RESUMO
Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein, which binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full length human protein tau and other negatively charged binding substrates, as revealed by differential-interference-contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes exhibited distinct types of morphologies. This included, large ≈20-30 micron tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles, and tau-mediated finite-size assemblies of small liposomes. As the ionic strength of the solution was increased to near and above physiological salt concentrations for 1:1 electrolytes (≈150 mM), AL-tau complexes remained stable while tau self-coacervate droplets were found to dissolve indicative of breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen facing monolayer of the axon plasma membrane suggesting the possibility of transient yet robust interactions at physiologically relevant ionic strengths.
RESUMO
The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. Tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in varying concentrations of Mg2+ or Ca2+ divalent cations in mixtures containing αß-tubulin, full-length tau, and GTP at 37 °C in a physiological buffer. A concentration-time kinetic phase diagram generated by synchrotron SAXS reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. SAXS with TEM of plastic-embedded samples provides evidence of a viscoelastic intervening network (IN) of complexes of tubulin oligomers and tau stabilizing MT bundles. In this model, αß-tubulin oligomers in the IN are crosslinked by tau's MT binding repeats, which also link αß-tubulin oligomers to αß-tubulin within the MT lattice. The model challenges whether the cross-bridging of MTs is attributed entirely to MAPs. Tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau, promoting nucleation and growth of tau fibrils in tauopathies.
Assuntos
Tubulina (Proteína) , Proteínas tau , Microtúbulos/metabolismo , Espalhamento a Baixo Ângulo , Proteínas tau/metabolismo , Tubulina (Proteína)/metabolismo , Difração de Raios X , HumanosRESUMO
Aggregated and hyperphosphorylated Tau is one of the pathological hallmarks of Alzheimer's disease. Tau is a polyampholytic and intrinsically disordered protein (IDP). In this paper, we present for the first time experimental results on the ionic strength dependence of the radius of gyration (Rg) of human Tau 4RS and 4RL isoforms. Synchrotron X-ray scattering revealed that 4RS Rg is regulated from 65.4 to 58.5 Å and 4RL Rg is regulated from 70.9 to 57.9 Å by varying ionic strength from 0.01 to 0.592 M. The Rg of 4RL Tau is larger than 4RS at lower ionic strength. This result provides an insight into the ion-responsive nature of intrinsically disordered and polyampholytic Tau, and can be implicated to the further study of Tau-Tau and Tau-tubulin intermolecular structure in ionic environments.
Assuntos
Proteínas Intrinsicamente Desordenadas , Síncrotrons , Humanos , Raios XRESUMO
The emergence of the SARS-CoV-2 Omicron variant in 2021 is associated with a global surge of cases in late 2021 and early 2022. Identifying the introduction of novel SARS-CoV-2 variants to a population is imperative to inform decisions by clinicians and public health officials. Here, we describe a quantitative reverse transcription PCR-based assay (RT-qPCR) targeting unique mutations in the Omicron BA.1/BA1.1 and BA.2 viral genomes. This assay accurately and precisely detect the presence of these Omicron variants in patient samples in less than four hours. Using this assay, we tested 270 clinical samples and detected the introduction of Omicron BA.1/BA1.1 and BA.2 in the Santa Barbara County (SBC) population in December 2021 and February 2022, respectively. Identifying Omicron variants using this RT-qPCR assay showed complete concordance with whole viral genome sequencing; both assays indicated that Omicron was the dominant variant in SB County. Our data substantiate that RT-qPCR-based virus detection assays offer a fast and inexpensive alternative to NGS for virus variant-specific detection approach, which allows streamlining the detection of Omicron variants in patient samples.
RESUMO
Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Oligopeptídeos/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Sistema Nervoso Periférico/efeitos dos fármacos , Moduladores de Tubulina/toxicidade , Tubulina (Proteína)/metabolismo , Transporte Axonal/efeitos dos fármacos , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Microtúbulos/patologia , Mitose/efeitos dos fármacos , Oligopeptídeos/metabolismo , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Ligação Proteica , Medição de Risco , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Moduladores de Tubulina/metabolismoRESUMO
By virtue of their native structures, tubulin dimers are protein building blocks that are naturally preprogrammed to assemble into microtubules (MTs), which are cytoskeletal polymers. Here, polycation-directed (i.e., electrostatically tunable) assembly of tubulins is demonstrated by conformational changes to the tubulin protofilament in longitudinal and lateral directions, creating tubulin double helices and various tubular architectures. Synchrotron small-angle X-ray scattering and transmission electron microscopy reveal a remarkable range of nanoscale assembly structures: single- and double-layered double-helix tubulin tubules. The phase transitions from MTs to the new assemblies are dependent on the size and concentration of polycations. Two characteristic scales that determine the number of observed phases are the size of polycation compared to the size of tubulin (≈4 nm) and to MT diameter (≈25 nm). This work suggests the feasibility of using polycations that have scissor- and glue-like properties to achieve "programmable breakdown" of protein nanotubes, tearing MTs into double-stranded tubulins and building up previously undiscovered nanostructures. Importantly, a new role of tubulins is defined as 2D shape-controllable building blocks for supramolecular architectures. These findings provide insight into the design of protein-based functional materials, for example, as metallization templates for nanoscale electronic devices, molecular screws, and drug delivery vehicles.
Assuntos
Microtúbulos , Tubulina (Proteína) , Citoesqueleto , PolímerosRESUMO
In this minireview, which is part of a special issue in honor of Jacob N. Israelachvili's remarkable research career on intermolecular forces and interfacial science, we present studies of structures, phase behavior, and forces in reaction mixtures of microtubules (MTs) and tubulin oligomers with either intrinsically disordered protein (IDP) Tau, cationic vesicles, or the polyamine spermine (4+). Bare MTs consist of 13 protofilaments (PFs), on average, where each PF is made of a linear stack of αß-tubulin dimers (i.e., tubulin oligomers). We begin with a series of experiments which demonstrate the flexibility of PFs toward shape changes in response to local environmental cues. First, studies show that MT-associated protein (MAP) Tau controls the diameter of microtubules upon binding to the outer surface, implying a shape change in the cross-sectional area of PFs forming the MT perimeter. The diameter of a MT may also be controlled by the charge density of a lipid bilayer membrane that coats the outer surface. We further describe an experimental study where it is unexpectedly found that the biologically relevant polyamine spermine (+4e) is able to depolymerize taxol-stabilized microtubules with efficiency that increases with decreasing temperature. This MT destabilization drives a dynamical structural transition where inside-out curving of PFs, during the depolymerization peeling process, is followed by reassembly of ring-like curved PF building blocks into an array of helical inverted tubulin tubules. We finally turn to a very recent study on pressure-distance measurements in bundles of MTs employing the small-angle X-ray scattering (SAXS)-osmotic pressure technique, which complements the surface-forces-apparatus technique developed by Jacob N. Israelachvili. These latter studies are among the very few which are beginning to shed light on the precise nature of the interactions between MTs mediated by MAP Tau in 37 °C reaction mixtures containing GTP and lacking taxol.
Assuntos
Biopolímeros/química , Proteínas Intrinsicamente Desordenadas/química , Microtúbulos/química , Tubulina (Proteína)/química , Proteínas tau/química , Cátions , Paclitaxel/química , Conformação ProteicaRESUMO
The microtubule (MT)-associated protein tau regulates the critical growing and shortening behaviors of MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Although tau is known to promote net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear. Here, we have used the slowly-hydrolyzable GTP analog, guanylyl-(α,ß)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. The addition of both four-repeat (4R) and three-repeat (3R) tau isoforms to pre-formed GMPCPP MTs resulted in the formation of extended, multiprotofilament-wide projections at MT ends. Furthermore, at temperatures too low for assembly of bona fide MTs, both tau isoforms promoted the formation of long spiral ribbons from GMPCPP tubulin heterodimers. In addition, GMPCPP MTs undergoing cold-induced disassembly in the presence of 4R tau (and to a much lesser extent 3R tau) also formed spirals. Finally, three pathological tau mutations known to cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize MT disassembly intermediates. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin-tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants.
Assuntos
Microtúbulos/química , Tubulina (Proteína)/química , Proteínas tau/química , Demência/metabolismo , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.
Assuntos
Antineoplásicos/toxicidade , Neuropatia Ciática , Animais , Modelos Animais de Doenças , Epotilonas/toxicidade , Feminino , Furanos/toxicidade , Filamentos Intermediários/metabolismo , Cetonas/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Paclitaxel/toxicidade , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Proteínas S100/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Neuropatia Ciática/induzido quimicamente , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of disability in cancer survivors. CIPN investigations in preclinical model systems have focused on either behaviors or acute changes in nerve conduction velocity (NCV) and amplitude, but greater understanding of the underlying nature of axonal injury and its long-term processes is needed as cancer patients live longer. In this study, we used multiple independent endpoints to systematically characterize CIPN recovery in mice exposed to the antitubulin cancer drugs eribulin, ixabepilone, paclitaxel, or vinorelbine at MTDs. All of the drugs ablated intraepidermal nerve fibers and produced axonopathy, with a secondary disruption in myelin structure within 2 weeks of drug administration. In addition, all of the drugs reduced sensory NCV and amplitude, with greater deficits after paclitaxel and lesser deficits after ixabepilone. These effects correlated with degeneration in dorsal root ganglia (DRG) and sciatic nerve and abundance of Schwann cells. Although most injuries were fully reversible after 3-6 months after administration of eribulin, vinorelbine, and ixabepilone, we observed delayed recovery after paclitaxel that produced a more severe, pervasive, and prolonged neurotoxicity. Compared with other agents, paclitaxel also displayed a unique prolonged exposure in sciatic nerve and DRG. The most sensitive indicator of toxicity was axonopathy and secondary myelin changes accompanied by a reduction in intraepidermal nerve fiber density. Taken together, our findings suggest that intraepidermal nerve fiber density and changes in NCV and amplitude might provide measures of axonal injury to guide clinical practice.Significance: This detailed preclinical study of the long-term effects of widely used antitubulin cancer drugs on the peripheral nervous system may help guide clinical evaluations to improve personalized care in limiting neurotoxicity in cancer survivors. Cancer Res; 78(3); 817-29. ©2017 AACR.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Recuperação de Função Fisiológica/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Moduladores de Tubulina/toxicidade , Doença Aguda , Animais , Células Cultivadas , Feminino , Gânglios Espinais/lesões , Gânglios Espinais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microtúbulos/patologia , Doenças do Sistema Nervoso Periférico/patologia , Células de Schwann/patologia , Nervo Isquiático/lesões , Nervo Isquiático/patologiaRESUMO
The proliferation of successful, cell-free reconstitutions of cytoskeletal networks have prompted measurements of forces between network elements via induced osmotic pressure by the addition of depletants. Indeed, it was through osmotic pressurization that Tau, an intrinsically disordered protein found in neuronal axons, was recently discovered to mediate two distinct microtubule (MT) bundle states, one widely spaced and a second tightly packed, separated by an energy barrier due to polyelectrolyte repulsions between opposing Tau projection domains on neighboring MT surfaces. Here, we compare interfilament force measurements in Tau coated MT bundles using PEO20k (poly(ethylene oxide), Mw = 20000), a commonly used inert depletant, and recently published measurements with PEO102k. While force measurements with either depletant reveals the transition between the two bundled states, measurements with PEO20k cannot recapitulate the correct critical pressure (Pc) at which widely spaced MT bundles transition to tightly packed MT bundles due to depletant penetration into widely spaced bundles below Pc. Surprisingly, upon transitioning to the tightly packed bundle state data from both depletants are in quantitative agreement indicative of expulsion of the smaller PEO20k depletant, but only at distances comparable or less than the PEO20k radius of gyration, significantly smaller than the effective diameter of PEO20k. While PEO102k (with size larger than the wall-to-wall distance between MTs in bundles) can more accurately capture the force response behavior at low to intermediate pressures (<104 Pa), measurements with PEO20k, beyond the overlap regime with PEO102k, extend the achievable osmotic pressure range into the higher-pressure regime (â¼5 × 104 Pa). The data underscores the importance of the use of polymeric depletants of different sizes to elucidate force response behavior of cytoskeletal filamentous networks over a more complete extended pressure range.
RESUMO
The early oligomerization of amyloid ß-protein (Aß) is a crucial step in the etiology of Alzheimer's disease (AD), in which soluble and highly neurotoxic oligomers are produced and accumulated inside neurons. In search of therapeutic solutions for AD treatment and prevention, potent inhibitors that remodel Aß assembly and prevent neurotoxic oligomer formation offer a promising approach. In particular, several polyphenolic compounds have shown anti-aggregation properties and good efficacy on inhibiting oligomeric amyloid formation. 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a large polyphenol that has been shown to be effective at inhibiting aggregation of full-length Aß1-40 and Aß1-42, but has the opposite effect on the C-terminal fragment Aß25-35. Here, we use a combination of ion mobility coupled to mass spectrometry (IMS-MS), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations to elucidate the inhibitory effect of PGG on aggregation of full-length Aß1-40 and Aß1-42. We show that PGG interacts strongly with these two peptides, especially in their N-terminal metal binding regions, and suppresses the formation of Aß1-40 tetramer and Aß1-42 dodecamer. By exploring multiple facets of polyphenol-amyloid interactions, we provide a molecular basis for the opposing effects of PGG on full-length Aß and its C-terminal fragments.
RESUMO
In this chapter, we describe methods for the purification of both untagged and polyhistidine-tagged tau protein. These protocols utilize a bacterial expression system to produce the tau isoform of interest, followed by heat treatment and column chromatography to separate tau from impurities. These techniques yield a biochemically pure protein with which to pursue any number of questions regarding the mechanisms of tau action.
Assuntos
Cromatografia de Afinidade/métodos , Histidina/metabolismo , Proteínas tau/isolamento & purificação , Proteínas tau/metabolismo , Histidina/química , Histidina/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas tau/genéticaRESUMO
Tau, a neuronal protein known to bind to microtubules and thereby regulate microtubule dynamic instability, has been shown recently to not only undergo conformational transitions on the microtubule surface as a function of increasing microtubule coverage density (i.e., with increasing molar ratio of Tau to tubulin dimers) but also to mediate higher-order microtubule architectures, mimicking fascicles of microtubules found in the axon initial segment. These discoveries would not have been possible without fine structure characterization of microtubules, with and without applied osmotic pressure through the use of depletants. Herein, we discuss the two primary techniques used to elucidate the structure, phase behavior, and interactions in microtubule/Tau mixtures: transmission electron microscopy and synchrotron small-angle X-ray scattering. While the former is able to provide striking qualitative images of bundle morphologies and vacancies, the latter provides angstrom-level resolution of bundle structures and allows measurements in the presence of in situ probes, such as osmotic depletants. The presented structural characterization methods have been applied both to equilibrium mixtures, where paclitaxel is used to stabilize microtubules, and also to dissipative nonequilibrium mixtures at 37°C in the presence of GTP and lacking paclitaxel.
Assuntos
Microscopia Eletrônica/métodos , Microtúbulos/química , Espalhamento a Baixo Ângulo , Síncrotrons/instrumentação , Tubulina (Proteína)/química , Difração de Raios X/métodos , Proteínas tau/química , Humanos , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismoRESUMO
The proper organization and function of the mammalian nervous system relies on neuronal processes or "neurites," extended morphological projections that include axons and dendrites. Tau is a structural microtubule-associated protein that is widely expressed in the nervous system that mediates the establishment of cell polarity, neurite outgrowth, and axonal transport. A useful model for studying the establishment and maintenance of these neuronal structures are rat neuronal PC12 cells, which can be induced to express tau and project neurites by treating the cells with nerve growth factor. Here, we present a simple method for continuously measuring the rate of neurite outgrowth and retraction over time by neurite length and neurite area analyses. This method uses freely available ImageJ software and widely available phase-contrast imaging.
Assuntos
Microscopia de Contraste de Fase/métodos , Neuritos/ultraestrutura , Crescimento Neuronal , Proteínas tau/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Diferenciação Celular , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Neuritos/metabolismo , Células PC12 , RatosRESUMO
Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of anticancer treatment with microtubule-targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced activating transcription factor 3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and end binding protein 1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence, and recovery from CIPN.
Assuntos
Antineoplásicos/toxicidade , Furanos/uso terapêutico , Cetonas/uso terapêutico , Paclitaxel/uso terapêutico , Neuropatia Ciática/induzido quimicamente , Neuropatia Ciática/patologia , Células Receptoras Sensoriais/efeitos dos fármacos , Fator 3 Ativador da Transcrição/metabolismo , Animais , Corpo Celular , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: Microtubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αß-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius
Assuntos
Microtúbulos/metabolismo , Paclitaxel/farmacologia , Proteínas tau/metabolismo , Animais , Bovinos , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent 'active' microtubule bundles at 37 °C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw-w≈25-41 nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw-w≈16-23 nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau's projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge-charge attractions mediated by weakly penetrating opposing PDs. This landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.
Assuntos
Segmento Inicial do Axônio/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Animais , Segmento Inicial do Axônio/ultraestrutura , Bovinos , Microtúbulos/ultraestrutura , Pressão Osmótica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Termodinâmica , Difração de Raios X , Proteínas tau/químicaRESUMO
Despite extensive structure-function analyses, the molecular mechanisms of normal and pathological tau action remain poorly understood. How does the C-terminal microtubule-binding region regulate microtubule dynamics and bundling? In what biophysical form does tau transfer trans-synaptically from one neuron to another, promoting neurodegeneration and dementia? Previous biochemical/biophysical work led to the hypothesis that tau can dimerize via electrostatic interactions between two N-terminal 'projection domains' aligned in an anti-parallel fashion, generating a multivalent complex capable of interacting with multiple tubulin subunits. We sought to test this dimerization model directly. Native gel analyses of full-length tau and deletion constructs demonstrate that the N-terminal region leads to multiple bands, consistent with oligomerization. Ferguson analyses of native gels indicate that an N-terminal fragment (tau(45-230) ) assembles into heptamers/octamers. Ferguson analyses of denaturing gels demonstrates that tau(45-230) can dimerize even in sodium dodecyl sulfate. Atomic force microscopy reveals multiple levels of oligomerization by both full-length tau and tau(45-230) . Finally, ion mobility-mass spectrometric analyses of tau(106-144) , a small peptide containing the core of the hypothesized dimerization region, also demonstrate oligomerization. Thus, multiple independent strategies demonstrate that the N-terminal region of tau can mediate higher order oligomerization, which may have important implications for both normal and pathological tau action. The microtubule-associated protein tau is essential for neuronal development and maintenance, but is also central to Alzheimer's and related dementias. Unfortunately, the molecular mechanisms underlying normal and pathological tau action remain poorly understood. Here, we demonstrate that tau can homo-oligomerize, providing novel mechanistic models for normal tau action (promoting microtubule growth and bundling, suppressing microtubule shortening) and pathological tau action (poisoning of oligomeric complexes).