Potential Method of Autophagy Imaging with Cationized Gelatin Nanospheres Incorporating Molecular Beacon.

The objective of this research is to develop an imaging method with cationized gelatin nanospheres incorporating molecular beacon (cGNSMB) to visualize an autophagy activity in living cells. Cationized gelatin nanospheres (cGNS) were prepared by the conventional coacervation method, and then molecular beacon (MB) was incorporated into them. The cGNSMB prepared were internalized into cells at a high efficiency. In this study, a starvation medium of serum and amino acids-free was used to induce autophagy. The autophagy activity was confirmed by an immunofluorescence staining for microtubule-associated proteins light chain 3B (LC3B) of an autophagy specific protein. With the autophagy induction time, the number of LC3 fluorescent dots increased, which indicated an increased autophagy activity. As the autophagy-related genes, sequestosome 1 (SQSTM1) and cathepsin F (CTSF), which up-regulate after autophagy induction, were chosen as the targets of cGNSMB. The fluorescence intensity of cGNSMB targeting to SQSTM1 and CTSF increased with the starvation treatment time, which well corresponded with the gene expression results. When applied to cells in different autophagy conditions, the cGNSMB visualized the autophagy activity corresponding with the autophagy condition of cells. From the results obtained, it was concluded that the cGNSMB provide a promising method to visualize the autophagy of cells. The advantage of cGNSMB visualization is to obtain the temporal and spatial information without destroying sample cells.

Extracellular vesicles synchronize cellular phenotypes of differentiating cells.

During embryonic development, cells differentiate in a coordinated manner, aligning their fate decisions and differentiation stages with those of surrounding cells. However, little is known about the mechanisms that regulate this synchrony. Here we show that cells in close proximity synchronize their differentiation stages and cellular phenotypes with each other via extracellular vesicle (EV)-mediated cellular communication. We previously established a mouse embryonic stem cell (ESC) line harbouring an inducible constitutively active protein kinase A (CA-PKA) gene and found that the ESCs rapidly differentiated into mesoderm after PKA activation. In the present study, we performed a co-culture of Control-ESCs and PKA-ESCs, finding that both ESC types rapidly differentiated in synchrony even when PKA was activated only in PKA-ESCs, a phenomenon we named 'Phenotypic Synchrony of Cells (PSyC)'. We further demonstrated PSyC was mediated by EVs containing miR-132. PKA-ESC-derived EVs and miR-132-containing artificial nano-vesicles similarly enhanced mesoderm and cardiomyocyte differentiation in ESCs and ex vivo embryos, respectively. PSyC is a new form of cell-cell communication mediated by the EV regulation of neighbouring cells and could be broadly involved in tissue development and homeostasis.

Complexation design of cationized gelatin and molecular beacon to visualize intracellular mRNA.

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/µg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/µg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.