Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions

Microtubules gate tau condensation to spatially regulate microtubule functions.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule

Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization

Cytoplasmic dynein-1 is a minus-end-directed motor protein that transports cargo over long distances and organizes the intracellular microtubule (MT) network. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, resulting in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, suggesting that minus-end accumulations of motors produce network-scale contractile stresses. Our data provide a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the cytoskeleton, for example, during mitotic spindle assembly

Structure and Mechanics of Dynein Motors

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia

Microtubules gate tau condensation to spatially regulate microtubule functions.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins.

Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive 'envelopes' around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2

Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions

Inhibiting Insulin-Mediated &bgr;2-Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction

BackgroundType 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity.MethodsHigh-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking β2-adrenergic receptor (β2AR) or β-arrestin2. Wild-type mice fed with high-fat diet were treated with a β-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function.ResultsHigh-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and β-arrestin2-dependent transactivation of a β2AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of β2AR or GRK2, or genetic deletion of β2AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction.ConclusionsThese studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β2AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM

Inhibiting Insulin-Mediated β 2

BACKGROUND: Type-2 diabetes and obesity independently increases the risk of heart failure via incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in hearts of subjects with type-2 diabetes and obesity. METHODS: High fat diet feeding was used to induce obesity and diabetes in wild type mice or mice lacking β(2)-adrenergic receptor (β(2)AR) or β-arrestin2. Wild type mice fed with high fat diet were treated with β-blocker carvedilol or G-protein receptor kinase 2 (GRK2) inhibitor. We examined the signaling and cardiac contractile function. RESULTS: High fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced PKA phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with diabetes. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and (GRK2) and β-arrestin2-dependent transactivation of a β(2)AR-ERK signaling cascade. Thus pharmacological inhibition of β(2)AR or GRK2, or genetic deletion of β(2)AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of ERK and PDE4D induction, to prevent diabetes-related contractile dysfunction. CONCLUSIONS: These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β(2)AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type-2 diabetes