Stimulate Microtubule Assembly Research Articles

The βγ subunit of heterotrimeric G proteins interacts with actin filaments during neuronal differentiation

The βγ subunit of heterotrimeric G proteins, a key molecule in the G protein-coupled receptors (GPCRs) signaling pathway, has been shown to be an important factor in the modulation of the microtubule cytoskeleton. Gβγ has been shown to bind to tubulin, stimulate microtubule assembly, and promote neurite outgrowth of PC12 cells. In this study, we demonstrate that in addition to microtubules, Gβγ also interacts with actin filaments, and this interaction increases during NGF-induced neuronal differentiation of PC12 cells. We further demonstrate that the Gβγ-actin interaction occurs independently of microtubules as nocodazole, a well-known microtubule depolymerizing agent did not inhibit Gβγ-actin complex formation in PC12 cells. A confocal microscopic analysis of NGF-treated PC12 cells revealed that Gβγ co-localizes with both actin and microtubule cytoskeleton along neurites, with specific co-localization of Gβγ with actin at the distal end of these neuronal processes. Furthermore, we show that Gβγ interacts with the actin cytoskeleton in primary hippocampal and cerebellar rat neurons. Our results indicate that Gβγ serves as an important modulator of the neuronal cytoskeleton by interacting with both microtubules and actin filaments, and is likely to participate in various aspects of neuronal differentiation including axon and growth cone formation.

Structural insight into TPX2-stimulated microtubule assembly.

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.

Centrosomal Protein 70 Is a Mediator of Paclitaxel Sensitivity.

Centrosome aberrations have been implicated in the development and progression of breast cancer. Our previous worked show that centrosomal protein 70 (Cep70) regulates breast cancer growth and metastasis. However, it remains elusive whether Cep70 is implicated in the sensitivity of the anti-microtubule drug paclitaxel in breast cancer. Here we provide evidence that Cep70 is a mediator of paclitaxel sensitivity in breast cancer. Cell proliferation assays show that Cep70 expression correlates with paclitaxel sensitivity in breast cancer cell lines. In addition, paclitaxel sensitivity varies when altering Cep70 expression level. Mechanistic studies reveal that Cep70 interacts with tubulin, and promotes the ability of paclitaxel to stimulate microtubule assembly. These data demonstrate that Cep70 mediates paclitaxel sensitivity in breast cancer.

GIT1 enhances neurite outgrowth by stimulating microtubule assembly.

GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

The active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics.

The pathological accumulation of abnormally hyperphosphorylated and aggregated tau, a neuronal microtubule (MT)-associated protein that functions to maintain MT stability, is implicated in a number of hereditary and sporadic neurodegenerative diseases including frontotemporal dementia and Alzheimer's disease. Targeting tau for the treatment of these diseases is an area of intense interest and toward that end, modulation of cellular molecular chaperones is a potential therapeutic target. In particular, the constitutive Hsp70 isoform, Hsc70, seems highly interconnected with tau, preserving tau protein levels and synergizing with it to assemble MTs. But the relationship between tau and Hsc70, as well as the impact of this interaction in neurons and its therapeutic implications remain unknown. Using a human dominant negative Hsc70 that resembles isoform selective inhibition of this important chaperone, we found for the first time that Hsc70 activity is required to stimulate MT assembly in cells and brain. However, surprisingly, active Hsc70 also requires active tau to regulate MT assembly in vivo, suggesting that tau acts in some ways as a co-chaperone for Hsc70 to coordinate MT assembly. This was despite tau binding to Hsc70 as substrate, as determined biochemically. Moreover, we show that while chronic Hsc70 inhibition damaged MT dynamics, intermittent treatment with a small molecule Hsp70 inhibitor lowered tau in brain tissue without disrupting MT integrity. Thus, in tauopathies, where MT injury would be detrimental to neurons, the unique relationship of tau with the Hsc70 machinery can be exploited to deplete tau levels without damaging MT networks.

The subtle allostery of microtubule dynamics.

New high-resolution structures of microtubules reveal that GTP and taxol each stimulate microtubule assembly by inducing straight protofilaments and promoting extension of the interdimer spacing. However, these effects take place through different mechanisms: GTP directly extends loops around the active site, whereas taxol works like a remote lever.

Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.

The octarepeat region of hamster PrP (PrP51–91) enhances the formation of microtubule and antagonize Cu<sup>2+</sup>-induced microtubule-disrupting activity

Prion protein (PrP) is considered to associate with microtubule and its major component, tubulin. In the present study, octarepeat region of PrP (PrP51-91) was expressed in prokaryotic-expressing system. Using GST pull-down assay and co-immunoprecipitation, the molecular interaction between PrP51-91 and tubulin was observed. Our data also demonstrated that PrP51-91 could efficiently stimulate microtubule assembly in vitro, indicating a potential effect of PrP on microtubule dynamics. Moreover, PrP51-91 was confirmed to be able to antagonize Cu(2+)-induced microtubule-disrupting activity in vivo, partially protecting against Cu(2+) intoxication to culture cells and stabilize cellular microtubule structure. The association of the octarepeat region of PrP with tubulin may further provide insight into the biological function of PrP in the neurons.

Interaction of CDK5RAP2 with EB1 to Track Growing Microtubule Tips and to Regulate Microtubule Dynamics

Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2-EB1 complex regulates microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of microtubules. The CDK5RAP2-EB1 complex induces microtubule bundling and acetylation when expressed in cell cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics.

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

BackgroundYB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.ResultsWe show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.ConclusionThese results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.

NAP, A Neuroprotective Drug Candidate in Clinical Trials, Stimulates Microtubule Assembly in the Living Cell

NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP) provides neuroprotection in vitro and in vivo against a wide variety of neurotoxic substances. To further understand the mechanism by which NAP provides broad neuroprotection it was essential to find NAP's binding partners. Previous results, using affinity chromatography coupled with mass spectrometry, identified tubulin, the subunit protein of microtubules, as the major NAP binding protein in neurons and glial cells. Here, following microtubule depolymerization in the presence of nocodazole, NAP treatment enhanced rapid microtubule assembly and stimulated neurite outgrowth. Nocodazole is an established inhibitor of axoplasmic transport and cell division that exerts its effect by depolymerizing microtubules. NAP shows selectivity in interacting with brain tubulin and does not affect dividing cells. This data demonstrates that NAP functions as a neuroprotectant, at least in part, through its interaction with tubulin with a resulting increase in microtubule assembly.

Site‐specific effects of tau phosphorylation on its microtubule assembly activity and self‐aggregation

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains with Alzheimer's disease. The phosphorylation sites of tau are mainly localized in the proline-rich (residues 172-251) and C-terminal tail (residues 368-441) regions, which flank the microtubule-binding repeats. Here, we investigated the effects of tau phosphorylation at these distinct sites/regions on its activity of stimulating microtubule assembly and its self-aggregation. We found that tau phosphorylation at the proline-rich region by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A inhibited its microtubule assembly activity moderately and promoted its self-aggregation slightly. Tau phosphorylation at the C-terminal tail region by glycogen synthase kinase-3beta increased its activity and promoted its self-aggregation markedly. Tau phosphorylation at both regions plus the microtubule-binding region by cAMP-dependent protein kinase diminished its activity (approximately 70% inhibition) and disrupted microtubules. These studies reveal the differential regulation of tau's biological activity and self-aggregation by phosphorylation at various sites/regions.

Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities.

The guanosine tri-phosphatase Ran stimulates assembly of microtubule spindles. However, it is not known what aspects of the microtubule cytoskeleton are subject to regulation by Ran in mitosis. Here we show that Ran-GTP stimulates microtubule assembly by increasing the rescue frequency of microtubules three- to eightfold. In addition to changing microtubule dynamics, Ran-GTP also alters the balance of motor activities, partly as a result of an increase in the amount of motile Eg5, a plus-end-directed microtubule motor that is essential for spindle formation. Thus, Ran regulates multiple processes that are involved in spindle assembly.

Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly.

Fetal or adult rat-brain cytosol and fetal rat-brain microtubules contain a high-affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30-50 nM range. The best competitors (in decreasing order) are pregnenolone sulfate, progesterone, Delta5-pregnene-3beta,20alpha-diol, and 3beta-hydroxy-5alpha-pregnan-20-one. It was hypothesized that the pregnenolone-binding protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and gel-filtration columns yielded two fractions, one of very high molecular mass, >200 kDa, and the other of 40-60 kDa, enriched in [(3)H]pregnenolone-binding activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubules, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [(3)H]pregnenolone binding with an affinity very close to that of rat-brain microtubules, but with a much larger concentration of binding sites (16 pmol/mg MAP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin. Finally, steroid effects on microtubule-assembly kinetics were assayed. Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, counteracted the stimulatory effect of pregnenolone. Electron microscopic analysis confirmed that pregnenolone-increased assembly of microtubules produced a completely normal structure. The stimulatory effect on MAP2-tubulin interaction was also observed in fetal rat-brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging.

G Protein β1γ2 Subunits Promote Microtubule Assembly

alpha and betagamma subunits of G proteins are thought to transduce signals from cell surface receptors to intracellular effector molecules. Galpha and Gbetagamma have also been implicated in cell growth and differentiation, perhaps due to their association with cytoskeletal components. In this report Gbetagamma is shown to modulate the cytoskeleton by regulation of microtubule assembly. Specificity among betagamma species exists, as beta1gamma2 stimulates microtubule assembly, and beta1gamma1 is without any effect. Furthermore, a mutant beta1gamma2, beta1gamma2(C68S), which does not undergo prenylation and subsequent carboxyl-terminal processing on the gamma subunit, does not stimulate the formation of microtubules. beta immunoreactivity was detected exclusively in the microtubule fraction after assembly in the presence of beta1gamma2, suggesting a preferential association with microtubules rather than soluble tubulin. Crude microtubule fractions from ovine brain contain Gbetagamma, and electron microscopy reveals a specific association with microtubules. The decoration of microtubules by Gbetagamma appears to be strikingly similar to the periodic pattern observed for microtubule-associated proteins, suggesting a similar site of activation of microtubule assembly by both agents. It is suggested that reformation of the cytoskeleton represents an additional cellular process mediated by Gbetagamma.

GS-164, a small synthetic compound, stimulates tubulin polymerization by a similar mechanism to that of Taxol.

During our search for new microtubule effectors as anticancer agents, we have found that a small synthetic molecule designated GS-164 interferes with the assembly of porcine microtubule proteins and has cytotoxic activity against a wide range of human tumor cell lines. In this study, we investigated mode of action of the compound in comparison with Taxol and colcemid. To gain an insight into the mode of action of GS-164, we used an in vitro microtubule polymerization assay and a flow-cytometric measurement technique. Microtubule organization and the level of tubulin polymerization in HeLa cells were also examined by immunofluorescence microscopy and cytoskeletal protein analyses, respectively. GS-164 stimulated assembly of microtubule proteins in vitro in a concentration-dependent and a GTP-independent manner. Furthermore, as with Taxol, the microtubule polymerization induced by GS-164 was antagonized by podophyllotoxin, a tubulin polymerization inhibitor, and microtubules formed by GS-164 were resistant to disassembly by calcium or low temperatures. GS-164 in the micromolar range arrested the cell cycle of HeLa cells in the mitotic phase leading to cell death. GS-164 also increased the amounts of cellular microtubules in HeLa cells, resulting in the formation of microtubule bundles. These results indicate that GS-164 stimulates microtubule assembly by a similar mechanism to that of Taxol. A comparative conformational analysis of GS-164 and Taxol suggested that the structure of the former mimics the minimum essential sites of Taxol required to exert the Taxol-like activities described above. Although the cytotoxicity of GS-164 against human tumor cells was 1000-fold lower than that of Taxol and GS-164 was one-tenth as active as Taxol in vitro, these findings pave the way for synthesizing clinically useful anticancer agents using GS-164 as a lead compound.

Influence of microtubule-associated proteins on the differential effects of paclitaxel and docetaxel.

Microtubules are complex structures arising in part from the polymerization of tubulin dimers. Tubulin binds to a wide range of drugs which have been used as probes for tubulin conformation and assembly properties. There is some evidence that taxol and taxotere have differing effects on tubulin conformation. Previous work has shown that MAP2 and Tau, although they both induce microtubule assembly, have qualitatively different effects on tubulin's behavior. Since most microtubules in vivo are likely to be associated with MAPs, we decided to characterize the differential effects of MAP2, Tau, taxol, and taxotere on tubulin polymerization with the aim of understanding the mechanisms through which these agents stimulate microtubule assembly. Furthermore, the inhibitive effect of calcium has been used to elucidate the ability of the two drugs to force tubulin assembly. These observations suggest that docetaxel, in addition to its greater efficiency in tubulin assembly, may have the capacity to differently alter certain classes of microtubules. Tau and MAP2 accessory proteins may represent important cofactors modulating the effects of taxoids.

Temperature sensitivity of vinblastine-induced tubulin polymerization in the presence of microtubule-associated proteins.

The antitumor drug vinblastine has been a useful probe for examining the interaction of tubulin with the microtubule-associated proteins (MAPs), specifically with tau and MAP 2. Although tau and MAP 2 can stimulate microtubule assembly in vitro, their specific interactions with tubulin are known to differ. For example, in the presence of vinblastine, both tau and MAP 2 cause tubulin to form spirals, but tau causes formation of clustered spirals of high turbidity, while MAP 2 causes formation of loose spirals of low turbidity [Ludueña et al., J. Biol. Chem. 259, 12890-12898 (1984)]. Although cold temperatures can inhibit microtubule assembly, cold has no effect on vinblastine-induced tubulin spiral formation. Consequently, we used the vinblastine-tubulin system to examine the interactions of tau and MAP 2 with tubulin at low temperatures. We found that tau-tubulin-vinblastine complexes form about as well at 0 degree C as at 37 degrees C. In contrast, MAP 2-tubulin-vinblastine complexes form much less well at 0 degrees C than at 37 degrees C. We find, however, that MAP 2, at 0 degrees C, will strongly inhibit, and even reverse, formation of the tau-tubulin-vinblastine complex. This suggests that the temperature-sensitive factor is the MAP 2-stimulated tubulin-tubulin interaction rather than the MAP 2-tubulin interaction per se; this raises the possibility that the tubulin-tubulin interactions stimulated by tau differ in their temperature sensitivity from those stimulated by MAP 2.

Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated protein 4

We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.