Actin stress in cell reprogramming

Abstract

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin-cpstFRET-actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFPtagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals.actin | force probe | stem cell | reprogramming | cell mechanics C ells have only three sources of free energy-chemical, electrical, and mechanical potential (1)-and life is driven by the flow of energy between them. The flow of mechanical energy has not been well studied in living cells outside of muscle, yet all cells are subject to endogenous and exogenous mechanical forces. The lack of progress in measuring these forces (stress) has primarily been due to a lack of suitable probes, but that has now been remedied with the development of genetically coded FRETbased force sensors (2). The literature shows many macroscopic effects of mechanical stress on cellular processes including cell motility, embryogenesis, stem cell replication and differentiation (3, 4), bone and muscle homeostasis, gene expression, protein folding (5), and membrane potential (6). However, these studies lacked the ability to measure stress in specific structural proteins, and the analyses have tended to treat cytoskeletal stresses as uniform, which we now know is not true (2, 7). We, and other groups (8), have shown that the distribution of forces is nonuniform in both time and space and protein specificity (9).The force sensors consist of a FRET pair (typically CFP/ YFP) connected by a protein linker, a biological analog of a mechanic...

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“…However, it was more recently shown that this might not be necessary insofar as cell lines growing in a matrix can result in high rates of reversion to stem-like cells and this could mean that pluripotency can be produced by modification of the ECM [151], a finding that remains to be replicated.…”

Regulation of Intracellular Structural Tension by Talin in the Axon Growth and Regeneration

“…An angular-dependent probe termed Bcircularly permuted stretch-sensitive Förster resonance energy transfer( cpstFRET) was created by tandem linkage of circularly permutated yellow fluorescent protein (YFP)/Venus and circularly permutated cyan fluorescent protein (CFP)/Cerulean according to our previous reports [27]. The cpstFRET gene was inserted into the pEGFP-C1 vector (Clontech Laboratories, Mountain View, CA, USA) without the enhanced green fluorescent protein (EGFP) gene.…”

“…Genetically encoded optical-force probes have enabled measurement of intracellular tension in real time. Here, a probe named actin-cpstFRETactin (AcpA) was adopted according to our previous reports [27,30]. This probe is a dipole orientation-based FRET probe composed of a pair of CFP/YFP (cpCerulean/cpVenus), and can be inserted into microfilament skeletons naturally when microfilaments are under assembly in cells.…”

“…Traditional methods such as atomic force microscopy cannot be utilized to measure intracellular or intramolecular forces in real time at the subcellular level [26][27][28]. Therefore, we created Förster resonance energy transfer (FRET)-based tension probes that could be integrated into cytoskeletal proteins and turn changes in structural forces into optical signals to estimate intracellular forces [29][30][31].…”

“…Therefore, we created Förster resonance energy transfer (FRET)-based tension probes that could be integrated into cytoskeletal proteins and turn changes in structural forces into optical signals to estimate intracellular forces [29][30][31]. Hence, we could monitor structural forces and analyze the underlying mechanism during growth and regeneration of axons [27,31].…”