Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.

Subcellular localization of neuronal nitric oxide synthase in the superficial gray layer of the rat superior colliculus.

The superficial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase (NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic reticulum, in pre- and postsynaptic profiles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal elements.

Short- and long-term influences of calcitonin gene-related peptide on the synthesis of acetylcholinesterase in mammalian myotubes.

The present study analyses the short- (15 min - 2 h) and long-term (24 - 48 h) influences of calcitonin gene-related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. To assess the effect of CGRP on AChE synthesis, myotubes were pre-exposed to the irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 - 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP. The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 - 30 microM forskolin. In contrast, pre-treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment. Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. These findings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time-dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP-dependent mechanism. The decreased AChE synthesis observed after long-term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down-regulation, that might function as an important adaptative mechanism of the muscle fibre in response to long-term changes in neuromuscular transmission.

Tubulin carboxypeptidase/microtubules association can be detected in the distal region of neural processes.

The association of tubulin carboxypeptidase with microtubules has been demonstrated in crude brain extracts and in living non-nervous cells. Here, we studied this phenomenon in cultured brain cells. To determine the association of the enzyme with neural microtubules we isolated the cytoskeletons (detergent-extraction under microtubule-stabilizing conditions) and measured the content of Tyr, Glu, and delta2 tubulin as a function of the in vitro incubation time of the cytoskeletons. The carboxypeptidase was found associated with microtubules in 2 days-cultured cells but not in 7 days-cultured cells. Quantitative analysis of digitized images after immunofluorescent staining revealed that detyrosination during the incubation of the cytoskeletons occurred preferentially in the distal regions of the neural processes. Prolonged taxol-treatment of the cells promoted higher detyrosination but Tyr tubulin was not depleted suggesting the existence of a subset of microtubules that has not associated carboxypeptidase and therefore cannot be detyrosinated even after prolonged taxol-treatment. This hypothesis was supported, although not conclusively, by additional experiments.

Pyruvate decarboxylase filaments are associated with the cortical cytoskeleton of asci and spores over the sexual cycle of filamentous ascomycetes.

We show that pyruvate decarboxylase (PDC) 8- to 10-nm-diameter filaments, first described in vegetative cells of Neurospora crassa, are ubiquitously present in filamentous fungi. The cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetes N. crassa, N. tetraesperma, Podospora anserina, and Sordaria macrospora. PDC-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and spindle pole bodies. Nocodazole-induced depolymerization of the cortical microtubules results in the loss of PDC-filaments. Moreover, a S. macrospora mutant defective in cortical MT distribution shows abnormal PDC organization. Neurospora asci generated on various metabolic conditions, which modify the presence and relative abundance of PDC-filaments in vegetative cells, have identical patterns of subcellular distribution of these structures. A N. crassa mutant (snowflake) that accumulates giant bundles of PDC-filaments during vegetative growth, shows normal distribution of the filaments during ascogenesis. Thus, the regulation conditioning the presence and supramolecular assembly of PDC-filaments in Neurospora differs between vegetative and sexual cells. Taken together, these results suggest that PDC in filamentous fungi may perform two functions, intervening as an enzyme in vegetative metabolism and as a structural protein associated with the cytoskeleton during sexual development.

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes.

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the alpha-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75-78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.

Morphogenesis of the flagellum in the spermatids of Coelomera lanio (Coleoptera, Chrysomelidae): ultrastructural and cytochemical studies.

The flagellum of Coelomera lanio has a single axoneme with a 9 + 9 + 2 axoneme pattern and the microtubules are linked to the E-PTA positive dinein arms. The electron-dense fibres between microtubules show a positive acid phosphatase reaction. A single E-PTA positive accessory body is seen flanking the axoneme, from which extends a puff-like corpuscle. On the opposite side of the accessory body, away from the axoneme, exists another puff-like corpuscle. The mitochondrial complex gives rise to two mitochondrial derivatives of different shapes and sizes. The major derivative possesses a paracrystalline corpuscle and the minor has an electron-dense area in the juxta-axonemal region. All these structures are involved in the mechanism of flagellar motility.

Ultrastructural localization of acid phosphatase in spermatic cells of Ceratitis capitata (Diptera).

The cytochemical study of acid phosphatase in spermatic cells of Ceratitis capitata defines the enzimatically active sites, relating this enzyme with morphological modifications of the cell components during spermiogenesis. In the axoneme, acid phosphatase is associated with the metabolism of phosphates which promote flagellar motility. The enzymatic activity verified on the cytoplasmic membranes demonstrates the importance of this enzyme in the process of cellular differentiation.