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The Nucleotide-binding State of Microtubules Modulates Kinesin Processivity and the Ability of Tau to Inhibit Kinesin-mediated Transport*

Open AccessPublished:October 27, 2011DOI:https://doi.org/10.1074/jbc.M111.292987
      The ability of Tau to act as a potent inhibitor of kinesin's processive run length in vitro suggests that it may actively participate in the regulation of axonal transport in vivo. However, it remains unclear how kinesin-based transport could then proceed effectively in neurons, where Tau is expressed at high levels. One potential explanation is that Tau, a conformationally dynamic protein, has multiple modes of interaction with the microtubule, not all of which inhibit kinesin's processive run length. Previous studies support the hypothesis that Tau has at least two modes of interaction with microtubules, but the mechanisms by which Tau adopts these different conformations and their functional consequences have not been investigated previously. In the present study, we have used single molecule imaging techniques to demonstrate that Tau inhibits kinesin's processive run length in an isoform-dependent manner on GDP-microtubules stabilized with either paclitaxel or glycerol/DMSO but not guanosine-5′-((α,β)-methyleno)triphosphate (GMPCPP)-stabilized microtubules. Furthermore, the order of Tau addition to microtubules before or after polymerization has no effect on the ability of Tau to modulate kinesin motility regardless of the stabilizing agent used. Finally, the processive run length of kinesin is reduced on GMPCPP-microtubules relative to GDP-microtubules, and kinesin's velocity is enhanced in the presence of 4-repeat long Tau but not the 3-repeat short isoform. These results shed new light on the potential role of Tau in the regulation of axonal transport, which is more complex than previously recognized.

      Introduction

      Neurons are electrically excitable cells responsible for receiving and transmitting information throughout the nervous system. These cells have a distinct polarity and a unique architecture, consisting of a complicated system of input processes known as dendrites and a single extremely long output process known as the axon, which can be up to a meter in length in extreme cases. Such extraordinarily long distances pose a unique set of problems for neurons, because most of the proteins, organelles, and other cellular materials required for axonal function are produced in the neuronal cell body (
      • Hirokawa N.
      • Noda Y.
      ). Because these distances are far too great for diffusion to move cargo efficiently along the length of the axon, neurons take advantage of the microtubule-based molecular motors kinesin and dynein to facilitate anterograde and retrograde axonal transport, respectively. Kinesin is particularly well suited for this function, because it is a highly processive motor capable of transporting cargo produced in the cell body over long distances down the axon without dissociating from the underlying microtubule track. However, this presents a new challenge as to how the processive behavior of kinesin can be modulated to ensure that cargo is delivered to the appropriate locations within the axon, either at intermediate points along its length (e.g. nodes of Ranvier in myelinated nerve cells) or at presynaptic terminals, which may require navigating through numerous axonal branch points. Given the number of heterogeneous intracellular cargo and destinations within the axon, there are likely to be several different mechanisms for regulating kinesin's motile function in vivo, including posttranslational modifications of specific kinesin subunits (
      • Morfini G.
      • Pigino G.
      • Brady S.T.
      ) and the underlying microtubule track (e.g. polyglutamylation, tyrosination/detyrosination, and acetylation) (
      • Dunn S.
      • Morrison E.E.
      • Liverpool T.B.
      • Molina-París C.
      • Cross R.A.
      • Alonso M.C.
      • Peckham M.
      ,
      • Konishi Y.
      • Setou M.
      ,
      • Reed N.A.
      • Cai D.
      • Blasius T.L.
      • Jih G.T.
      • Meyhofer E.
      • Gaertig J.
      • Verhey K.J.
      ). Recent work has indicated that the nucleotide-binding state (i.e. GTP versus GDP) of tubulin subunits in the microtubule may be important in modulating interactions with kinesin as well (
      • Nakata T.
      • Niwa S.
      • Okada Y.
      • Perez F.
      • Hirokawa N.
      ). A fourth possible level of regulation involves microtubule-associated proteins, which may directly or indirectly influence the function of kinesin. For example, Tau is a neuron-specific microtubule-associated protein that has previously been shown to inhibit kinesin mediated transport in an isoform-specific manner, both in vitro (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Vershinin M.
      • Carter B.C.
      • Razafsky D.S.
      • King S.J.
      • Gross S.P.
      ) and in vivo (
      • Stamer K.
      • Vogel R.
      • Thies E.
      • Mandelkow E.
      • Mandelkow E.M.
      ,
      • Stoothoff W.
      • Jones P.B.
      • Spires-Jones T.L.
      • Joyner D.
      • Chhabra E.
      • Bercury K.
      • Fan Z.
      • Xie H.
      • Bacskai B.
      • Edd J.
      • Irimia D.
      • Hyman B.T.
      ). However, mechanisms underlying the inhibition of kinesin-based axonal transport by Tau remain unknown (
      • Morfini G.
      • Pigino G.
      • Mizuno N.
      • Kikkawa M.
      • Brady S.T.
      ).
      In humans, there are six known Tau isoforms, which are found primarily in the axonal compartment of neurons (
      • Andreadis A.
      • Brown W.M.
      • Kosik K.S.
      ). Isoforms of Tau differ by possessing either three or four microtubule binding motifs in the C-terminal microtubule binding domain and by the presence or absence of one or two acidic inserts in their N-terminal projection domain (
      • Goode B.L.
      • Chau M.
      • Denis P.E.
      • Feinstein S.C.
      ). Tau has been shown to inhibit kinesin motility in vitro, with the 3-repeat short (3RS)
      The abbreviations used are: 3RS
      3-repeat short
      4RL
      4-repeat long
      GMPCPP
      guanosine-5′-((α,β)-methyleno)triphosphate
      TIRF
      total internal reflection fluorescence
      MAB
      motility assay buffer
      Qdot
      quantum dot.
      isoform (possessing three microtubule binding repeats and no acidic inserts) having a greater effect than the 4-repeat long (4RL) isoform (possessing four microtubule binding repeats and two acidic inserts) (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Vershinin M.
      • Carter B.C.
      • Razafsky D.S.
      • King S.J.
      • Gross S.P.
      ). Given these results, it is unclear how axonal transport can efficiently operate in neurons, because the 3- and 4-repeat isoforms of Tau are highly expressed at approximately equal levels in mature axons (
      • Aronov S.
      • Aranda G.
      • Behar L.
      • Ginzburg I.
      ,
      • Trojanowski J.Q.
      • Schuck T.
      • Schmidt M.L.
      • Lee V.M.
      ). Reports from more physiologically relevant model systems have been conflicting, with the overexpression of Tau disrupting mitochondrial transport in cortical neurons (
      • Stoothoff W.
      • Jones P.B.
      • Spires-Jones T.L.
      • Joyner D.
      • Chhabra E.
      • Bercury K.
      • Fan Z.
      • Xie H.
      • Bacskai B.
      • Edd J.
      • Irimia D.
      • Hyman B.T.
      ) but the addition of supraphysiological levels of Tau having no effect on fast axonal transport in extruded axoplasm (
      • Morfini G.
      • Pigino G.
      • Mizuno N.
      • Kikkawa M.
      • Brady S.T.
      ). One possible explanation that reconciles these disparate observations is that the manner in which Tau itself interacts with the microtubule lattice may be regulated, because there is both structural (
      • Kar S.
      • Fan J.
      • Smith M.J.
      • Goedert M.
      • Amos L.A.
      ) and biochemical (
      • Makrides V.
      • Massie M.R.
      • Feinstein S.C.
      • Lew J.
      ) evidence that Tau may have more than one binding site on the microtubule. In addition to the external binding site occupied by Tau when bound to preformed, paclitaxel-stabilized microtubules, there appears to be an interior (luminal side) binding site for Tau when copolymerized with free tubulin and stabilized with the slowly hydrolyzable GTP analog GMPCPP (
      • Kar S.
      • Fan J.
      • Smith M.J.
      • Goedert M.
      • Amos L.A.
      ). Consistent with these results, Tau copolymerized with tubulin in the presence of GTP has also been observed to exist in two populations, one being more stably bound than the other, whereas preformed microtubules stabilized with paclitaxel only possess the more dynamic population of Tau (
      • Makrides V.
      • Massie M.R.
      • Feinstein S.C.
      • Lew J.
      ).
      The existence of multiple populations of Tau opens up the intriguing possibility that, depending on its mode of interaction with the microtubule, Tau can adopt different conformations that result in different functions within the neuron. However, because most previous in vitro studies of the effect of Tau on kinesin motility have been done using paclitaxel-stabilized microtubules (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Vershinin M.
      • Carter B.C.
      • Razafsky D.S.
      • King S.J.
      • Gross S.P.
      ), the role of alternative Tau-tubulin complexes involving copolymerization or different nucleotide-binding states of the microtubule (i.e. GDP versus GTP) has not been investigated. Thus, in the current work, we have directly examined the processive run length and velocity of kinesin-quantum dot complexes on microtubules in different combinations of nucleotide-binding state (GDP or GMPCPP), paclitaxel-stabilization, isoforms of Tau (3RS and 4RL), and order of Tau addition (during or after tubulin polymerization). We show for the first time that Tau is not only a negative regulator of kinesin motility, as previously reported (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Vershinin M.
      • Carter B.C.
      • Razafsky D.S.
      • King S.J.
      • Gross S.P.
      ), but can adopt a non-inhibitory conformation on GMPCPP-microtubules and even enhance kinesin velocity in an isoform specific manner. These results have important implications for the process of axonal transport in nerve cells, which have recently been shown to be rich in GTP-tubulin (
      • Nakata T.
      • Niwa S.
      • Okada Y.
      • Perez F.
      • Hirokawa N.
      ), and suggest a mechanism by which changes to the microtubule lattice can dictate the function of microtubule binding partners, including kinesin and Tau, in complex and interesting ways.

      DISCUSSION

      Our results are consistent with previous reports of the ability of Tau to inhibit kinesin-mediated transport on paclitaxel-stabilized microtubules in an isoform-specific manner, with the 3RS-Tau isoform being more inhibitory than the 4RL-Tau isoform (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Vershinin M.
      • Carter B.C.
      • Razafsky D.S.
      • King S.J.
      • Gross S.P.
      ). In striking contrast, Tau loses all ability to reduce kinesin's processive run length on GMPCPP-stabilized microtubules over the physiologically relevant range of Tau concentrations used in these experiments. At supraphysiological concentrations of Tau (1:1 Tau/tubulin ratio), we do see abolishment of kinesin motility on GMPCPP-microtubules (data not shown); however, it has been shown that Tau forms aggregates on the microtubule surface at such high concentrations, which may result in a completely different form of inhibition (
      • Ackmann M.
      • Wiech H.
      • Mandelkow E.
      ,
      • Santarella R.A.
      • Skiniotis G.
      • Goldie K.N.
      • Tittmann P.
      • Gross H.
      • Mandelkow E.M.
      • Mandelkow E.
      • Hoenger A.
      ). Because we did observe inhibition of kinesin by Tau on microtubules formed in the presence of glycerol/DMSO as an alternative stabilizing agent to paclitaxel, we doubt that paclitaxel is somehow inducing the inhibitory state of Tau. To completely preclude this possibility, we monitored kinesin motility on microtubules stabilized by both GMPCPP and paclitaxel. Again, Tau has no apparent effect on kinesin motility under these conditions. GMPCPP-microtubules closely resemble microtubules in the GTP nucleotide state, whereas microtubules stabilized with paclitaxel or glycerol/DMSO are presumably more representative of the GDP nucleotide state (
      • Meurer-Grob P.
      • Kasparian J.
      • Wade R.H.
      ,
      • Munson K.M.
      • Mulugeta P.G.
      • Donhauser Z.J.
      ,
      • Vale R.D.
      • Coppin C.M.
      • Malik F.
      • Kull F.J.
      • Milligan R.A.
      ). We therefore conclude that Tau and/or kinesin can adopt a different mode of interaction with microtubules in the GTP state as compared with the GDP state, which abolishes the ability of Tau to inhibit kinesin motility.
      In addition to Tau losing its ability to inhibit kinesin-mediated transport on GMPCPP-microtubules, we observe that kinesin's characteristic run length was reduced by ∼20% on GMPCPP-microtubules as compared with paclitaxel- or glycerol/DMSO-stabilized microtubules. This indicates that kinesin, like Tau, also interacts with GMPCPP-microtubules in a different manner than with paclitaxel- or glycerol/DMSO-stabilized microtubules. Previous work has demonstrated that small changes in the microtubule lattice can affect kinesin-mediated transport, where post-translational modifications, such as acetylation, detyrosination, and polyglutamylation, influence kinesin binding and motility on microtubules (
      • Reed N.A.
      • Cai D.
      • Blasius T.L.
      • Jih G.T.
      • Meyhofer E.
      • Gaertig J.
      • Verhey K.J.
      ,
      • Bulinski J.C.
      ,
      • Hammond J.W.
      • Huang C.F.
      • Kaech S.
      • Jacobson C.
      • Banker G.
      • Verhey K.J.
      ). Kinesin has also been shown to have an increased velocity in gliding assays on GMPCPP-microtubules as compared with GDP-microtubules (
      • Vale R.D.
      • Coppin C.M.
      • Malik F.
      • Kull F.J.
      • Milligan R.A.
      ) and to bind GMPCPP-microtubules 3.7-fold tighter than paclitaxel-stabilized GDP-microtubules. This discrimination toward the GTP state is facilitated by kinesin loop 11, which mediates the strong binding of kinesin in the ATP or apo states of its enzymatic cycle (
      • Nakata T.
      • Niwa S.
      • Okada Y.
      • Perez F.
      • Hirokawa N.
      ). Interestingly, this is similar to what has been observed with kinesin binding to subtilisin-treated microtubules in which the C-terminal tail of tubulin has been enzymatically removed (
      • Skiniotis G.
      • Cochran J.C.
      • Müller J.
      • Mandelkow E.
      • Gilbert S.P.
      • Hoenger A.
      ). Not only does subtilisin treatment promote kinesin binding to microtubules, but it also leads to a reduction in kinesin's processive run length (
      • Wang Z.
      • Sheetz M.P.
      ) much like we observe on GMPCPP-microtubules (
      • Wang Z.
      • Sheetz M.P.
      ). Although we are not proposing direct structural links between microtubules in the GTP state and those missing their C-terminal tails, there may be a correlation between the stronger binding of kinesin on GMPCPP-stabilized microtubules and our observation that kinesin's characteristic run length is reduced under these conditions, just as in the case of subtilisin-treated microtubules.
      Interestingly, in addition to the observed effects of the underlying microtubule lattice on kinesin motility, we see a small but significant increase in the velocity of kinesin in the presence of 4RL-Tau relative to that observed in the absence of Tau or the presence of 3RS-Tau, which has not been reported previously (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Seitz A.
      • Kojima H.
      • Oiwa K.
      • Mandelkow E.M.
      • Song Y.H.
      • Mandelkow E.
      ). We cannot account for this discrepancy between our group and others; however, we consistently see this increase in velocity across all of our experimental conditions. We also observed a 20% increase in Vmax of the microtubule-activated ATPase activity of kinesin in the presence of 4RL-Tau compared with the 3RS-Tau and no Tau cases, which corresponds well with the 20% increase in velocity we saw in the motility assays in the presence of 4RL-Tau. Furthermore, it was recently shown in in vitro gliding assays of microtubules on a surface of kinesin that sliding velocity was reduced 17% in the presence of 4RS-Tau as compared with 3RS-Tau (
      • Peck A.
      • Sargin M.E.
      • LaPointe N.E.
      • Rose K.
      • Manjunath B.S.
      • Feinstein S.C.
      • Wilson L.
      ). The major difference between the 4RS-Tau used in the previous work and the 4RL-Tau used by our group in the current study is the inclusion of two 29-amino acid acidic inserts in the N-terminal projection domain of 4RL-Tau. Because we see an increase in velocity in our study, it is possible that these acidic inserts are influencing kinesin's interaction with the microtubule and are responsible for the isoform-specific effect on kinesin velocity and possibly reduction in processive run length. The N-terminal tails of Tau are acidic in general, and with the inclusion of the acidic inserts, the tails become highly acidic (
      • Goode B.L.
      • Chau M.
      • Denis P.E.
      • Feinstein S.C.
      ). Previous work has demonstrated that the acidic C-terminal tails of tubulin enhance kinesin processivity and velocity (
      • Wang Z.
      • Sheetz M.P.
      ,
      • Lakämper S.
      • Meyhöfer E.
      ). Because we see an increase in velocity with the 4RL-Tau isoform, it is tempting to speculate that when Tau is not in an inhibitory state, it may actually enhance kinesin motility via its acidic tail domain analogous to the C-terminal tails of tubulin.
      Our results present a novel role for the microtubule lattice in modulating the interaction between kinesin and Tau and have important implications for the regulation of axonal transport in different developmental and pathological states of the neuron. The traditional view is that microtubules, with the exception of their GTP caps, exist primarily in the GDP form within the cell. Thus, considering the results of the present work and those reported previously (
      • Dixit R.
      • Ross J.L.
      • Goldman Y.E.
      • Holzbaur E.L.
      ,
      • Vershinin M.
      • Carter B.C.
      • Razafsky D.S.
      • King S.J.
      • Gross S.P.
      ), one would expect Tau to bind microtubules mainly in its inhibitory conformation in vivo and, at the high levels at which it is expressed in neurons, significantly disrupt axonal transport. However, it has recently been demonstrated that there are significant populations of both GTP and GDP-tubulin in the axon of developing and mature neurons, and the microtubule lattice contains numerous segments predominantly composed of GTP-tubulin (
      • Nakata T.
      • Niwa S.
      • Okada Y.
      • Perez F.
      • Hirokawa N.
      ). In addition, kinesin localizes to these regions of GTP-tubulin, which may facilitate the localization of kinesin-1 to axons as opposed to dendrites (
      • Nakata T.
      • Niwa S.
      • Okada Y.
      • Perez F.
      • Hirokawa N.
      ). If GTP-tubulin is indeed the axonal localization cue for kinesin, it would be detrimental to the cell if Tau were inhibitory in regions of high GTP-tubulin content that promote kinesin binding, because kinesin could lose its ability to target cargo to their correct locations. This problem is potentially even more acute during the early stages of neuronal development when 3-repeat Tau, a more potent inhibitor of kinesin motility than 4-repeat Tau, is the predominant isoform expressed (
      • Takuma H.
      • Arawaka S.
      • Mori H.
      ). During this time, one would expect kinesin-mediated transport to be essential for delivering materials to the growing axon and ancillary branches (
      • Yu W.
      • Baas P.W.
      ). The existence of significant GTP-tubulin populations along the length of the axon, which are present in mature neurons and appear to be enriched at early stages of development (
      • Nakata T.
      • Niwa S.
      • Okada Y.
      • Perez F.
      • Hirokawa N.
      ) and to which we observe that Tau binds in a non-inhibitory conformation, would explain how axonal transport mediated by kinesin could proceed unimpeded in the presence of high levels of Tau expression in the neuron. Thus, regulation of kinesin-mediated transport in the axon by Tau is likely to be a complex process dependent on the structural state of the microtubule (i.e. GTP versus GDP). Our results demonstrate that Tau is not simply an inhibitor of kinesin motility but that it can function in a non-inhibitory manner or even enhance axonal transport, depending on the specific isoform involved and its interaction with the underlying microtubule lattice.

      Acknowledgments

      We thank Dr. Steven King for the 3RS- and 4RL-Tau cDNA constructs and the laboratory of Dr. Kathy Trybus for the kinesin cDNA and ongoing support throughout this project. We also thank Gabrielle Anderson, Dr. Justin Decarreau, Dr. Nicole E. LaPointe, Dr. Gerardo A. Morfini, Dr. Jason Stumpff, and Dr. Andrew Thompson for help and guidance during the preparation of the manuscript.

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