Biomechanical signals such as cell shape and spreading play an important role in controlling stem cell commitment. Cell shape, adhesion and spreading are also affected by calreticulin, a multifunctional calcium-binding protein, which influences several cellular processes, including adipogenesis. Here we show that cytoskeletal disruption in mouse embryonic stem cells using cytochalasin D or nocodazole promotes adipogenesis. While cytochalasin D disrupts stress fibres and inhibits focal adhesion formation, nocodazole depolymerises microtubules and promotes focal adhesion formation. Furthermore, cytochalasin D increases the levels of both total and activated calcium/calmodulin-dependent protein kinase II, whereas nocodazole decreases it. Nevertheless, both treatments significantly increase the adipogenic potential of embryonic stem cells in vitro. Both cytochalasin D and nocodazole exposure caused cell rounding suggesting that it is cell shape that causes the switch towards the adipogenic programme. Calreticulin-containing embryonic stem cells, under baseline conditions, show low adipogenic potential, have low activity of signalling via calcium/calmodulin-dependent protein kinase II and display normal adhesive properties and cellular spreading in comparison to the highly adipogenic but poorly spread calreticulin-deficient ES cells. We conclude that forced cell rounding via cytoskeletal disruption overrides the effects of calreticulin, an ER chaperone, thus negatively regulating adipogenesis via focal adhesion-mediated cell spreading.

Calreticulin is an endoplasmic reticulum-resident multifunctional protein, which has been shown to influence numerous cellular processes, including cell adhesion. In this study, we characterized the adhesive properties of embryonic stem cells (ESCs) lacking calreticulin and showed that adipogenesis from ESCs is directly and reciprocally controlled by the adhesive status of a cell, which in turn is modulated by calreticulin. Calreticulin-deficient ESCs are not only highly adipogenic but also show elevated calmodulin/CaMKII signaling and poor adhesiveness compared with the wild-type ESCs. Calreticulin deficiency leads to a disorganized cytoskeleton and low levels of focal adhesionrelated proteins, such as vinculin, paxillin, and phosphorylated focal adhesion kinase, which cause limited focal adhesion formation and limited fibronectin deposition. Moreover, differentiation on nonadhesive substrata, which hinder cell spreading, promoted adipogenesis in the wildtype ESCs that normally have low adipogenic potential, causing a decrease in focal adhesion protein expression and an increase in calmodulin/CaMKII signaling. In contrast, inhibition of CaMKII effectively increased focal adhesion protein levels and inhibited adipogenesis in calreticulin-deficient ESCs, causing them to behave like the low adipogenic, wild-type ESCs. Thus, the adipogenic potential of ESCs is proportional to their calmodulin/ CaMKII activity but is inversely related to their focal adhesion protein levels and degree of adhesiveness/spreading.

Cell adhesion is regulated by a variety of Ca2+-regulated pathways that depend on Ca2+-binding proteins. One such protein is calreticulin, an ER-resident protein. Calreticulin signalling from within the ER can affect processes outside the ER, such as expression of several adhesion-related genes, most notably vinculin and fibronectin. In addition, changes in the expression level of calreticulin strongly affect tyrosine phosphorylation of cellular proteins, which is known to affect many adhesion-related functions. While calreticulin has been localized to cellular compartments other than the ER, it appears that only the ER-resident calreticulin affects focal-contact-dependent adhesion. In contrast, calreticulin residing outside the ER may be involved in contact disassembly and other adhesion phenomena. Here, we review the role of calreticulin in focal contact initiation, stabilization, and turnover. We propose that calreticulin may regulate cell-substratum adhesion by participating in an "ER-to-nucleus" signalling and in parallel "ER-to-cell surface" signalling based on posttranslational events.