MHC-IIB Filament Assembly and Cellular Localization Are Governed by the Rod Net Charge

Actin-dependent myosin II molecular motors form an integral part of the cell cytoskeleton. Myosin II molecules contain a long coiled-coil rod that mediates filament assembly required for myosin II to exert its full activity. The exact mechanisms orchestrating filament assembly are not fully understood., negatively-charged regions of the coiled-coil were found to play an important role by controlling the intracellular localization of native MHC-IIB. The entire positively-charged region is also important for intracellular localization of native MHC-IIB.A correct distribution of positive and negative charges along myosin II rod is a necessary component in proper filament assembly and intracellular localization of MHC-IIB

High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming

Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15–590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming

A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

Reprogramming of somatic cells to different extents has been reported using different methods. However, this is normally accompanied by the use of exogenous materials, and the overall reprogramming efficiency has been low. Chemicals and small molecules have been used to improve the reprogramming process during somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation. We report here the first application of a combined epigenetic and non-genetic approach for reprogramming somatic cells, i.e., DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and human embryonic stem cell (hESC) extracts. When somatic cells were pretreated with these inhibitors before exposure to hESC (MEL1) extracts, morphological analysis revealed a higher rate of hESC-like colony formation than without pretreatment. Quantitative PCR (qPCR) demonstrated that pluripotency genes were upregulated when compared to those of somatic cells or treated with hESC extracts alone. Overall changes in methylation and acetylation levels of pretreated somatic cells suggests that epigenetic states of the cells have an effect on reprogramming efficiency induced by hESC extracts. KnockOutserum replacement (KOSR™) medium (KO-SR) played a positive role in inducing expression of the pluripotency genes. hESC extracts could be an alternative approach to reprogram somatic cells without introducing exogenous materials. The epigenetic pre-treatment of somatic cells could be used to improve the efficiency of reprogramming process. Under differentiation conditions, the reprogrammed cells exhibited differentiation ability into neurons suggesting that, although fully reprogramming was not achieved, the cells could be transdifferentiated after reprogramming