cells (, D and E) where CA-Kinesin-1 was already concentrated in one neurite tip before Taxol treatment, a longer time frame (e.g., hours) was required for redistribution of the motor. In polarized stage 3 cells, most CA-Kinesin-1 motors were accumulated in the axon before Taxol treatment, but they began to accumulate in minor neurites after Taxol treatment, with gradual increases occurring over the 4-h imaging period (, D and E). Presumably, accumulated motors are more likely to reuse the Taxol-stabilized microtubule tracks in the axon rather than diffuse back to the cell body and choose microtubules leading to another neurite. Taxol treatment resulted in localization of CA-Kinesin-1 to the tips of 95% of axons and 66% of minor neurites compared with 98% of axons and 3% of minor neurites in control cells. Taxol treatment did not alter the polarized distribution of the MAPs Tau and MAP2 (axonal and dendritic markers, respectively; Supplemental Figure 4), demonstrating that the influence of Taxol on CA-Kinesin-1 motors is not due to Taxol-induced alterations in the association of MAPs with microtubules (Black, 1987
; Samsonov et al., 2004
; Kim et al., 2006
). We conclude that Taxol induces rapid increases in microtubule PTMs and results in mistargeting of CA-Kinesin-1 motors to minor neurites.
Inhibition of GSK3β Activity Also Results in Increased Microtubule Posttranslational Modifications and Misdirection of Kinesin-1
Taxol treatment of cultured neurons was recently reported to result in the formation of multiple axons at the expense of dendrites (Witte et al., 2008
). Our demonstration that Taxol treatment results in alterations in the selective axonal targeting of CA-Kinesin-1 motors and JIP cargoes is consistent with this effect. Another treatment that has been reported to disturb axon/dendrite formation in cultured neurons is the inhibition of GSK3β. Global inhibition of GSK3β results in the formation of multiple axons and/or increased axonal branching (Jiang et al., 2005
; Yoshimura et al., 2005
; Gartner et al., 2006
; Kim et al., 2006
). We wondered whether the formation of multiple axons upon GSK3β inhibition could be linked, like Taxol treatment, to enhanced microtubule PTMs and a change in the selectivity of Kinesin-1.
We first compared the ability of Taxol, GSK3β inhibitors, and deacetylase inhibitors to induce the formation of supernumerary axons. Primary hippocampal neurons were incubated in low levels of Taxol, SB216763 (GSK3β inhibitor), or tubacin (HDAC6 inhibitor) for 6 d in vitro, when both axons and dendrites are actively growing, and then we fixed and stained them with antibodies to Tau and MAP2. Both Taxol treatment and GSK3β inhibition led to the formation of multiple axons whereas tubacin treatment had no effect (Supplemental Figure 5). Taxol treatment resulted in multiple axon formation in a large percentage of cells (85.9 ± 1.6%) compared with control (DMSO-treated) cells (13.1 ± 1.4%). SB216763 treatment also led to a significant, yet smaller, increase in the percentage of cells with multiple axons (36.7 ± 2.5%) consistent with previous reports (Jiang et al., 2005
; Yoshimura et al., 2005
; Gartner et al., 2006
We then tested whether inhibition of GSK3β results in an alteration of microtubule PTMs and a corresponding misdirection of CA-Kinesin-1 motors and JIP1 cargoes. Polarized (stage 3) neurons were treated with SB216763 and the levels of specific PTMs were analyzed by Western blotting of the cell lysates. SB216763 treatment resulted in increased levels of microtubule acetylation, detyrosination, and polyglutamylation (A). Inhibition of GSK3β activity by SB216763 treatment also resulted in a misdirection of CA-Kinesin-1 motors into minor neurites rather than only to axons, as seen in control cells (, B and C). Similar results were obtained in mature (stage 5) neurons where SB216763 treatment caused a significant increase in the amount of CA-Kinesin-1 motors that accumulated at the tips of dendrites (C). Thus, GSK3β inhibition in polarized cells caused the formation of supernumerary axons as well as increased levels of microtubule PTMs and mistargeting of CA-Kinesin-1 motors. Interestingly, inhibition of GSK3β was not as effective in altering the selective targeting of CA-Kinesin-1 motors and JIP1 cargoes in unpolarized (stage 2) cells. In this case, SB216763 treatment caused a small, but still significant, misdirection of CA-Kinesin-1 motors (, D and F) and JIP1 cargoes (, E and F) to more neurites than in control DMSO-treated cells.
Figure 7. Inhibition of GSK3β results in increased levels of tubulin PTMs and disrupts the polarized sorting of CA-Kinesin-1 and JIP1 in polarized stage 3 and unpolarized stage 2 cells. (A–C) Effects of GSK3β inhibition in polarized stage (more ...)
Under some circumstances, GSK3β activity contributes to the regulation of microtubule stability in cultured cells (Owen and Gordon-Weeks, 2003
; Eng et al., 2006
). In polarized stage 3 hippocampal neurons, SB216763 treatment caused a small increase in the fraction of stable microtubules and in the calculated half-life of stable microtubules in the shaft regions of both axons and minor neurites, as measured by the fluorescence decay of photoconverted dendra2-α-tubulin (Supplemental Figure 3), although the differences between treated and control cells were not statistically significant. We conclude that inhibition of GSK3β promotes an “axonal signal” that may be comprised, at least in part, of microtubule PTMs that can influence Kinesin-1 translocation in primary hippocampal neurons.