Archives of Dermatology and Skin Care ISSN 2638-4914 Volume 2, Issue 1, 2019, PP: 01-07 Non-Visual Opsin 3 Localizes with Microtubules During Mitosis and is Essential for Cytokinesis F. Labarrade1, C. Gondran*2, K. Cucumel3 123 [email protected], [email protected], [email protected] Ashland Specialties France, Global Skin Research Center 655 route du Pin Montard, 06904 Sophia Antipolis, France. *Corresponding Author: Dr. Catherine Gondran, Ashland Specialties France, Global Skin Research Center 655 route du Pin Montard Sophia Antipolis, 06904, France. Abstract Opsin photoreceptors are responsible for the absorption of light, transduce information about daily lighting conditions and provide vision. Opsins are also involved in diverse non-visual functions such as the circadian clock. The non-visual opsin 3, also known as encephalopsin, is expressed in skin and its functions remain poorly defined. The expression of opsin 3 in human skin tissue was evaluated by immunohistochemistry, in keratinocytes and melanocytes by immunodetection. Opsin 3 and β-tubulin colocalization was performed during the keratinocyte mitotic phase. Opsin 3-siRNA transfection was used in primary keratinocytes and successful transfection was confirmed by immunodetection. These results show that opsin 3 was detected in the epidermis of human skin, both in keratinocytes and melanocytes. Opsin 3 colocalize with β-tubulin during the mitotic phase and participate in keratinocyte cell division. Opsin 3 protein expression was reduced after transfection with opsin 3-siRNA; inhibition of opsin 3-siRNA reduces keratinocyte cell proliferation and promotes the apparition of binucleated cells. This study highlights an unconventional function of opsin 3 in extra-ocular cells. Opsin 3 appears to be involved in the regulation of late cytokinesis in keratinocytes, and therefore in the maintenance of epidermal homeostasis. Keywords: Skin; Keratinocyte; Melanocyte; Photoreceptors; Opsin 3; Mitosis; Cytokinesis. Introduction opsins has been described in the skin [4], suggesting photosensitivity properties to this tissue. Interestingly, Humans can see the world in color thanks to photon- opsin 3, also known as encephalopsin or panopsin, sensitive receptors called opsins that are present in the retina. With many millions of photo-sensitive in keratinocytes [4]. The encephalopsin name was rod and cone cells in the eye, photo transduction is derivedoriginally from identified their instrong the brain expression [5] is highly in expressedthe brain a mechanism activated by a source of light [1]. Many [6]. Opsin 3 has been suggested to play a role in the animals utilize light perception to regulate biological entrainment of circadian rhythm and the regulation processes, such as vision and circadian rhythms [2]. of pineal melatonin production [7]. Opsin 3 also has Opsins are seven-transmembrane-helix G protein- coupled receptors (GPCRs) that trap photons with a pigmentation processes [9]. retinal molecule in the heart of their architecture, thus been identified as involved in hair growth [8] and skin The skin epithelium continually self-renews and ‘sensing’ light [3]. can rapidly regenerate after damage. Epidermal In vertebrates, opsins sense light for visual and non-visual functions, but non-visual roles are less known. The expression of both visual and non-visual homoeostasis is maintained by a fine balance ofs keratinocyte proliferation and terminal differentiation Archives of Dermatology and Skin Care V2 . I1 . 2019 [10]. The influence of light on skin homeostasis ha1 Non-Visual Opsin 3 Localizes with Microtubules During Mitosis and is Essential for Cytokinesis previously been described, such as in the pigmentation 0.1% triton X100, then blocked in 1% BSA solution. process, synthesis of vitamin D, endorphins and the Cells were incubated with primary antibodies, diluted neuropeptide called substance P [11; 12; 13; 14]. The cell division process controls the renewal of followed by incubation with an Alexa Fluor conjugated proliferating epithelial progenitors and hence the secondaryin phosphate-buffered antibodies (Invitrogen). saline at room Image temperature, acquisition was performed using an Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany). Photos were extent of production of terminally differentiated captured with an EXI blue camera (Qimaging, Surrey, lineages. It has been shown that GPCRs are effectors during mitosis remains poorly understood. GPCRs BC, Canada) coupled to Volocity acquisition software in cell division [15], but the function of GPCRs mostly localize to the plasma membrane, transmit (Perkin Elmer, Waltham, MA, USA). extracellular stimuli into the cytosol and also enter the Immunohistological Fluorescence the expression of non-visual opsin 3 during the Normal human epithelial keratinocytes were isolated cytoplasm [15]. In the present work, we investigated mitosis process in human keratinocytes. Here we from skin obtained from healthy female donors who report the participation of opsin 3 in the regulation had undergone plastic surgery procedures and given written informed consent (Biopredic International). unexpected relationship between sensory GPCRs and After removal of subcutaneous fat, tissue was used to mitoticof keratinocyte cell divisions. division. These findings establish an Materials And Methods in formaldehyde and processed in an automated Shandonobtain 6 mmHypercenter punch biopsies, XP (Shandon which Ltd., were Runcor, then fixed UK) Antibodies and Probes The following antibodies were used: anti-opsin 3 were cut with a microtome (Shandon) and collected onfor paraffinpoly-lysine embedding. coated glass Sections slides of (Menzel4 μm thickness Gläser, (Abcam, Cambridge, UK). Alexa Fluor® coupled Braunschweig, Germany) for immunostaining. Heat- (LifeSpan BioSciences, Seattle, WA, USA), anti β tubulin secondary antibodies were used (Molecular Probes, mediated antigen retrieval was performed in citrate e incubation with opsin 3 antibody. MolecularEugene, OR, Probes). USA). An Alexa Fluor® 594 Phalloidin bufferStatistical befor Analysis probe was used for F-actin detection (A12381, Cell Culture All experiments have been repeated, statistical analyses were performed using JMP software (SAS, Cary, NC, Normal human epithelial keratinocytes were isolated USA). Normality testing of the data was performed from skin obtained from healthy female donors who had undergone plastic surgery procedures and given with the Shapiro-Wilk test. The one-way analysis of written informed consent (Biopredic International). variance (ANOVA) was used to determine whether Keratinocytes were cultured in a keratinocyte serum betweenthere was two any means significant was performed difference with between Student’s the means of two or more independent groups. Difference free medium, supplemented with 50 μg/ml bovine t-test. A p (Invivogen,pituitary extract, San Diego, 5 ng/ml CA, USA). epidermal growth factor p -value ≤0.05 was considered statistically (Gibco, Auckland, NZ), and 0.1 mg/ml Primocin™ and p RNA Interference significant (*), -value ≤0.01 as very significant (**) Opsin 3 siRNA (Invitrogen, Carlsbad, CA, USA) was Results-value ≤0.005 as highly significant (***). Patterns of Opsin 3 Expression in Human lipofectamine RNAiMAX (Invitrogen). As a negative Skin controltransfected Stealth with a RNAifinal concentrationNegative Control of 50 nM Duplex using (Invitrogen) was used as recommended by the Immunodetection of opsin 3 showed that the protein manufacturer. was broadly detectable in all compartments of the studied tissue. In human adult skin, Opn3 receptor Immunocyto Fluorescence expression was detected in all layers of the epidermis and in many cells located in the dermis (Figure 1). Archives of Dermatology and Skin Care V2 . I1 . 2019 Formaldehyde-fixed2 cells were permeabilized in Non-Visual Opsin 3 Localizes with Microtubules During Mitosis and is Essential for Cytokinesis Figure 1. Immunodetection of opsin 3 receptors in human skin. Representative images of paraffin-embedded sections, using polyclonal antibodies directed against the human opsin 3 receptor (green) and DNA stained with DAPI (blue) respectively (scale bar = 31 µm). Patterns of Opsin 3 Expression in Human staining (Figure 2A). However, in melanocytes, opsin Keratinocyte and Melanocyte 3 exhibited a granular staining pattern predominant In cultured keratinocytes, opsin 3 staining showed within the cell body as well as throughout the dendrites (Figure 2B). a cytoplasmic location, with a fluorescence punctate Figure 2. Immunodetection of opsin 3 receptors in human keratinocytes (A) and melanocytes. (B) Representative images of opsin 3 (red), β tubulin (green) and DNA stained with DAPI (blue) respectively (scale bar = 16 µm). Differential Localization of Opsin 3 and During the mitosis step corresponding from Tubulin During Keratinocyte Division prometaphase to anaphase, the opsin 3 localized to Rather than just being specialized for a sensory event, as the mitotic pole (Figure 2). The opsin 3 staining opsin 3 distribution analysis during cell division duringdifferent cytokinesis areas related and to latethe cytokinesisβ tubulin network, showed such a localization at the cleavage furrow, again in relation that could be observed in interphase cells (Figure2A). revealed a different pattern of localization (Figure3) Archives of Dermatology and Skin Care V2 . I1 . 2019with the β tubulin staining. 3 Non-Visual Opsin 3 Localizes with Microtubules During Mitosis and is Essential for Cytokinesis