Increased Microtubule Stability Research Articles

Microtubule stabilization specifies initial neuronal polarization

Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.

Functional Importance of the Anaphase-Promoting Complex-Cdh1-Mediated Degradation of TMAP/CKAP2 in Regulation of Spindle Function and Cytokinesis

Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G(1)/S and peaks at G(2)/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis.

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

Stable microtubules contribute to cardiac dysfunction in the streptozotocin-induced model of type 1 diabetes in the rat

Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. Here, we investigate the reason for increased microtubule stability, and the functional consequences of stable microtubule disruption. Ventricular myocytes were isolated from rats at 8-12 weeks after injection of STZ. A 10% increase in microtubule density, but no difference in the ratio of microtubule-associated protein 4 (MAP4) to tubulin was seen in myocytes from STZ rats. Functionally, STZ myocytes showed a tendency for reduced shortening and intracellular Ca2+ ([Ca2+]i) transient amplitude, and a significant prolongation of time to peak (ttp) shortening and [Ca2+]i. Although microtubules in STZ myocytes were less sensitive to the microtubule disruptor nocodazole (NOC; 33 microM) than control myocytes, we only saw marked functional consequences of microtubule disruption by NOC in myocytes from diabetic animals. NOC increased shortening and [Ca2+]i transient amplitude in STZ myocytes by 45 and 24%, respectively (compared with 4 and 6% in controls). Likewise, NOC decreased ttp shortening and [Ca2+]i only in STZ myocytes, such that these parameters were no longer different between the two groups. In conclusion, stable microtubules in diabetes are not associated with an increase in MAP4, but are functionally relevant to cardiac dysfunction in diabetes, regulating both [Ca2+]i and shortening.

Protein tyrosine nitration is associated with cold- and drug-resistant microtubules in neuronal-like PC12 cells

Among the myriad of cellular functions played by nitric oxide in the brain, there is increasing evidence that nitric oxide might be a primary player in the program of neurogenesis and neuronal differentiation. We have recently reported that tyrosine nitration of proteins is implicated in the signaling pathway triggered by nitric oxide during NGF-induced neuronal differentiation in PC12 cells. The cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and the cytoskeletal proteins α-tubulin and tau are two of the targets. Here, we have studied the association of nitrated proteins with the cytoskeletal fraction in differentiating PC12 cells following exposure to microtubule depolymerising treatments and found that nitration of the cytoskeleton correlates with the increased microtubule stability underlying the progression of neuronal differentiation. These results suggest a novel functional role for nitrated cytoskeletal proteins in the stabilisation of neurites occurring in differentiated neuronal cells.

688. The Effects of Cyclic Stretch on Microtubule Stability and Exogenous Gene Expression in the Alveolar Epithelium

Top of pageAbstract Cells in the lung undergo cyclic deformation under both normal breathing processes as well as mechanical ventilation. While large deformations due to disease or mechanical ventilation can cause cellular damage such as that seen in ventilator induced lung injury, our lab has found that smaller, physiologically relevant deformations have a positive effect on gene transfer in an in vitro pulmonary epithelium model, increasing reporter gene products up to 12-fold in A549 cells, a lung adenocarcinoma cell line frequently used in cellular models of the pulmonary epithelium. This result is not that surprising considering exogenous mechanical forces have been shown by a number of groups to alter the same barriers efficient gene transfer faces as a vector moves from the outside of the cell to the nucleus; namely alterations in the cytoskeleton and extracellular matrix, activation of cell signaling pathways and changes in transcription factor activation. We have previously shown that this increase in exogenous gene expression is due to increased trafficking of the plasmid DNA through the cytosol toward the nucleus, and that cytoskeletal rearrangement, including depolymerization of the microtubule network, is required for stretch-enhanced gene transfer and expression to occur. These results are somewhat puzzling as our lab and others have also shown that the cytoskeleton, specifically the microtubule network, is required for cytoplasmic trafficking of plasmid DNA. One type of post-polymerization modification that microtubules undergo is acetylation, an activity that is not well understood, but has been shown through the use of a variety of drugs to potentially increase microtubule stability against depolymerization from cold or drug-treatment. We wanted to see if this stabilization also applied to exogenous mechanical forces, and if this stabilization allowed for stretch-enhanced gene transfer and expression to occur. To do this, A549 cells were exposed to equibiaxial stretch (10% change in membrane surface area, 15 cycles per minute). Significant increases in gene expression were seen within 15 minutes of the applied stretch. Overexpression of acetylated microtubules through the addition of either tricostatin A, a drug known to inhibit all histone deacetylases (HDACs), or tubacin, a small-molecule inhibitor of only HDAC6, the specific deacetylase for microtubules, caused a significant increase in gene expression over untreated controls. Changes in acetylated microtubule levels were examined via western blot analysis and immunofluoresent microscopy in both cells treated with drugs that affected HDAC activity as well as cells that were stretched. These data demonstrated that a majority of the microtubules in cells that were stretched were also acetylated. Taken together, these results suggest that acetylated microtubules may be resistant to depolymerization due to mechanical forces, a result not previously demonstrated in the literature, and that acetylation may also enhance gene expression through enhanced trafficking of the plasmid DNA through the cytosol.

Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells

Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule–microtubule and microtubule–cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule–microtubule and microtubule–cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

Farnesyl transferase (FT) inhibitors (FTI) are anticancer agents developed to target oncogenic Ras proteins by inhibiting Ras farnesylation. FTIs potently synergize with paclitaxel and other microtubule-stabilizing drugs; however, the mechanistic basis underlying this synergistic interaction remains elusive. Here we show that the FTI lonafarnib affects the microtubule cytoskeleton resulting in microtubule bundle formation, increased microtubule stabilization and acetylation, and suppression of microtubule dynamics. Notably, treatment with the combination of low doses of lonafarnib with paclitaxel markedly enhanced tubulin acetylation (a marker of microtubule stability) as compared with either drug alone. This synergistic effect correlated with FT inhibition and was accompanied by a synergistic increase in mitotic arrest and cell death. Mechanistically, we show that the combination of lonafarnib and paclitaxel inhibits the in vitro deacetylating activity of the only known tubulin deacetylase, histone deacetylase 6 (HDAC6). In addition, the lonafarnib/taxane combination is synergistic only in cells lines expressing the wild-type HDAC6, but not a catalytic-mutant HDAC6, revealing that functional HDAC6 is required for the synergy of lonafarnib with taxanes. Furthermore, tubacin, a specific HDAC6 inhibitor, synergistically enhanced tubulin acetylation in combination with paclitaxel, similar to the combination of lonafarnib and paclitaxel. Taken together, these data suggest a relationship between FT inhibition, HDAC6 function, and cell death, providing insight into the putative molecular basis of the lonafarnib/taxane synergistic antiproliferative combination.

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

Tumor Cells Resistant to a Microtubule-Depolymerizing Hemiasterlin Analogue, HTI-286, Have Mutations in α- or β-Tubulin and Increased Microtubule Stability

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.

The fragile X protein controls microtubule-associated protein 1B translation and microtubule stability in brain neuron development.

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein implicated in regulating translation of its mRNA ligands. The absence of FMRP results in fragile X syndrome, one of the leading causes of inherited mental retardation. Delayed dendritic spine maturation was found in fragile X mental retardation patients as well as in Fmr1 knockout (KO) mice, indicating the functional requirement of FMRP in synaptic development. However, the biochemical link between FMRP deficiency and the neuronal impairment during brain development has not been defined. How FMRP governs normal synapse development in the brain remains elusive. We report here that the developmentally programmed FMRP expression represses the translation of microtubule associated protein 1B (MAP1B) and is required for the accelerated decline of MAP1B during active synaptogenesis in neonatal brain development. The lack of FMRP results in misregulated MAP1B translation and delayed MAP1B decline in the Fmr1 KO brain. Furthermore, the aberrantly elevated MAP1B protein expression leads to abnormally increased microtubule stability in Fmr1 KO neurons. Together, these results indicate that FMRP plays critical roles in controlling cytoskeleton organization during neuronal development, and the abnormal microtubule dynamics is a conceivable underlying factor for the pathogenesis of fragile X mental retardation.

A divergent canonical WNT-signaling pathway regulates microtubule dynamics: dishevelled signals locally to stabilize microtubules.

Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability.

Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas

We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays. Three phosphorylated forms and one unphosphorylated form of the Op18 protein were identified and found to be overexpressed in lung adenocarcinomas as compared with normal lung. The percentage of phosphorylated to total Op18 protein isoforms increased from 3.2% in normal lung to 7.9% in lung tumors. Both the phosphorylated and unphosphorylated Op18 proteins were significantly increased in poorly differentiated tumors as compared with moderately or well differentiated lung adenocarcinomas (p<0.03), suggesting that up-regulated expression of Op18 reflects a poor differentiation status and higher cell proliferation rates. This was further verified in A549 and SKLU1 lung adenocarcinoma cell lines by examining Op18 levels and phosphorylation status following treatment that altered either cell proliferation or differentiation. The increased expression of Op18 protein was significantly correlated with its mRNA level indicating that increased transcription likely underlies elevated expression of Op18. The overexpression of Op18 proteins in poorly differentiated lung adenocarcinomas and the elevated expression of the phosphorylated forms of Op18 may offer a new target for drug- or gene-directed therapy and may have potential utility as a tumor marker.

Signaling Mechanisms Underlying Reversible, Activity-Dependent Dendrite Formation

Neuronal activity and neurotrophins play a central role in the formation, maintenance, and plasticity of dendritic arbors. Here, we show that neuronal activity, mediated by electrical stimulation, KCl depolarization, or cholinergic receptor activation, promotes reversible dendrite formation in sympathetic neurons and that this effect is enhanced by NGF. Activity-dependent dendrite formation is accompanied by increased association of HMW MAP2 with microtubules and increased microtubule stability. Inhibition of either CaMKII or the MEK-ERK pathway, both of which phosphorylate MAP2, inhibits dendrite formation, but inhibition of both pathways simultaneously is required for dendrites to retract. These data indicate that neuronal activity signals via CamKII and the ERKs to regulate MAP2:microtubule interactions and hence reversible dendrite stability, and to provide a mechanism whereby activity and neurotrophins converge intracellularly to dynamically regulate dendritic morphology.

Heterozygosity in MAT locus affects stability and function of microtubules in yeast

The dynamic nature of microtubules is important for their cellular function and is tightly regulated by the cell cycle machinery and through other pathways that act on microtubule-associated proteins. Recently, it was reported that simultaneous expression of MATa and MATα genes in haploid cells of Saccharomyces cerevisiae increases microtubule stability 〚Mol. Gen. Genet. 264 (2000) 300〛. In order to investigate the effect of zygosity in the MAT loci on microtubules independent of the effect of the actual ploidy, we compared microtubule stability and function in homozygous ( MATa/ MATa) and heterozygous ( MATa/ MATα) wild-type diploid cells. It was found that homozygosity in the MAT locus decreases stability of both cytoplasmic and nuclear microtubules. This was expressed in a more symmetrical distribution in the placement of the spindle relative to the neck, a delay in cytokinesis and reduced fidelity of chromosome segregation in these cells compared to the heterozygotes. Our results suggest that expression of both MAT loci initiates a pathway that results in an increase in microtubule stability and the fidelity of chromosome segregation in diploids. This pathway is independent of the pathway that determines the budding pattern in haploids and diploids, which is also initiated by simultaneous expression of MATa and MATα genes.

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3β phosphorylation

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.

Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes.

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.

Prostaglandins protect human intestinal cells against ethanol injury by stabilizing microtubules: role of protein kinase C and enhanced calcium efflux.

Prostaglandins (PG) protect gastrointestinal cells against damage induced by ethanol (EtOH) and other noxious agents, a process termed cytoprotection. The present study investigated the relationships between microtubule (MT) stability, protein kinase C (PKC) activation, and calcium efflux as a possible mechanism of PG's protective action using a human colonic cell line (Caco-2) exposed to known damaging concentrations of EtOH (7.5% and 10%). Preincubation of Caco-2 cells with 16,16-dimethyl-PGE2 (PG, 2.6 microM) significantly increased PKC activity in these cells. Pretreatment of Caco-2 cells with 50 microM OAG (a synthetic diacylglycerol and PKC activator) or 30 nM TPA (a direct PKC activator) prior to exposure to 7.5% or 10% EtOH for 5 min significantly reduced cell injury, as determined by trypan blue exclusion, and increased MT stability, as confirmed by confocal microscopy. Pretreatment of Caco-2 cells with 4 alpha-PDD (an inactive phorbol ester, 20 nM) failed to prevent cell injury and disruption of the MT cytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM) abolished the protective effects of PG in cells exposed to 7.5% and 10% EtOH. Incubation of Caco-2 cells with A23187 (a Ca2+ ionophore), similar to 10% EtOH, caused a significant reduction in cell viability and MT stability. Preincubation with A23187 in combination with PG or OAG prior to subsequent exposure to EtOH significantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatment with OAG, TPA, or PG resulted in significant increases in calcium-45 efflux, which correlated with increased stability of the MT cytoskeleton. These data suggest that PG possesses direct protective effects against EtOH injury in Caco-2 cells and may act by stabilizing MT through the PKC signal transduction pathway and/or stimulation of calcium efflux from the cells.

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412–419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5–9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412–419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca 2+-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (S p-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and S p-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.

Multiple centrosomal microtubule organising centres and increased microtubule stability are early features of VP-16-induced apoptosis in CCRF-CEM cells

Microtubular reorganisation contributing to apoptotic morphology occurs in normal and neoplastic cells undergoing apoptosis induced by cytotoxic drugs [1–3]. The aim of this study was to correlate the changes in the microtubules (MTs) with behaviour of the centrosome in apoptotic cells, and to see whether post-translational changes in tubulin occurred with the emergence of apoptotic MT bands. Apoptosis was induced in the human T-cell leukaemia line (CCRF-CEM) by treatment with 17 μM etoposide over a 4 h period. The time course of changes was assessed using flow cytometry (FCM) and immunocytochemistry in cells labelled for a centrosomal antigen (CSP-α) or α-tubulins. One hour following treatment we observed multiple centrosomal microtubule organising centres (MTOCs) associated with the nucleus and the transient appearance of a subset of stable MTs detected with an antibody specific for acetylated α-tubulin, as the bands of MTs which lobulate the nucleus are formed. The altered properties of the MTs thus reflect changes in function as apoptosis progresses.