Mechanisms of black and white stripe pattern formation in the cuticles of insect larvae

Molecular mechanisms that produce pigment patterns in the insect cuticle were studied. Larvae of the armyworm Pseudaletia separata have stripe patterns that run longitudinally along the body axis. The pattern in the cuticle became clear by being emphasized by the increasing contrast between the black and white colors of the lines after the last larval molt. We demonstrated that dopa decarboxylase (DDC) mRNA as well as protein are expressed specifically in the epidermal cells under the black stripes. The pigmentation on the stripes was clearly diminished by injection of a DDC inhibitor (m-hydroxybenzylhydrazine) to penultimate instar larvae for 1 day before molting, suggesting that DDC contributes to the production of melanin. Further, electron microscopic observation showed that the epidermal cells under the gap cuticle region (white stripe) between the black stripes contain many uric acid granules, which gives a white color. Our findings suggest that the spatially regulated expression of DDC in the epidermal cells produces the black stripes while abundant granules of uric acid in the cells generate the white stripes in the cuticle. Based on these results, we concluded that this heterogeneity in the epidermal cells forms cuticular stripe patterns in the armyworm larvae.     

Insect cytokine growth-blocking peptide signaling cascades regulate two separate groups of target genes

Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found in lepidopteran insects that has diverse biological activities, such as larval growth regulation, paralysis induction, cell proliferation, and stimulation of immune cells. GBP also enhances expression of the tyrosine hydroxylase (TH, EC 1.14.16.2) and 3,4-dihydroxy-l-phenylalanine (Dopa) decarboxylase (DDC, EC 4.1.1.26) genes, which elevate dopamine levels in insect epidermal cells. We used insect epidermis and cultured cells to define the role of the GBP signaling pathway in the enhancement of TH and DDC gene expression. It has been recently reported that robust expression of the DDC gene requires activation of extracellular signal-regulated kinase (ERK) in epidermal cells of wounded Drosophila embryos. This study confirmed that GBP activates ERK, but this activation is not directly linked to the enhancement of TH and DDC gene expression. One of the GBP pathway components is phospholipase C, whose activation is essential for the activation of ERK and elevation of expression of both enzyme genes. The downstream signaling pathways diverge to ERK activation through activated protein kinase C and expression of the enzyme genes through inositol triphosphate receptor-mediated Ca2+ influx from extracellular fluid. Our data indicate that the diverged GBP signaling pathways enable GBP to exert completely different biological functions, even in a single cell type.     

MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM,BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p^B), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p^S) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA‐induced damage.     

Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts

Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

Characterization of receptors of insect cytokine, growth-blocking peptide, in human keratinocyte and insect Sf9 cells

Insect cytokine, growth-blocking peptide (GBP), enhances cell proliferation of human keratinocyte cells with a potency almost equivalent to that of human epidermal growth factor (EGF). GBP consists of 25 amino acid residues containing a core region that shows a striking similarity to the C-terminal beta-loop domain of EGF and disordered N and C termini. The present study demonstrates that, although GBP lacks the N-terminal half-portion of EGF molecule, at least five amino acids of the disordered N-terminal six-amino acid region are indispensable for affecting the cell growth activity of GBP. Upon stimulating mitogenesis in keratinocyte cells, GBP directly binds and activates their EGF receptors. GBP also effects proliferative activity on insect Sf9 cells through the binding and activation of the specific receptor, which consists of a heterodimeric complex: a binding subunit (60 kDa) and a tyrosine phosphorylation subunit (58 kDa). These results indicate that GBP enhances cell proliferation of human keratinocyte and insect Sf9 cells through the activation of EGF and GBP receptors, respectively.     

Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell

Rhizobium-made Nod factors induce rapid changes in both Ca(2+) and gene expression. Mutations and inhibitors that abolish Nod-factor-induced Ca(2+) spiking block gene induction, indicating a specific role for Ca(2+) spiking in signal transduction. We used transgenic Medicago truncatula expressing a "cameleon" Ca(2+) sensor to assess the relationship between Nod-factor-induced Ca(2+) spiking and the activation of downstream gene expression. In contrast to ENOD11 induction, Ca(2+) spiking is activated in all root-hair cells and in epidermal or pre-emergent root hairs cells in the root tip region. Furthermore, cortical cells immediately below the epidermal layer also show slow Ca(2+) spiking and these cells lack Nod-factor-induced ENOD11 expression. This indicates a specialization in nodulation gene induction downstream of Nod-factor perception and signal transduction. There was a gradient in the frequency of Ca(2+) spiking along the root, with younger root-hair cells having a longer period between spikes than older root hairs. Using a Ca(2+)-pump inhibitor to block Ca(2+) spiking at various times after addition of Nod factor, we conclude that about 36 consecutive Ca(2+) spikes are sufficient to induce ENOD11-GUS expression in root hairs. To determine if the length of time of Ca(2+) spiking or the number of Ca(2+) spikes is more critical for Nod-factor-induced ENOD11 expression, jasmonic acid (JA) was added to reduce the rate of Nod-factor-induced Ca(2+) spiking. This revealed that even when the period between Ca(2+) spikes was extended, an equivalent number of Ca(2+) spikes were required for the induction of ENOD11. However, this JA treatment did not affect the spatial patterning of ENOD11-GUS expression suggesting that although a minimal number of Ca(2+) spikes are required for Nod-factor-induced gene expression, other factors restrict the expression of ENOD11 to a subset of responding cells.     

Expression of polycystin-1 C-terminal fragment enhances the ATP-induced Ca2+ release in human kidney cells

Polycystin-1 (PC1) is a membrane protein expressed in tubular epithelia of developing kidneys and in other ductal structures. Recent studies indicate this protein to be putatively important in regulating intracellular Ca(2+) levels in various cell types, but little evidence exists for kidney epithelial cells. Here we examined the role of the PC1 cytoplasmic tail on the activity of store operated Ca(2+) channels in human kidney epithelial HEK-293 cell line. Cells were transiently transfected with chimeric proteins containing 1-226 or 26-226 aa of the PC1 cytoplasmic tail fused to the transmembrane domain of the human Trk-A receptor: TrkPC1 wild-type and control Trk truncated peptides were expressed at comparable levels and localized at the plasma membrane. Ca(2+) measurements were performed in cells co-transfected with PC1 chimeras and the cytoplasmic Ca(2+)-sensitive photoprotein aequorin, upon activation of the phosphoinositide pathway by ATP, that, via purinoceptors, is coupled to the release of Ca(2+) from intracellular stores. The expression of TrkPC1 peptide, but not of its truncated form, enhanced the ATP-evoked cytosolic Ca(2+) concentrations. When Ca(2+) assays were performed in HeLa cells characterized by Ca(2+) stores greater than those of HEK-293 cells, the histamine-evoked cytosolic Ca(2+) increase was enhanced by TrkPC1 expression, even in absence of external Ca(2+). These observations indicate that the C-terminal tail of PC1 in kidney and other epithelial cells upregulates a Ca(2+) channel activity also involved in the release of intracellular stores.     

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.     

Functional consequences of activating store-operated CRAC channels

Store-operated CRAC channels, which are activated by the emptying of the endoplasmic reticulum Ca(2+) stores, are an important and widespread route for triggering rises in cytoplasmic Ca(2+). The cellular responses that are activated in response to Ca(2+) entry through CRAC channels are being dissected out, and recent evidence has established that CRAC channels can induce both short-term (safeguarding the Ca(2+) content of the endoplasmic reticulum, maintenance of cytoplasmic Ca(2+) oscillations, enzyme activation, secretion) and long-term (gene expression) changes in cells. CRAC channel activation is therefore capable of evoking a range of temporally distinct responses, highlighting the versatility of this ubiquitous Ca(2+) entry pathway.     

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1?μM tetrodotoxin decreased TH expression beginning around 20?h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300?nM apamin increased TH mRNA and protein between 12 and 24?h later in slices of adult midbrain. Two-week infusions of 300?nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ～20?h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.     

Regulating cytoplasmic calcium homeostasis can reduce aluminum toxicity in yeast

Our previous study suggested that increased cytoplasmic calcium (Ca) signals may mediate aluminum (Al) toxicity in yeast (Saccharomyces cerevisiae). In this report, we found that a yeast mutant, pmc1, lacking the vacuolar calcium ion (Ca(2+)) pump Ca(2+)-ATPase (Pmc1p), was more sensitive to Al treatment than the wild-type strain. Overexpression of either PMC1 or an anti-apoptotic factor, such as Bcl-2, Ced-9 or PpBI-1, decreased cytoplasmic Ca(2+) levels and rescued yeast from Al sensitivity in both the wild-type and pmc1 mutant. Moreover, pretreatment with the Ca(2+) chelator BAPTA-AM sustained cytoplasmic Ca(2+) at low levels in the presence of Al, effectively making the cells more tolerant to Al exposure. Quantitative RT-PCR revealed that the expression of calmodulin (CaM) and phospholipase C (PLC), which are in the Ca(2+) signaling pathway, was down-regulated under Al stress. This effect was largely counteracted when cells overexpressed anti-apoptotic Ced-9 or were pretreated with BAPTA-AM. Taken together, our results suggest that the negative regulation of Al-induced cytoplasmic Ca signaling is a novel mechanism underlying internal resistance to Al toxicity.     

Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca(2+) concentration. In Vicia faba, however, guard cell anion channels activate in a Ca(2+)-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca(2+) concentration. ABA triggered transient rises in cytoplasmic Ca(2+) in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca(2+) closely matched the activation of anion channels, while the Ca(2+) rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca(2+) level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca(2+) concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca(2+) in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca(2+)-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca(2+)-independent pathway.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Regulation of 20-hydroxyecdysone on the larval pigmentation and the expression of melanin synthesis enzymes and yellow gene of the swallowtail butterfly, Papilio xuthus

The swallowtail butterfly, Papilio xuthus, changes its larval body pattern dramatically during the fourth ecdysis. Cuticular pigmentation occurs with precise timing just before ecdysis. We previously found that the cuticular pigmentation was regulated by three melanin synthesis genes, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. We discovered that yellow is strongly expressed in the presumptive black markings earlier than TH and DDC. Because the ecdysis is triggered by 20-hydroxyecdysone (20E), the effects of 20E on the pigmentation and expression of the melanin synthesis genes were examined. Here, we established a method for the topical application of 20E to molting specimens, so that 20E has only a partial effect, resulting in successful ecdysis. When we applied 20E during the mid-phase of the molting period, when the 20E titer is declining, cuticular pigmentation was completely inhibited. The cessation of hormonal treatments caused delayed pigmentation. yellow expression was promoted by a high titer of 20E, whereas the expression of TH, DDC, and ebony was suppressed, suggesting that a decline in the 20E concentration is necessary for the induction of the expression of the latter three genes. These results indicate that cuticular pigmentation is controlled by the exposure to 20E and its removal.     

Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo

The calcium sensing receptor (CaSR) has emerged as an important mediator of a wide range of Ca(2+)-dependent physiological responses (Ca(2+) signaling) in various tissues. To explore the role of CaSR in the epidermis, we utilised the keratin 14 promoter to express CaSR cDNA constitutively in the basal cells of the stratified squamous epithelium of transgenic mice. Analysis of the transgenic mice revealed that a sensitized response to CaSR signaling accelerates the epidermal differentiation program with the precocious formation of the epidermal permeability barrier (EPB) during development and an accelerated hair growth at birth. Our observations indicate that overexpression of CaSR in the undifferentiated basal cells leads to changes in the differentiation program of the transgenic epidermis, including the stimulation of keratins 1 and 6 as well as the overexpression of several markers of terminal differentiation such as filaggrin, loricrin and involucrin. Our data suggest that the observed modifications in the differentiation pathway are a consequence of a CaSR-induced enhancement of Ca(2+) signaling involving cross-talk with other signaling pathways (e.g. EGF and Wnt/Ca(2+)). These studies provide new insights into the role of CaSR in epidermal differentiation including EPB development and hair follicle morphogenesis.     

Calcium signalling in sarcoplasmic reticulum, cytoplasm and mitochondria during activation of rabbit aorta myocytes

This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell configuration to remove cytoplasmic indicator. When the intracellular solution contained millimolar EGTA or BAPTA, activation of SR Ca(2+)release through IP(3)or ryanodine receptors induced a decrease in the [Ca(2+)] reported by Mag-Fura-2. However, when EGTA was present at < or =100 microM, the same stimuli caused an increase in the [Ca(2+)] reported by Mag-Fura-2. The increase in [Ca(2+)] caused by phenylephrine or caffeine was delayed, and prolonged, with respect to the cytoplasmic Ca(2+)transient. Evidence is presented that this Mag-Fura-2 signal reflected a rise in mitochondrial [Ca(2+)]. Agents that inhibit mitochondrial function, such as FCCP or cyanide in combination with oligomycin B, converted the increase in organelle Mag-Fura-2 fluorescence to a decrease, while also prolonging the cytoplasmic Ca(2+)transient. There was considerable similarity between the localization of Mag-Fura-2 fluorescence and the mitochondria-selective indicator tetramethylrhodamine ethyl ester. Thus, we propose that there is close functional integration between the SR and mitochondria in aorta smooth muscle cells, with mitochondria taking up Ca(2+)from the cytoplasm following cell activation.