The Expression and Role of Caveolin-1 in Different Development Periods of Drosophila melanogaster.
Caveolin-1 is an iconic structural molecule of Caveolae. Because of its pivotal role in the process of cell proliferation, apoptosis and other related signaling pathways of human tumors, caveolin-1 has been extensively studied. It has been found that caveolin-1 expresses in various types of invertebrates and vertebrates and is highly conserved in evolution. However, there has been no report about the caveolin-1 of insect so far. In the present study, caveolin-1 was amplified in the larvae and adults of Drosophila melanogaster to observe their expression levels. The in vivo distributions of caveolin-1 were observed in larvae, pupae and adults through immunofluorescence and confocal technology. Furthermore, the expression of caveolin-1 was detected in larvae, pupae and adults through Western blotting.
The results showed that caveolin-1 exists in D. melanogaster, and its expression and distribution varied in different developmental stages. The in vivo distribution of caveolin-1 was mainly in the brain, muscles and gut wall in adult, a little in the brain and fat bodies in pupa and a little in the fat bodies in larva. The expression levels of caveolin-1 were significantly higher than in adults in the larvae and pupae. The expression of the protein was significantly higher in the female adult than in the adult male.
Key words: Caveolin-1; Drosophila melanogaster; expression.
Lipid raft was a micro region containing special lipid and protein in membrane lipid bilayer (Gajate and Mollinedo, 2015). Caveolae, which is a special lipid raft structure formed from cell membrane invagination, was enriched in cholesterol and sphingomyelin, and primarily played the important role in membrane transport, signal transduction, substrate transport and endocytosis (Martin, 2013). Caveolins and flotillins are two families of the membrane proteins correlating with Caveolae of the mammals (Bickel et al., 1997). Caveolins are integral membrane proteins with molecular weight of 21 - 24 kDa, and as the scaffold structure, they recruit a lot of signal molecules. So far, 3 members of the caveolin family have been found in mammals: caveolin-1, caveolin-2 and caveolin-3. Among these, caveolin-1 had two subtypes - alpha and beta (Williams and Lisanti, 2004).
Co-expression of caveolin-1 and caveolin-2 is widely distributed in various types of tissues and cells, such as epithelial cells, endothelial cells, adipocytes, fibroblasts, central nervous system and the placental tissues. It is generally believed that caveolin-2 forms heterooligomeric complex with caveolin-1 involved in the transport process of the later (Chen and Che, 2014). Caveolin-3 is only specifically expressed in skeletal muscle cells, myocardial cells and smooth muscle cells, and more so in the first two muscle cells (Marx, 2001; Park et al., 2002).
Caveolin-1 is the most important protein in the caveolin family (Frank et al., 2008). It is involved in the formation of caveolae, endocytosis, signal transduction, angiogenesis and other physiological process (Chidlow and Sessa, 2010). It also plays an important role in cholesterol homeostasis and vesicular transport, but could also combine with a variety of signaling molecules which would activate or inhibit different intracellular signaling pathways, and regulate the expression of related genes (Fridolfsson et al., 2014; Liu et al., 2012). At present, caveolin-1 has been studied as key molecule in the signaling pathways of cell proliferation and apoptosis in the process of human tumorigenesis (Shatz and Liscovitch, 2008; Ubaldo et al., 2010).
Caveolin-1 which is highly conserved in evolution has been found to express in many invertebrate such as nematode pole worm, hookworm, animalcule, sea urchins and vertebrate species. Parker et al. (2007) reported that the caveolin-1 of Caenorhabditis elegans was homologous with caveolin-3 of human, and is expressed in nerve cells and muscle of body wall. Sato et al. (2006) and Scheel et al. (1999) reported that it could dynamically regulate the maturation of the germ cell line of the androgynous adult. Hernandez-Bello et al. (2008) cloned the caveolin-1 gene of Trichinella spiralis and used fragment specific cDNA probes, and reported its sex-specific expression in the oocyte maturation and early embryo development process. Caveolin-1 monoclonal antibodies have been prepared for the experimental study of nematode, which laid the foundation for the study of the caveolin-1 of model species (Hadwiger et al., 2010).
The caveolin-1 gene of Artemia sinica an arthropoda has been cloned. Zhang et al. (2011) have shown that the caveolin-1 protein of Artemia sinica has the same physiological function as in vertebrate. Li et al. (2014) cloned the full-length sequence of the caveolin-1 gene of Artemia sinica, and obtained the nucleotide sequence of 675bp. There is no report on the caveolin-1 of insect.
In this study the expression levels of caveolin-1 in different developmental stages of D. melanogaster were observed by RT-PCR, immunofluorescence and Western blot. It is being reported for the first time that caveolin-1 exists in D. melanogaster and the expression of this protein in D. melanogaster is highly variable in different developmental stages, which lays foundation for further study on caveolin-1 of D. melanogaster and other insects.
MATERIALS AND METHODS
Materials and reagents
The wild type D. melanogaster was supplied by the Genetics Laboratory of Liaoning Normal University. DEPC was purchased from Amresco Co. (America); M- MLV reverse transcriptase, 5xM-MLV buffer, dNTP mixture, random primer-hexadeoxyribonucleotide mixture [pd(N)6] were all purchased from TaKaRa (Dalian, China). Taq enzyme, dNTP mixture and 10xPCR buffer were all purchased from Tiangen Biotech (Beijing) Co., Ltd. Caveolin-1 Mouse IgG2b 7C8 was purchased from Santa Cruz Biotechnology Co. (America). Hoechst 33342 and Anti-Mouse IgG-TRITC were purchased from Sigma Co. (America). Anti-fluorescence quencher was purchased from Shanghai biyotime Co. Goat anti rabbit IgG with horseradish enzyme and Goat anti mouse IgG with horseradish enzyme were purchased from Beijing Jinqiao Bio Technology Co., Ltd. ECLTM Chemiluminescence Kit was purchased from Thermo Scientific Co. (America).
Caveolin-1 monoclonal anti rabbit antibody was purchased from Cell signaling Co. (America). The Artemia anti rabbit antibody was prepared by the Laboratory of Lin Hou. Other reagents such as bromine phenol blue, xylene, ethanol, formaldehyde, acetic acid, picric acid, orchid agent, bleaching agent, hematoxylin, eosin and ultra pure water were domestic and analytical pure.
Culture of D. melanogaster
The specimens of D. melanogaster were cultured in the maize medium at room temperature, and the culture medium was prepared by agar (10g/L), sugar (100g/L), maize (90g/L) and propionate (7mL/L).
RT-PCR of caveolin-1
The Caveolin-1 sequences of Zebrafish, Xenopus and Nematoda were searched from the NCBI database, and the PCR primers were synthesized in high homology. The upstream primer was "5'-GGACGGTATCACGGCAAGA-3'", the downstream primer was "5'-TACCACTCGTCAGCCTCA-3'".
The total RNA of the larvae and adults of the D. melanogaster was extracted by TRIzol reagent, and the integrity of RNA was detected by agarose gel electrophoresis. This RNA was used for RT-PCR with random primers. The reaction conditions were as follows: denaturation at 35degC for 10min, annealing at 42degC for 1h and elongation at 70degC for 15 min, followed by 1 cycle. Amplificated the target gene fragment with the above synthetic primers, the reaction conditions were as follows: initial denaturation at 94degC for 5min, followed by 35 cycles of denaturation at 94degC for 30s, annealing at 56degC for 30s and elongation at 72degC for 30s, with a final extension at 72degC for 10min. Observed through agarose gel electrophoresis and gel imaging system.
Preparation of paraffin section
Larva, pupa and adult D. melanogaster were put into Bouin's liquid and fixed for several hours, followed by moving directly into the 70% alcohol for rinse, until the materials are not yellow anymore. The samples were dehydrated, embedded in paraffin, and 4 um thick sections were cut and stored at room temperature. The paraffin sections were stained with hematoxylin and eosin.
Thirty eight paraffin sections of larva of D. melanogaster were prepared, out of which No. 5, 16 and 27 were stained with H and E. Twenty paraffin sections of pupa of D. melanogaster were prepared. Out of which No. 4, 8 and 12 were stained with H and E. Thirty one paraffin sections of adult of D. melanogaster were prepared. Out of which No. 7, 15 and 23 were stained with H and E.
Immunofluorescence and confocal technology
The paraffin sections of D. melanogaster were dewaxed and dewatered in xylene and ethanol concentration gradient solutions; washed and blocked with 1% BSA at 37degC for 30min avoid light and incubated with caveolin-1 antibody at 4degC for 48h; washed and incubated for 1.5h with IgG-TRITC secondary antibody at room temperature; then washed and incubated with Hoechst 33342 in dark for 5min at room temperature, washed with PBS and double distilled water; added anti fluorescence quencher and mounted the sections; observed the sections with laser confocal microscope.
Total proteins were extracted respectively from larva, pupa and adult samples of D. melanogaster with radio immunoprecipitation assay (RIPA) lysis buffer and quantified using the Bradford method. Each protein sample was fractionated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% evaporated skimmed milk for 1h at room temperature. According to the best titer of different antibodies, PBST was used to dilute the primary antibodies and then the blocked PVDF membranes were incubated overnight at 4degC. Next day, the membranes were incubated with HRP-conjugated secondary antibody for 90 min at room temperature and exposed in the darkroom. The western blots were photographed and the images were analyzed with Image J software.
Image gray scale analysis method in the Image J documentation was used to convert the data to column charts. The expression intensities of caveolin-1 specific bands were normalized against the Gapdh-specific bands.
All experiments were repeated more than three times independently, data were analyzed using t-test and analysis of variance by SPSS 14.0 software. The data were expressed with mean +- SD, *P<0.05 referred to significant difference, and **Pthose of the pupa and larva.
The results of western blot also showed that the caveolin-1 expressed mainly in adult of D. melanogaster. Moreover, the expression levels of female was significantly higher than that of the male, suggesting that caveolin-1 might be related to the gender development in D. melanogaster.
Since the expression of caveolin-1 was mainly evident in the adult stage of D. melanogaster compared to larval and the pupal stages it was speculated that caveolin-1 mainly affected the growth and physiological functions of the adult. The previous studies of our lab have indicated that caveolin-1 was related to the physiological and pathological changes of mammalian brain function, and played important role in the neuron development and synaptic plasticity (Wang et al., 2007; Zou et al., 2006). In this experiment, we observed obvious expression of caveolin-1 in the brain of D. melanogaster which suggests that caveolin-1 might have important role in the development of brain of insects. In addition, caveolin-1 also showed strong expression in the muscles of thorax, suggesting that caveolin-1 was also very closely related to the development and function of the muscles in D. melanogaster.
This work was supported by the National Natural Science Foundation of China  and the Key laboratory project of the Education Department of Liaoning province [LS2010095].
The expression level and distribution of caveolin-1 varied in Drosophila melanogaster. The in vivo distribution of Caveolin-1 was mainly found in the brain, muscles and gut wall in adult. The expression level of caveolin-1 in adults were significantly higher in adults than in the larvae and pupae. Moreover, the expression was significantly higher in the female adult than in the male adult.
Statement of conflict of interest
The authors have declared no conflict of interest.
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|Author:||Zhang, Yejun; Wang, Ying; Qu, Changqing; Huang, Hui; Li, Hongyan; Li, Yan; Cui, Yuying; Zou, Wei|
|Publication:||Pakistan Journal of Zoology|
|Date:||Oct 31, 2016|
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