Thermoresponsive block copolymer brush for temperature-modulated hepatocyte separation.

Hepatic tissue engineering may be an effective approach for the treatment of liver disease; however, its practical application requires hepatic cell separation technologies that do not involve cell surface modification and maintain cell activity. In this study, we developed hepatocyte cell separation materials using a thermoresponsive polymer and a polymer with high affinity to hepatocytes. A block copolymer of poly(N-p-vinylbenzyl-O-ß-D-galactopyranosyl-(1→4)-D-gluconamide) (PVLA) and poly(N-isopropylacrylamide) (PNIPAAm) [PVLA-b-PNIPAAm] was prepared through two steps of atom transfer radical polymerization. On the prepared PVLA-b-PNIPAAm brush, HepG2 cells (model hepatocytes) adhered at 37 °C and detached at 20 °C, attributed to the temperature-modulated affinity between PVLA and HepG2. Cells from the immortalized human hepatic stellate cell line (TWNT-1) did not adhere to the copolymer brush, and RAW264.7 cells (mouse macrophage; model Kupffer cells) adhered to the copolymer brush, regardless of temperature. Using the difference in cell adhesion properties on the copolymer brush, temperature-modulated cell separation was successfully demonstrated. A mixture of HepG2, RAW264.7, and TWNT-1 cells was seeded on the copolymer brush at 37 °C for adherence. By reducing the temperature to 20 °C, adhered HepG2 cells were selectively recovered with a purity of approximately 85% and normal activity. In addition, induced pluripotent stem (iPS) cell-derived hepatocytes adhered on the PVLA-b-PNIPAAm brush at 37 °C and detached from the copolymer brush at 20 °C, whereas the undifferentiated iPS cells did not adhere, indicating that the prepared PVLA-b-PNIPAAm brush could be utilized to separate hepatocyte differentiated and undifferentiated cells. These results indicated that the newly developed PVLA-b-PNIPAAm brush can separate hepatic cells from contaminant cells by temperature modulation, without affecting cell activity or modifying the cell surface. Thus, the copolymer brush is expected to be a useful separation tool for cell therapy and tissue engineering using hepatocytes.

Modification of exosomes with carbonate apatite and a glycan polymer improves transduction efficiency and target cell selectivity.

Exosomes are secreted from a variety of cells and transmit parental cell-derived biomolecules, such as nucleic acids and proteins, to recipient cells in distant organs. In addition to their important roles in both physiological and pathological conditions, exosomes are expected to serve as natural drug carriers without any cytotoxicity, immunogenicity, or tumorigenicity. However, the use of exosomes as drug delivery tools is limited due to the low uptake efficiency of the target cells, insufficient release of the contents from the endosome to the cytosol, and possible adverse effects caused by the delivery to non-target cells. In the present study, we examined the effects of the modification of exosomes with carbonate apatite or a lactose-carrying polymer. Using newly generated monitoring exosomes that contain either firefly luciferase or fused mCherry/enhanced green fluorescent protein, we demonstrated that the modification of exosomes with carbonate apatite improved their release from the endosome into the cytosol in recipient cells. Meanwhile, the modification of exosomes with a lactose-carrying polymer enhanced the selective delivery to parenchymal hepatocytes. These modified exosomes may provide an efficient strategy for macromolecule therapy for incurable diseases that cannot be treated with conventional small-molecule compounds.

Direct visualization of the extracellular binding structure of E-cadherins in liquid.

E-cadherin is a key Ca-dependent cell adhesion molecule, which is expressed on many cell surfaces and involved in cell morphogenesis, embryonic development, EMT, etc. The fusion protein E-cad-Fc consists of the extracellular domain of E-cadherin and the IgG Fc domain. On plates coated with this chimeric protein, ES/iPS cells are cultivated particularly well and induced to differentiate. The cells adhere to the plate via E-cad-Fc in the presence of Ca2+ and detach by a chelating agent. For the purpose of clarifying the structures of E-cad-Fc in the presence and absence of Ca2+, we analyzed the molecular structure of E-cad-Fc by AFM in liquid. Our AFM observations revealed a rod-like structure of the entire extracellular domain of E-cad-Fc in the presence of Ca2+ as well as trans-binding of E-cad-Fc with adjacent molecules, which may be the first, direct confirmation of trans-dimerization of E-cadherin. The observed structures were in good agreement with an X-ray crystallographic model. Furthermore, we succeeded in visualizing the changes in the rod-like structure of the EC domains with and without calcium. The biomatrix surface plays an important role in cell culture, so the analysis of its structure and function may help promote cell engineering based on cell recognition.

An anti-oxidative cell culture dish inhibits intracellular reactive oxygen species accumulation and modulates pluripotency-associated gene expression in mesenchymal stem cells.

Maintenance of the pluripotent state of mesenchymal stem cells (MSCs) during in vitro expansion is an important factor for the successful proliferation of MSCs possessing high differentiation capacity. However, the differentiation potential of MSCs can easily be lost during in vitro expansion, particularly at late passages. Reactive oxygen species (ROS) are signaling molecules that help to maintain MSC function; however, excessive ROS generation can induce senescence and impair both the differentiation capacity and proliferation of MSCs. In this study, we have designed an amphiphilic block copolymer (redox copolymer), which possesses ROS scavenging capacity in the hydrophobic site. When this redox copolymer was coated on cell culture dishes coupled with human E-cadherin chimeric antibody (hE-cad-Fc), it had an antioxidative effect on cultured MSCs. We also confirmed that the redox polymer construct poly(ethylene glycol) tethered chain on the surface prevented nonspecific cell binding, whereas the co-immobilized surface allowed high adhesion of E-cadherin-positive MSCs. Interestingly, the intracellular ROS level was significantly decreased by the prepared cell culture dish, despite ROS being scavenged only on the surface of the dish, on the cell exterior. Consequently, the cultured MSCs retained high expression levels of pluripotency-associated genes, including SOX2.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Asymmetricity Between Sister Cells of Pluripotent Stem Cells at the Onset of Differentiation.

Various somatic stem cells divide asymmetrically; however, it is not known whether embryonic stem cells (ESCs) divide symmetrically or asymmetrically, not only while maintaining an undifferentiated state but also at the onset of differentiation. In this study, we observed single ESCs using time-lapse imaging and compared sister cell pairs derived from the same mother cell in either the maintenance or differentiation medium. Mouse ESCs were cultured on E-cadherin-coated glass-based dishes, which allowed us to trace single cells. The undifferentiated cell state was detected by green fluorescent protein (GFP) expression driven by the Nanog promoter, which is active only in undifferentiated cells. Cell population analysis using flow cytometry showed that the peak width indicating distribution of GFP expression broadened when cells were transferred to the differentiation medium compared to when they were in the maintenance medium. This finding suggested that the population of ESCs became more heterogeneous at the onset of differentiation. Using single-cell analysis by time-lapse imaging, we found that although the total survival ratio decreased by changing to differentiation medium, the one-live-one-dead ratio of sister cell pairs was smaller compared with randomly chosen non-sister cell pairs, defined as an unsynchronized cell pair control, in both media. This result suggested that sister cell pairs were more positively synchronized with each other compared to non-sister cell pairs. The differences in interdivision time (the time interval between mother cell division and the subsequent cell division) between sister cells was smaller than that between non-sister cell pairs in both media, suggesting that sister cells divided synchronously. Although the difference in Nanog-GFP intensity between sister cells was smaller than that between non-sister cells in the maintenance medium, it was the same in differentiation medium, suggesting asymmetrical Nanog-GFP intensity. These data suggested that ESCs may divide asymmetrically at the onset of differentiation resulting in heterogeneity.

Radial Glial Fibers Promote Neuronal Migration and Functional Recovery after Neonatal Brain Injury.

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Fabrication of 3D-culture platform with sandwich architecture for preserving liver-specific functions of hepatocytes using 3D bioprinter.

The development of new three-dimensional (3D) cell culture system that maintains the physiologically relevant signals of hepatocytes is essential in drug discovery and tissue engineering research. Conventional two-dimensional (2D) culture yields cell growth, proliferation, and differentiation. However, gene expression and signaling profiles can be different from in vivo environment. Here, we report the fabrication of a 3D culture system using an artificial scaffold and our custom-made inkjet 3D bioprinter as a new strategy for studying liver-specific functions of hepatocytes. We built a 3D culture platform for hepatocytes-attachment and formation of cell monolayer by interacting the galactose chain of galactosylated alginate gel (GA-gel) with asialoglycoprotein receptor (ASGPR) of hepatocytes. The 3D geometrical arrangement of cells was controlled by using 3D bioprinter, and cell polarity was controlled with the galactosylated hydrogels. The fabricated GA-gel was able to successfully promote adhesion of hepatocytes. To observe liver-specific functions and to mimic hepatic cord, an additional parallel layer of hepatocytes was generated using two gel sheets. These results indicated that GA-gel biomimetic matrices can be used as a 3D culture system that could be effective for the engineering of liver tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1583-1592, 2017.

Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/ß-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications.

Construction of a Defined Biomimetic Matrix for Long-Term Maintenance of Mouse Induced Pluripotent Stem Cells.

The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3ß (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro.

Human VE-Cadherin Fusion Protein as an Artificial Extracellular Matrix Enhancing the Proliferation and Differentiation Functions of Endothelial Cell.

In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/ß-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering.

Sugar-carrying Polystyrenes Facilitate Harvesting of APCs from MLRs: Possible Application of Sugar-carrying Polystyrenes to Immunotherapy.

Antigen-presenting cells (APCs) play a pivotal role in cancer immunotherapy. APCs in conventionally used flasks are harvested by enzymatic digestion or cell scraping for application to cancer immunotherapy. However, these methods may impair functional molecules expressed on the APC surface and reduce their effects in cancer immunotherapy. Recently, we found that APCs could be harvested by shaking at 4°C in flasks coated with poly[N-p-vinylbenzyl-O-2-acetoamide-2-deoxy-ß-D-glucopyranosyl-(1→4)-2-acetoamide-2-deoxy-ß-D-gluconamide] (PVGlcNAc) or a copolymer consisting of sulfonylurea (SU) linked to poly[N-p-vinyl-benzyl-4-O-ß-D-galactopyranosyl-D-gluconamide] [P(VLA-co-SU)]. In the present study, we compared the functions of cytotoxic T-lymphocytes (CTLs) induced by APCs generated in PVGlcNAc- or P(VLA-co-SU)-coated flasks and conventional flasks. APCs from PVGlcNAc- or P(VLA-co-SU)-coated flasks showed higher expression of cluster of differentiation (CD)80/86, CD11c, and major histocompatibility complex class II alloantigen I-A(d), and higher cytotoxicity than APCs from conventional flasks. These results suggest that the use of PVGlcNAc- or P(VLA-co-SU)-coated flasks is optimal for harvesting APCs. The generated APCs also have a higher antigen-presenting ability compared to those generated in conventional flasks. Our results may contribute to the development of effective cancer immunotherapies.

Surface modification with E-cadherin fusion protein for mesenchymal stem cell culture.

To effectively expand human mesenchymal stem cells (hMSCs) in vitro without affecting their innate biological properties, a fusion protein (hE-cad-Fc) consisting of a human E-cadherin extracellular domain and an immunoglobulin G Fc region was fabricated and used as a biomimetic matrix for MSC culture surface modification. The results showed that cells cultured on hE-cad-Fc-modified polystyrene surfaces exhibited improved proliferation and paracrine functions compared with cells cultured on unmodified and collagen-modified polystyrene surfaces. Meanwhile, surfaces modified with hE-cad-Fc effectively inhibited cell apoptosis even under the serum deprivation conditions. Additionally, the hE-cad-Fc not only up-regulated the expression of ß-catenin in MSCs and stimulated the cellular membrane complex of E-cadherin/ß-catenin, but also effectively activated the intracellular signals such as EGFR, AKT and ERK phosphorylation. Therefore, hE-cad-Fc appeared to be a promising candidate for biological surface modification and stem cell culture.

An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and ß-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency.

Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-ß-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose-asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.

Innovative delivery of siRNA to solid tumors by super carbonate apatite.

RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.

AFM characterization of chemically treated corneal cells.

We present a characterization of chemically treated cells using atomic force microscopy (AFM) which can observe changes in morphology and elasticity of cells. Since AFM has the significant advantage that it does not require fixation of samples, the method is simple and can capture various properties of living cells. In this study, corneal epithelial and endothelial cells were examined. The topography images of the corneal cells without glutaraldehyde (GA) fixation were successfully obtained. The images showed a natural three-dimensional shape of these cells, which scanning electron microscope (SEM) images could not provide. The AFM images of GA-fixed cells were taken and compared with a SEM image reported in the literature. Our results show that longer time for GA fixation makes the surface of the corneal endothelial tissue stiffer. Also, longer treatment results in relatively large structural variation in samples. Combined with conventional histochemical methods, this approach helps us gain an overall understanding of the influence of such chemical treatment.

Ligand-induced internalization, recycling, and resensitization of adrenomedullin receptors depend not on CLR or RAMP alone but on the receptor complex as a whole.

Adrenomedullins (AM) is a multifaceted distinct subfamily of peptides that belongs to the calcitonin gene-related peptide (CGRP) superfamily. These peptides exert their functional activities via associations of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs) RAMP2 and RAMP3. Recent studies established that RAMPs and CLRs can modify biochemical properties such as trafficking and glycosylation of each other. However there is very little or no understanding regarding how RAMP or CLR influence ligand-induced events of AM-receptor complex. In this study, using pufferfish homologs of CLR (mfCLR1-3) and RAMP (mfRAMP2 and mfRAMP3), we revealed that all combinations of CLR and RAMP quickly underwent ligand-induced internalization; however, their recycling rates were different as follows: mfCLR1-mfRAMP3>mfCLR2-mfRAMP3>mfCLR3-mfRAMP3. Functional receptor assay confirmed that the recycled receptors were resensitized on the plasma membrane. In contrast, a negligible amount of mfCLR1-mfRAMP2 was recycled and reconstituted. Immunocytochemistry results indicated that the lower recovery rate of mfCLR3-mfRAMP3 and mfCLR1-mfRAMP2 was correlated with higher proportion of lysosomal localization of these receptor complexes compared to the other combinations. Collectively our results indicate, for the first time, that the ligand-induced internalization, recycling, and reconstitution properties of RAMP-CLR receptor complexes depend on the receptor-complex as a whole, and not on individual CLR or RAMP alone.