The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.     

Protein Synthesis and Rapid Axonal Transport During Regrowth of Dorsal Root Axons

Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two-dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes in proteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.     

The Ras/Rac guanine nucleotide exchange factor mammalian Son-of-sevenless interacts with PACSIN 1/syndapin I, a regulator of endocytosis and the actin cytoskeleton

Mammalian Son-of-sevenless (mSos) functions as a guanine nucleotide exchange factor for Ras and Rac, thus regulating signaling to mitogen-activated protein kinases and actin dynamics. In the current study, we have identified a new mSos-binding protein of 50 kDa (p50) that interacts with the mSos1 proline-rich domain. Mass spectrometry analysis and immunodepletion studies reveal p50 as PACSIN 1/syndapin I, a Src homology 3 domain-containing protein functioning in endocytosis and regulation of actin dynamics. In addition to PACSIN 1, which is neuron-specific, mSos also interacts with PACSIN 2, which is expressed in neuronal and nonneuronal tissues. PACSIN 2 shows enhanced binding to the mSos proline-rich domain in pull-down assays from brain extracts as compared with lung extracts, suggesting a tissue-specific regulation of the interaction. Proline to leucine mutations within the Src homology 3 domains of PACSIN 1 and 2 abolish their binding to mSos, demonstrating the specificity of the interactions. In situ, PACSIN 1 and mSos1 are co-expressed in growth cones and actin-rich filopodia in hippocampal and dorsal root ganglion neurons, and the two proteins co-immunoprecipitate from brain extracts. Moreover, epidermal growth factor treatment of COS-7 cells causes co-localization of PACSIN 1 and mSos1 in actin-rich membrane ruffles, and their interaction is regulated through epidermal growth factor-stimulated mSos1 phosphorylation. These data suggest that PACSINs may function with mSos1 in regulation of actin dynamics.     

Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons

The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP–tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin–microtubule balance. This indicates that tau''s dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.     

Microtubule fascicles in the stem processes of cultured sensory ganglion cells

Summary Microtubule fascicles, resembling those characterizing the initial segment of multipolar neurons, have been observed by electron microscopy within and close to the origin of the stem process of some unipolar ganglion cells in explant cultures of embryonic chick dorsal root ganglia. Each fascicle comprised 2–6 closely spaced parallel microtubules linked by electron dense cross-bridges. Since similar observations have been made on stem processes in vivo, the possibility that linked microtubules occur commonly in this site is considered. The observations are discussed in relation to a possible correlation between the presence of microtubule fascicles and the initiation of action potentials.We thank Messrs. S. Waterman, P. Felton and D. Fraser for technical assistance and Prof. D.W. James for laboratory facilities     

The regulatory effect of Tau protein on polymerization of MCF7 microtubules in vitro

Growing evidence continues to point toward the critical role of beta tubulin isotypes in regulating some intracellular functions. Changes that were observed in the microtubules’ intrinsic dynamics, the way they interact with some chemotherapeutic agents, or differences on translocation specifications of some molecular motors along microtubules, were associated to their structural uniqueness in terms of beta tubulin isotype distributions. These findings suggest that the effects of microtubule associated proteins (MAPs) may also vary on structurally different microtubules. Among different microtubule associated proteins, Tau proteins, which are known as neuronal MAPs, bind to beta tubulin, stabilize microtubules, and consequently promote their polymerizations.In this study, in a set of well controlled experiments, the direct effect of Tau proteins on the polymerization of two structurally different microtubules, porcine brain and breast cancer (MCF7), were tested and compared. Remarkably, we found that in contrast with the promoted effect of Tau proteins on brain microtubules’ polymerization, MCF7 expressed a demoted polymerization while interacting with Tau proteins. This finding can potentially be a novel insight into the mechanism of drug resistance in some breast cancer cells.It has been reported that microtubules show destabilizing behavior in some MCF7 cells with overexpression of Tau protein when treated with a microtubules’ stabilizing agent, Taxol. This behavior has been classified by others as drug resistance, but it may instead be potentially caused by a competition between the destabilizing effect of the Tau protein and the stabilizing effect of the drug on MCF7 microtubules. Also, we quantified the polarization coefficient of MCF7 microtubules in the presence and absence of Tau proteins by the electro-orientation method and compared the values. The two significantly different values obtained can possibly be one factor considered to explain the effect of Tau proteins on the polymerization of MCF7 microtubules.     

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.     

PACSIN proteins bind tubulin and promote microtubule assembly

PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.     

PAR‐1 promotes microtubule breakdown during dendrite pruning in Drosophila

Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. Here, we show that the kinase PAR‐1 is required for pruning and dendritic microtubule breakdown. Our data show that neurons lacking PAR‐1 fail to break down dendritic microtubules, and PAR‐1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR‐1 is a known Tau kinase, and genetic interactions suggest that PAR‐1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR‐1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. Our results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.     

The MAP2/Tau family of microtubule-associated proteins

Microtubule-associated proteins (MAPs) of the MAP2/Tau family include the vertebrate proteins MAP2, MAP4, and Tau and homologs in other animals. All three vertebrate members of the family have alternative splice forms; all isoforms share a conserved carboxy-terminal domain containing microtubule-binding repeats, and an amino-terminal projection domain of varying size. MAP2 and Tau are found in neurons, whereas MAP4 is present in many other tissues but is generally absent from neurons. Members of the family are best known for their microtubule-stabilizing activity and for proposed roles regulating microtubule networks in the axons and dendrites of neurons. Contrary to this simple, traditional view, accumulating evidence suggests a much broader range of functions, such as binding to filamentous (F) actin, recruitment of signaling proteins, and regulation of microtubule-mediated transport. Tau is also implicated in Alzheimer's disease and other dementias. The ability of MAP2 to interact with both microtubules and F-actin might be critical for neuromorphogenic processes, such as neurite initiation, during which networks of microtubules and F-actin are reorganized in a coordinated manner. Various upstream kinases and interacting proteins have been identified that regulate the microtubule-stabilizing activity of MAP2/Tau family proteins.     

Protein Synthesis and Axonal Transport During Nerve Regeneration

Abstract— Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [³⁵S]-methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two-dimensional polyacryl-amide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.     

TRPV1 at nerve endings regulates growth cone morphology and movement through cytoskeleton reorganization

While the importance of Ca(2+) channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton. Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retraction, and collapse and formation of varicosities along neurites. These changes were due to TRPV1-activation-mediated disassembly of microtubules and are partly Ca(2+)-independent. Prolonged activation with very low doses (1 nM) of NADA results in shortening of neurites in the majority of isolectin B4-positive dorsal root ganglia neurones. We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules. This might have a role in the chronification of pain.     

The Arabidopsis microtubule-associated protein AtMAP65-1: molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.     

Dynactin Subunit p150Glued Is a Neuron-Specific Anti-Catastrophe Factor

Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150^Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150^Glued may also modulate the dynamics of microtubules, we hypothesized that p150^Glued might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150^Glued is a potent anti-catastrophe factor for microtubules. p150^Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150^Glued are required in tandem for this activity. p150^Glued is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150^Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150^Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150^Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease.     

CNP/cGMP signaling regulates axon branching and growth by modulating microtubule polymerization

The peptide hormone CNP has recently been found to positively regulate axon branching and growth via activation of cGMP signaling in embryonic dorsal root ganglion (DRG) neurons, but the cellular mechanisms mediating the regulation of these developmental processes have not been established. In this study, we provide evidence linking CNP/cGMP signaling to microtubule dynamics via the microtubule regulator CRMP2. First, phosphorylation of CRMP2 can be suppressed by cGMP activation in embryonic DRG neurons, and non‐phosphorylated CRMP2 promotes axon branching and growth. In addition, real time analysis of growing microtubule ends indicates a similar correlation of CRMP2 phosphorylation and its activity in promoting microtubule polymerization rates and durations in both COS cells and DRG neuron growth cones. Moreover, direct activation of cGMP signaling leads to increased assembly of dynamic microtubules in DRG growth cones. Finally, low doses of a microtubule depolymerization drug nocodazole block CNP/cGMP‐dependent axon branching and growth. Taken together, our results support a critical role of microtubule dynamics in mediating CNP/cGMP regulation of axonal development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 673–687, 2013     

The Balance Between τ Protein's Microtubule Growth and Nucleation Activities: Implications for the Formation of Axonal Microtubules

Abstract: The microtubule-associated protein τ is found primarily in neuronal tissues and is highly enriched in the axon. It promotes microtubule assembly in vitro and stabilizes microtubules in cells. To study how τ protein might be involved in the unique features of axonal microtubules, we have analyzed the effect of E. coli -synthesized τ protein using an in vitro centrosome-mediated microtubule regrowth assay over a wide range of τ/tubulin ratios. We report that microtubule assembly promoted by τ protein exhibits characteristic changes dependent on the τ/tubulin ratio. Above a threshold level, nucleation of new microtubules is favored over growth of existing ones, τ isoform variation does not change this phase transition in microtubule assembly. We discuss how τ might participate in the elaboration of axonal morphology based on our results and present evidence that the phase transition from microtubule growth to nucleation is critical for axonal development.     

Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules

Tau is one of the most abundant microtubule-associated proteins involved in kinetic stabilization and bundling of axonal microtubules. Although intense research has revealed much about tau function and its involvement in Alzheimer's disease during the past years, it still remains unclear how exactly tau binds on microtubules and if the kinetic stabilization of microtubules by tau is accompanied, at least in part, by a mechanical reinforcement of microtubules. In this paper, we have used atomic force microscopy to address both aspects by visualizing and mechanically analyzing microtubules in the presence of native tau isoforms. We could show that tau at saturating concentrations forms a 1 nm thick layer around the microtubule, but leaves the protofilament structure well visible. The latter observation argues for tau binding mainly along and not across the protofilaments. The radial elasticity of microtubules was almost unaffected by tau, consistent with tau binding along the tops of the protofilaments. Tau did increase the resistance of microtubules against rupture. Finite-element calculations confirmed our findings.     

Retrograde Axonal Transport of Locally Synthesized Phosphoinositides in the Rat Sciatic Nerve

Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo-[3H]inositol into the rat sciatic nerve (50-55 mm distal to L4 and L5 dorsal root ganglia), a time-dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10-12-fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water-soluble label in the dorsal root ganglia still remained approximately 1.0, whereas the corresponding ratio in the chloroform/methanol-soluble fraction was approximately 20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injection site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia.     

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus‐end directed kinesin and minus‐end directed dynein motors. Microtubules are decorated by microtubule‐associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimer's disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single‐molecule assays indicate that kinesin‐1 is more strongly inhibited than kinesin‐2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin‐1, kinesin‐2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus‐end in a dose‐dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin‐1, kinesin‐2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor‐specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus‐ and minus‐end directed transport.      

Novel diffusion barrier for axonal retention of Tau in neurons and its failure in neurodegeneration

Missorting of Tau from axons to the somatodendritic compartment of neurons is a hallmark of Alzheimer's disease, but the mechanisms underlying normal sorting and pathological failure are poorly understood. Here, we used several Tau constructs labelled with photoconvertible Dendra2 to analyse its mobility in polarized neurons. This revealed a novel mechanism of sorting-a retrograde barrier in the axon initial segment (AIS) operating as cellular rectifier. It allows anterograde flow of axonal Tau but prevents retrograde flow back into soma and dendrites. The barrier requires binding of Tau to microtubules but does not require F-actin and thus is distinct from the sorting of membrane-associated proteins at the AIS. The barrier breaks down when Tau is phosphorylated in its repeat domain and detached from microtubules, for example, by the kinase MARK/Par1. These observations link the pathological hallmarks of Tau missorting and hyperphosphorylation in neurodegenerative diseases.