Physical properties and actin organization in embryonic stem cells depend on differentiation stage

Abstract

max 300 words)The cellular cytoskeleton provides the cell with mechanical rigidity and mediates mechanical interaction between cells and with the extracellular environment. The actin structure plays a key role in regulating cellular behaviors like motility, cell sorting, or cell polarity. From the earliest stages of development, in naïve stem cells, the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. The ultrastructure of the earliest embryonic stem cells has not been investigated in living cells despite the fact that it is wellknown that cells undergo morphological shape changes during the earliest stages of development. Here, we provide 3D investigations of the actin cytoskeleton of naïve mouse embryonic stem cells (ESCs) in clusters of sizes relevant for early stage development using super resolution optical reconstruction microscopy (STORM). We quantitatively describe the morphological, cytoskeletal and mechanical changes appearing between cells in small clusters at the earliest stages of inner cell mass differentiation, as recapitulated by cells cultured under two media conditions, 2i and Serum/LIF, thus promoting the naïve and first primed state, respectively. High resolution images of living stem cells showed that the peripheral actin structure undergoes a dramatic change between the two media conditions. The actin organization changed from being predominantly oriented parallel to the cell surface in 2i medium to a more radial orientation in Serum/LIF. Finally, using an optical trapping based technique, we detected micro-rheological differences in the cell periphery between the cells cultured in these two media, with results correlating well with the observed nano-architecture of the ESCs in the two different differentiation stages. These results pave the way for linking physical properties and cytoskeletal architecture to the development from naïve stem cells to specialized cells. Statement of Significance (max 120 words)Cells receive mechanical signals and must provide mechanical feedback, therefore, physical properties are instrumental for cell-cell interactions. Mechanical signals mediated through the cell surface can significantly affect transport of signaling molecules and can influence biological processes like transcriptional regulation. To achieve a deeper insight into how the cytoskeletal structure is responsible for cell shape and material properties at the earliest stages of development, we employ super-resolution microscopy to image actin fibers in clusters of embryonic stem cells mimicking early development. By modification of the culturing conditions, we investigate how the actin cytoskeleton and micro-rheological properties of ESCs change between the naïve ground state and the stage primed towards epiblast, thus revealing a correlation between differentiation stage and cytoskeletal structure.

Pluripotency of embryonic stem cells lacking clathrin-mediated endocytosis cannot be rescued by restoring cellular stiffness

“…Studies using superresolution microscopy have demonstrated that mESCs are softer and have a low-density meshwork of F-actin, with large pore sizes, compared with stiffer, differentiated cells ( 21 ). Stem cells transitioning from the naive to the primed state of pluripotency also display a reorganization of the actin cytoskeleton ( 23 ). Our data show that the loss of CME in mESCs results in the presence of stress fibers, which are largely absent in WT mESCs ( Fig.…”

“…Inhibition of actin polymerization in mESCs has also been shown to result in a decrease in differentiation toward the mesodermal lineage coupled with an increase in differentiation toward the endodermal lineage ( 22 ). Naive mESCs had few radial actin structures, whereas primed mESCs showed an increase in stiffness with more actin bundles near the cell periphery ( 23 ). Signaling molecules such as β-catenin have also been shown to be involved in regulating a change in cytoskeletal organization, membrane tension, and subsequently endocytosis during ESC differentiation ( 24 ).…”

“…Previous studies have demonstrated that differentiating mESCs have a high stiffness and higher Young's modulus compared with pluripotent mESCs ( 11 ). Additionally, a transition from the naive state to the primed state is also accompanied by a reorganization of the actin cytoskeleton and an alteration in viscoelastic properties ( 23 ). As a follow-up to our previous study ( 8 ), we asked whether the loss of CME was also accompanied by changes in the stiffness of these cells, similar to other differentiating cells, and whether the actin cytoskeleton played a role under these conditions.…”