Foley, A. C. | bioRxiv Science

bioRxiv2023

Tbx5 overexpression in embryoid bodies increases TAK1 expression but does not enhance the differentiation of sinoatrial node cardiomyocytes.

Genetic studies place Tbx5 at the apex of the sinoatrial node (SAN) transcriptional program. To understand its role in SAN differentiation, clonal embryonic stem (ES) cell lines were made that conditionally overexpress Tbx5, Tbx3, Tbx18, Shox2, Islet-1 and Map3k7/TAK1. and cardiac cells differentiated using embryoid bodies (EBs). EBs overexpressing Tbx5, Islet1, and TAK1 beat faster than cardiac cells differentiated from control ES cell lines suggesting possible roles in SAN differentiation. Tbx5 overexpressing EBs showed increased expression of TAK1, but cardiomyocytes did not differentiate as SAN cells. They showed no increase in the expression of Shox2, or Islet1 and decreased expression of HCN4. EBs constitutively overexpressing TAK1 direct cardiac differentiation to the SAN fate, but also have decreased phosphorylation of its targets, p38, and Jnk. This opens the possibility that blocking the phosphorylation of TAK1 targets may have the same impact as forced overexpression. To test this, we treated EBs with 5z-7-Oxozeanol (OXO), an inhibitor of TAK1 phosphorylation. Like TAK1 overexpressing cardiac cells, cardiomyocytes differentiated in the presence of OXO beat faster and showed increased expression of SAN genes (Shox2, HCN4, and Islet1). This suggests that activation of the SAN transcriptional network can be accomplished by blocking the phosphorylation of TAK1.

developmental biology↗

bioRxiv2019

A novel technique for mapping viscosity in discrete subcellular locations with a BODIPY based fluorescent probe

Numerous cellular processes, including enzyme behaviour, signalling, and protein folding and transport are highly influenced by the local microviscosity environment within living cells. Molecular rotors are fluorescent molecules that respond to the viscosity of their environment through changes in both the intensity and lifetime of their fluorescence. We have synthesised a novel benzyl-guanine derivatized boron-dipyrromethene (BODIPY) molecular rotor that is a substrate for the SNAP-tag targeting system (named BG-BODIPY), allowing us to target the rotor to discrete locations within the living cell. We demonstrate that BG-BODIPY reports viscosity, and that this can be measured either through fluorescence lifetime or intensity ratiometric measurements. The relative microviscosities within the ER, Golgi, mitochondrial matrix, peroxisomes, lysosomes, cytoplasm, and nucleoplasm were significantly different. Additionally, this approach permitted fluorescence lifetime imaging microscopy (FLIM) to determine the absolute viscosity within both mitochondria and stress granules, showcasing BG-BODIPYs usefulness in studying both membrane-bound and membraneless organelles. These results highlight targeted BG-BODIPYs broad usefulness for making measurements of cellular viscosity both with FLIM and conventional ratiometric confocal microscopy, the latter option greatly extending the accessibility of the technique although limited to relative meassurements. Statement of SignificanceLocal viscosity affects molecular behaviour from diffusion and conformational changes to enzyme kinetics and has important implications for cell and tissue function. Mechanical methods for measurement of viscosity average over large volumes and long times and are thus unsuitable for rapid changes on small scales that are biologically relevant. This paper reports a novel optical fluorescence method using genome edited cells to deliver a viscosity reporter to tightly defined locations inside living cells, from which non-destructive organelle-specific measurements can be repeatedly made. The local viscosity of seven separate organelles in living cultured human cells is shown for the first time, together with the viscosity behaviour of a membraneless organelle as it is induced in cells by stress.

cell biology↗