20-Hydroxyecdysone induces the expression of one beta-tubulin gene in Drosophila Kc cells

The expression of 56D and 60C beta-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with beta-tubulin subclones show that the 56D beta-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3'-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a beta-tubulin subunit (P4); this subunit is the so-called beta 3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced beta 3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.     

20-hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone (20-HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20-HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20-HE by injecting a pulse of 20-HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil-associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20-HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development. © 1995 John Wiley & Sons, Inc.     

Microtubular proteins of Chlamydomonas reinhardtii. An immunochemical study based on the use of an antibody specific for the beta-tubulin subunit.

An immunochemical assay for tubulin subunits is described. The method is applied directly to homogenates of Chlamydomonas reinhardtii solubilized in sodium dodecyl sulfate (Na dodecyl-SO4), and it makes use of a two-dimensional electrophoresis system; the first separation is carried out by Na dodecyl-SO4-polyacrylamide gel electrophoresis and the second by electrophoresis into an agarose gel containing antibodies. Tubulin is precipitated in the form of a "rocket" and the method is made quantitative through the use of cells labeled with [35S]sulfate. The antiserum used in this assay was prepared in rabbits using beta subunit of tubulin purified from Chlamydomonas flagella by two preparative Na dodecyl-SO4-polyacrylamide gel electrophoreses. This antiserum and an antiserum to alpha subunit of tubulin from porcine brain, prepared for comparative study, were extensively characterized. Both antisera show specificity for the polypeptide used as antigen and react with the native dimeric tubulin. The antiserum to beta subunit from Chlamydomonas flagella also forms immunoprecipitates with native brain tubulin and its beta subunit when used at high titer. In contrast, the antiserum to alpha subunit from porcine brain does not cross-react with Chlamydomonas tubulin. The immunochemical assay was applied to Chlamydomonas cells synchronized by a 12-h light/dark cycle. In cells collected during the light period (late G1), after removal of flagella, the content of tubulin is estimated to be 0.3% of total protein. As cells enter the dark period there is a striking increase in tubulin content which reaches a maximum just before cell division.     

Ecdysteroid control of ionic current development in Manduca sexta motoneurons

The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca²⁺ and K⁺ current densities. To determine whether 20-HE controls the changes in Ca²⁺ and K⁺ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca²⁺ and K⁺ currents were measured after 1–4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca²⁺ currents of pupal leg motoneurons increased from day 1 to day 4 in vitro. Thus, at culture day 4 the pupal Ca²⁺ current levels were larger in 20-HE–treated than in untreated cells. By contrast, 20-HE did not affect the Ca²⁺ current amplitudes of larval leg motoneurons. Whole-cell K⁺ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca²⁺ currents in leg motoneurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 211–223, 1998     

20-OH-ecdysone regulates 60 C beta tubulin gene expression in Kc cells and during Drosophila development

Cultured Kc cells of Drosophila melanogaster are sensitive to the insect moulting hormone, 20-hydroxy-ecdysone (20-OH-E). Morphological changes of Kc-treated cells were observed and electron microscopic analysis of pseudopodia shows a large increase in the number of microtubules, all arranged in the same orientation. The 60 C beta tubulin gene which is expressed only in 20-OH-E-treated cells encodes a 2.6-kb mRNA which is essentially cytoplasmic and polyadenylated. The corresponding premessenger is 7 kb in length and is absent in untreated cells. Two peaks of expression of the 60 C beta tubulin gene are observed during Drosophila development: at midembryogenesis (stage 8-13 h) and at the late third instar larvae-early pupae stage. By use of the Ecdysone 1 mutant, 60 C beta tubulin gene expression was demonstrated to be regulated in part by 20-OH-E during Drosophila development. Through these two complementary biological models of study, the mode and role of beta tubulin gene regulation are discussed.     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. We then explore the role microRNA plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3'UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation.     

Ecdysteroid mediated fat body acid phosphatase activity during larval development of rice moth, Corcyra cephalonica (Lepidoptera)

20-hydroxyecdysone (20-HE) stimulates acid phosphatase activity in the fat body of ligated late-last instar larvae. This effect is time dependent and the specific activity of enzyme increases significantly in hormone treated insects. 20-HE also stimulates general protein synthesis. Cycloheximide treatment either in conjunction with 20-HE or after hormone treatment blocks the increase in enzyme activity as well as increase in protein content. However, actinomycin D treatment does not alter the enzyme activity while it blocks the increase in total RNA as well as increase in protein content.     

Localization of the ATP binding site on alpha-tubulin

The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNai knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNa, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNa when exposed to micromolar levels of 20-HE. We then explore the role microRNa plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3′UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNa control of antimicrobial peptide translation.Key words: microRNA, ecdysone, innate immunity, let-7, diptericin, inflammation     

Some biochemical properties of higher plant tubulins

Tubulin was isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose) and ion exchange (DEAE-Sephacel) chromatography from mung bean and cultured carrot suspension cells. SDS-PAGE (Blose 1981) of mung bean tubulin has shown it to consist of two major subunits (MBT1 and MBT2) and a minor subunit (MBT3). Tubulin isolated from carrot cells was resolved into only two bands on SDS-PAGE (slow moving subunit was named CT1). However, the faster moving subunit on SDS-PAGE was resolved into two bands (CT2 and CT3) on SDS-4M urea-PAGE. On SDS-4M urea-PAGE, CT1 migrated faster than CT2, CT3. By contrast in SDS-4M urea-PAGE, mung bean tubulin remains unresolved. Mammalian tubulin could be resolved into alpha and beta-subunits in both electrophoretic systems. Monoclonal antibodies to mammalian alpha and beta-tubulin subunits (MCA-T alpha and MCA-T beta, respectively) and Western blot analysis clearly demonstrated a cross-reactivity of MCA-T alpha with MBT2, MBT3, CT2 and CT3, while MCA-T beta showed cross-reactivity with MBT1 and CT1. Although MBT2, MBT3, CT2 and CT3 are immunologically related to the alpha-subunit of mammalian tubulin, their migration on SDS-PAGE was reversed with respect to MBT1 or CT1, which were immunologically related to the beta-subunit of mammalian tubulin. Peptide mapping patterns also supported above the results.     

Induction and inhibition of an apparent neuronal phenotype in Spodoptera frugiperda insect cells (Sf21) by chemical agents

The goal of this research was to induce neuron-like properties in Sf21 cells, an insect ovarian cell line, which could lead to a new high-throughput insecticide screening method and a way to mass produce insect neuronal material for basic research. This study applied differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, a maximum of ca. 30?% of Sf21 cells expressed an apparent neuronal morphology of unipolar, bipolar, or multipolar axon-like processes within 2?C3?days. Maximal differentiation occurred after 2?days in the presence of 50???M 20-HE or 3?days in 10???M insulin. Both 20-HE and insulin displayed time- and concentration-dependent differentiation with biphasic curves, suggesting that two binding sites or processes were contributing to the observed effects. In addition, combinations of 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system stimulant, inhibited induction of elongated processes by 20-HE and/or insulin, with an IC₅₀ of 9 nM for 20-HE, and the inhibition was incomplete, resulting in about one-quarter of the differentiated cells remaining, even at high concentrations (up to 1?mM). The ability to induce a neural phenotype simplifies the studies of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined.     

Induction of Sarcophaga central nervous system remodeling by 20-hydroxyecdysone in vitro

Proliferation and apoptosis of neural cells were found to be induced simultaneously when larval brains of Sarcophaga peregrina were cultured in the presence of 20-hydroxyecdysone (20-HE) for 24 h. The locations of proliferating cells and apoptotic cells in the brain hemispheres were different. The morphology of brains exposed to 20-HE for a short period proceeded to change sequentially when culture was continued for 2 days even in the absence of 20-HE. These changes mainly consisted of enlargement of the brain hemispheres and extension of the interval between two hemispheres, which closely paralleled the morphological changes of brains that occur in the early pupal stage, suggesting that ecdysteroid alone is sufficient to induce the remodeling of the central nervous system of holometabolous insects. Synthesis of a protein with a molecular mass of 66 kDa was shown to be selectively repressed when brains were cultured in the presence of 20-HE.     

Subtilisin cleavage of tubulin heterodimers and polymers.

Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the alpha and beta subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, alpha' and beta', under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native alpha,beta tubulin to 7-9 for subtilisin-cleaved alpha',beta' tubulin. When tubulin heterodimers were cleaved, a single major beta' isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major beta' isotypes were found. Monoclonal antibodies that recognize a beta carboxyl-terminal peptide, residues 410-430, reacted with both major beta' isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major beta' isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the beta subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form.     

Reduction in S100 protein beta subunit mRNA in C6 rat glioma cells following treatment with anti-microtubular drugs

S100 protein is a calcium-binding protein found in vertebrate nervous tissue. Synthesis of S100 protein in the rat glioma cell line, C6, is inhibited by the addition of anti-microtubular drugs. We have cloned a cDNA for the beta subunit of S100 protein from rat brain in a lambda gt 11 expression vector and used this cDNA to measure the amounts of S100 beta subunit mRNA in C6 cells after treatment with anti-microtubular drugs. Levels of alpha-tubulin and beta-actin mRNAs were also measured. All measurements were performed using RNA-RNA hybridization techniques at high stringency with rat mRNA-specific probes. After 24 h of treatment, the S100 beta subunit mRNA was reduced to levels of 25% by colchicine and 32% by vinblastine when compared to untreated controls. In contrast, the levels of tubulin and actin mRNAs were only slightly changed by these treatments. These studies demonstrate that disruption of the microtubular cytoskeleton causes a specific reduction in the level of S100 protein mRNA in C6 cells.     

Hormonal regulation of macromolecular synthesis in testes and accessory reproductive glands of Spodoptera litura during post-embryonic and adult development

In S. litura testicular growth during the last larval instar and early pupal stage is associated with significant increase in DNA, RNA and protein contents. DNA synthesis is stimulated by 20-hydroxyecdysone (20-HE) in the penultimate instar testes. 20-HE injection in ligated late last instars increases the testicular weight and protein content. Accessory reproductive gland (ARG) development takes place during the mid and late pupal stages. Protein synthesis in the pharate adult ARG is stimulated by 20-HE. Juvenile hormone has no effect on ARG protein synthesis.     

Changes in translatable mRNAs during the larval-pupal transformation of the epidermis of the tobacco hornworm

At the initiation of metamorphosis when exposed to ecdysteroid in the absence of juvenile hormone (JH), the lepidopteran epidermis changes its commitment from one for larval differentiation to one for pupal differentiation. Changes in mRNA populations during this change both in vivo and in vitro were followed by a one-dimensional SDS-gel electrophoretic analysis of translation products made in a mRNA-dependent rabbit reticulocyte lysate system. The larval epidermal cell was found to lose its translatable mRNAs for larval cuticular proteins and the larval-specific pigment insecticyanin during the change in commitment; these never reappeared. For Class I cuticular proteins and for insecticyanin, this loss occurred during the exposure to ecdysteroid, each with a differing time course. By contrast, Class II cuticular mRNAs first increased during this time, then also disappeared by the time the cells were pupally committed. In vitro these mRNAs appeared in only trace amounts in response to 20-hydroxyecdysone (20-HE). The pupally committed cell (late in the wandering stage) contained mRNAs for three low-molecular-weight proteins which were precipitable with the pupal cuticular antiserum. The remainder of the pupal cuticular mRNAs were not translatable until the third day after wandering, a time when pupal cuticle is being deposited in response to a molting surge of ecdysteroid. The pupally committed cell also had at least one new noncuticular mRNA which coded for a 34K protein and which was absent from both larval and pupal epidermal cells making cuticle. Since its appearance in response to 20-HE in vitro is repressed by JH, it is called a pupal commitment-specific protein. Thus, during the change of commitment 20-HE inactivates larval-specific genes irreversibly in a sequential cascade of events. The activation of most pupal-specific genes then requires a subsequent exposure to more ecdysteroid.     

Occurrence of nuclear beta(II)-tubulin in cultured cells

Microtubules are cylindrical organelles that play critical roles in cell division. Their subunit protein, tubulin, is a target for various antitumor drugs. Tubulin exists as various forms, known as isotypes. In most normal cells, tubulin occurs only in the cytosol and not in the nucleus. However, we have recently reported the finding of the beta(II) isotype of tubulin in the nuclei of cultured rat kidney mesangial cells. Mesangial cells, unlike most normal cell lines, have the ability to proliferate rapidly in culture. In efforts to determine whether nuclear beta(II)-tubulin occurred in other cell lines, we examined the distribution of the beta(I), beta(II), and beta(IV) mammalian tubulin isotypes in a variety of normal and cancer human cell lines by immunofluorescence microscopy. We have found that, in the normal cell lines, all three isotypes are present only in the cytoplasm. However, the beta(II) isotype of tubulin is located not only in the cytoplasm, but also in the nuclei of the following cell lines: LNCaP prostate carcinoma, MCF-7, MDA-MB-231, MDA-MB-435, and Calc18 breast carcinoma, C6 and T98G glioma, and HeLa cells. In contrast, the beta(I) and beta(IV) isotypes, which are also synthesized in cancer cells, are not localized to the nucleus but are restricted to the cytoplasm. We have also seen beta(II) in breast cancer excisions. In most of these cells, beta(II) appears to be concentrated in the nucleoli. These results suggest that transformation may lead to localization of beta(II)-tubulin in cell nuclei, serving an as yet unknown function, and that nuclear beta(II) may be a useful marker for detection of tumor cells.     

Regulation of Five Tubulin Isotypes by Thyroid Hormone During Brain Development

Nucleic acid probes derived from the 3' noncoding region of five tubulin cDNAs were used to study the effects of thyroid hormone deficiency on the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2)- and three beta (beta 2, beta 4, and beta 5)-tubulin isotypes in the developing cerebral hemispheres and cerebellum. The content of alpha 1, which markedly declines during development in both brain regions, is maintained at high levels in the hypothyroid cerebellum, whereas it is decreased in the cerebral hemispheres. The alpha 2 level also declines during development and is decreased in both regions by thyroid hormone deficiency, but only during the two first postnatal weeks. Thyroid hormone deficiency slightly increases at all stages the beta 2 level in the cerebellum, whereas a decrease is observed at early stages in the cerebral hemispheres. The beta 5 level seems to be independent of thyroid hormone in the cerebral hemispheres, whereas it decreases at early stages in the hypothyroid cerebellum. Finally, the expression of the brain-specific beta 4 isotype is markedly depressed by thyroid hormone deficiency, particularly in the cerebellum. These data suggest that the genes encoding the tubulin isotypes are, directly or not, differently regulated by thyroid hormone during brain development. This might contribute to abnormal neurite outgrowth seen in the hypothyroid brain and therefore to impairment in brain functions produced by thyroid hormone deficiency.