Reorganization Of Cellular Cytoskeleton Research Articles

Folate-directed zinc (II) phthalocyanine loaded polymeric micelles engineered to generate reactive oxygen species for efficacious photodynamic therapy of cancer.

Targeted and effective drug transport is becoming an attractive option in cancer therapy since it can improve drug efficacy and reduce drugs' side effects in normal tissues. In addition to using specific surface ligand molecules, the selective drug delivery can be accomplished via enhanced permeability and retention effect. Therefore, in our studies, we entrapped zinc (II) phthalocyanine (ZnPc) - a second generation photosensitizer - in folate-functionalized micelles of the biocompatible, FDA-approved for biomedical application diblock copolymer methoxypoly(ethylene oxide)-b-poly(L-lactide) (mPEG-b-PLLA) and its derivative with folate (FA) attached to the end of PEG chain (FA-PEG-b-PLLA). Dynamic light scattering (DLS) measurements confirmed the micellar size to be <150 nm in diameter, a low polydispersity index, and good colloid stability of the studied nanocarriers, while atomic force microscopy (AFM) was used to study their morphology. The application potential of the resulting micelles was evaluated in cyto- and photocytotoxicity studies in conjunction with intracellular distribution and accumulation imaging of the photosensitizer delivered to ovarian carcinoma (SKOV-3) and metastatic melanoma (Me45) cell lines. Reactive oxygen species generation study was performed after photodynamic reaction, and cellular cytoskeleton reorganization was visualized after undergoing a photodynamic reaction. The results demonstrated that the functionalized polymeric micelles are promising nanocarriers for photodynamic therapy procedures and can be used in anticancer drug delivery.

Gold nanoparticles functionalized with angiogenin-mimicking peptides modulate cell membrane interactions.

Angiogenin is a protein crucial in angiogenesis, and it is overexpressed in many cancers and downregulated in neurodegenerative diseases, respectively. The protein interaction with actin, through the loop encompassing the 60-68 residues, is an essential step in the cellular cytoskeleton reorganization. This, in turn, influences the cell proliferation and migration processes. In this work, hybrid nanoassemblies of gold nanoparticles with angiogenin fragments containing the 60-68 sequence were prepared and characterized in their interaction with both model membranes of supported lipid bilayers (SLBs) and cellular membranes of cancer (neuroblastoma) and normal (fibroblasts) cell lines. The comparison between physisorption and chemisorption mechanisms was performed by the parallel investigation of the 60-68 sequence and the peptide analogous containing an extra cysteine residue. Moreover, steric hindrance and charge effects were considered with a third analogous peptide sequence, conjugated with a fluorescent carboxyfluorescein (Fam) moiety. The hybrid nanobiointerface was characterized by means of ultraviolet-visible, atomic force microscopy and circular dichroism, to scrutinize plasmonic changes, nanoparticles coverage and conformational features, respectively. Lateral diffusion measurements on SLBs "perturbed" by the interaction with the gold nanoparticles-peptides point to a stronger membrane interaction in comparison with the uncoated nanoparticles. Cell viability and proliferation assays indicate a slight nanotoxicity in neuroblastoma cells and a proliferative activity in fibroblasts. The actin staining confirms different levels of interaction between the hybrid assemblies and the cell membranes.

Optimization of differentiation time of mesenchymal-stem-cell to tenocyte under a cyclic stretching with a microgrooved culture membrane and selected measurement cells.

There is a need for efficient stem cell-to-tenocyte differentiation techniques for tendon tissue engineering. More than 1 week is required for tenogenic differentiation with chemical stimuli, including co-culturing. Research has begun to examine the utility of mechanical stimuli, which reduces the differentiation time to several days. However, the precise length of time required to differentiate human bone marrow-derived mesenchymal stem cells (hBMSCs) into tenocytes has not been clarified. Understanding the precise time required is important for future tissue engineering projects. Therefore, in this study, a method was developed to more precisely determine the length of time required to differentiate hBMSCs into tenocytes with cyclic stretching stimulus. First, it had to be determined how stretching stimulation affected the cells. Microgrooved culture membranes were used to suppress cell orientation behavior. Then, only cells oriented parallel to the microgrooves were selected and evaluated for protein synthesis levels for differentiation. The results revealed that growing cells on the microgrooved membrane and selecting optimally-oriented cells for measurement improved the accuracy of the differentiation evaluation, and that hBMSCs differentiated into tenocytes in approximately 10 h. The differentiation time corresponded to the time required for cellular cytoskeleton reorganization and cellular morphology alterations. This suggests that cells, when subjected to mechanical stimulus, secrete mRNAs and proteins for both cytoskeleton reorganization and differentiation.

Growth arrest specific 2-like protein 1 expression is upregulated in podocytes through advanced glycation end-products.

Growth arrest specific 2-like protein 1 (GAS2L1) protein is a member of the GAS2 family of proteins, known to regulate apoptosis and cellular cytoskeleton reorganization in different cells. Recently we identified that Gas2l1 gene expression in podocytes is influenced by advanced glycation end product-bovine serum albumin(AGE-BSA). The study was performed employing cultured podocytes and diabetic ( db/db ) mice, a model of type 2 diabetes. Akbuminuria as wellas urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion as measured with specific ELISAs. Gene expression was analysed via semiquantitative and real-time polymerase chain reaction. The protein levels were determined by western blotting and immunostaining. We found that the Gas2l1 α isoform is expressed in podocytes. Treatment with AGE-BSA induced Gas2l1 α and Gas2 mRNA levels compared with controls incubated with non-glycated control BSA (Co-BSA). Moreover, application of the recombinant soluble receptor of AGEs (sRAGE), a competitor of cellular RAGE, reversed the AGE-BSA effect. Interestingly, AGE-BSA also increased the protein levels of GAS2L1α in a RAGE-dependent manner, but did not affect the GAS2 expression. Periodic acid-Schiff staining and albuminuria as well as urinary NGAL excretion revealed that db/db mice progressively developed diabetic nephropathy with renal accumulation of N ε -carboxy-methyl-lysine (immunohistochemistry, western blots). Analyses of GAS2L1α and GAS2 proteins in diabetic mice revealed that both were significantly elevated relative to their non-diabetic littermates. In addition, GAS2L1α and GAS2 proteins positively correlated with the accumulation of AGEs in the blood plasma of diabetic mice and the administration of sRAGE in diabetic mice reduced the glomerular expression of both proteins. We show for the first time that the protein expression of GAS2L1α in vitro and in vivo is regulated by the AGE-RAGE axis. The suppression of AGE ligation with their RAGE in diabetic mice with progressive nephropathy reversed the GAS2L1α expression, thus suggesting a role of GAS2L1α in the development of diabetic disease, which needs to be further elucidated.

Distinct Roles for the α, β and γ Isoforms of the Catalytic Subunit of Protein Phosphatase 1 in Outside-in Integrin αIIbβ3 Signaling.

Abstract 154Outside-in integrin αIIbγ3 signaling mediated by kinases and phosphatases regulate platelet adhesion and thrombus formation. The catalytic subunit of protein phosphatase 1 (PP1c), expressed as α, β and γ isoforms regulates a variety of cellular functions. We have previously demonstrated that fibrinogen-αIIbβ3 engagement during outside-in signaling dissociated integrin αIIb*β3 anchored PP1c, which was followed by PP1c activation and dephosphorylation of myosin light chain (MLC). However, it is unknown whether any functions regulated by outside-in integrin αIIbβ3 signaling are modulated by PP1c. To overcome the lack of specificity of pharmacological agents towards PP1c, we have pursued a genetic approach to inhibit PP1c in 293 cells overexpressing αIIbβ3. In this study, we demonstrated that PP1cα interacts with αIIbβ3 in platelets and 293 cells by co-immunoprecipitation and GST pull down assays. Knockdown of PP1cα by short interference RNA resulted in a 50-60% enhanced adhesion (p<0.003) of 293 cells to immobilized fibrinogen. Conversely, overexpression of PP1cα inhibited (p<0.002) αIIbβ3 adhesiveness to fibrinogen. Furthermore, the enhanced adhesiveness of PP1cα depleted 293 cells was also observed with von Willebrand factor, indicating that the differential adhesion due to the lack of PP1cα was not ligand specific. Signaling pathways implicated in the reorganization of cellular cytoskeleton, like extracellular-regulated kinase (ERK1/2) and AKT were activated in PP1cα depleted 293 cells. Such observations imply that PP1cα, knockdown of PP1cγ, which also associates with αIIbβ3, did not affect the adhesion to fibrinogen. Indeed, platelets from the PP1cγ null and wild type mice showed comparable adhesion to fibrinogen. Unexpectedly, depletion of PP1cβ isoform in 293 cells significantly (p<0.01) inhibited the ability of these cells to adhere to fibrinogen. Since MLC is a specific substrate of PP1cβ, it is likely that alterations in the MLC phosphorylation may underlie the loss of adhesiveness in PP1cβ depleted cells. Finally, depletion of all the PP1c isoforms also significantly (p<0.001) inhibited the adhesion of 293 cells to fibrinogen. These data illustrate that each isoform of PP1c contributes distinctly to the adhesive phenotype of integrin αIIbβ3 and implies that the control of outside-in integrin αIIbβ3 signaling response might be a composite of all PP1c isoforms. Perhaps, each isoforms have specific substrates that contribute to the process of adhesion, such that alterations in the phosphorylation of these substrates may underlie the distinct functional roles for PP1c. Disclosures:No relevant conflicts of interest to declare.