Nonmuscle myosin IIA and p120-catenin control E-cadherin–mediated cell–cell adhesions essential for hESC pluripotency and long-term survival.

Highlights d Sequential activity of pyramidal neurons emerges and stabilizes with motor training d SST interneurons regulate the establishment and stabilization of sequential activity d VIP interneurons regulate the establishment of sequential activity d The regulation of sequential activity involves CaMKIIdependent synaptic plasticity

Summary Podosomes are self-organized dynamic actin-containing structures that adhere to the extracellular matrix via integrins [1–5]. Yet it is not clear what regulates podosome dynamics and whether podosomes can function as direct mechanosensors like focal adhesions [6–9]. We show here that myosin IIs form circular structures outside and at the podosome actin ring to regulate podosome dynamics. Inhibiting myosin II-dependent tension dissipated podosome actin rings before dissipating the myosin ring structure. As podosome rings changed size or shape, tractions underneath the podosomes were exerted onto the substrate, which were abolished when myosin light chain activity was inhibited. The magnitudes of tractions were comparable to those generated underneath focal adhesions and increased with substrate stiffness. The dynamics of podosomes and of focal adhesions were different. Torsional tractions underneath the podosome rings were generated with rotations of podosome rings in a non-motile and non-rotating cell, suggesting a unique feature of these circular structures. Stresses applied via integrins at the apical surface directly displaced podosomes near the basal surface. Stress-induced podosome displacements increased nonlinearly with applied stresses. Our results suggest that podosomes are dynamic mechanosensors in which interactions of myosin tension and actin dynamics are crucial in regulating these self-organized structures in living cells.

Summary Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically-encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1–2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ~1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.

Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein ␤␥ (G␤␥) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G␤␥ and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G␤␥-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC␤ and Src. Rather, RACK1 selectively inhibits G␤␥-stimulated phosphatidylinositol 3-kinase ␥ (PI3K␥) and phospholipase C (PLC) ␤ activity, due to the competitive binding of RACK1, PI3K␥, and PLC␤ to G␤␥. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G␤␥-dependent activation of key effectors critical for chemotaxis.

Despite recent advances in our understanding of biochemical regulation of neutrophil chemotaxis, little is known about how mechanical factors control neutrophils' persistent polarity and rapid motility. Here, using a human neutrophil-like cell line and human primary neutrophils, we describe a dynamic spatiotemporal pattern of tractions during chemotaxis. Tractions are located at both the leading and the trailing edge of neutrophils, where they oscillate with a defined periodicity.Interestingly, traction oscillations at the leading and the trailing edge are out of phase with the tractions at the front leading those at the back, suggesting a temporal mechanism that coordinates leading edge and trailing edge activities. The magnitude and periodicity of tractions depend on the activity of nonmuscle myosin IIA. Specifically, traction development at the leading edge requires myosin light chain kinase-mediated myosin II contractility and is necessary for ␣5␤1-integrin activation and leading edge adhesion. Localized myosin II activation induced by spatially activated small GTPase Rho, and its downstream kinase p160-ROCK, as previously reported, leads to contraction of actin-myosin II complexes at the trailing edge, causing it to de-adhere. Our data identify a key biomechanical mechanism for persistent cell polarity and motility. (Blood. 2010;116(17):3297-3310)

Abbreviations used in this paper: bFGF, basic FGF; E-cadherin, epithelial cadherin; ESC, embryonic stem cell; hE/Fc, E-cadherin/Fc proteins; hESC, human ESC; hiPSC, human iPSC; iPSC, induced pluripotent stem cell; MEF, mouse embryonic fibroblast; mESC, mouse ESC; mTOR, mammalian target of rapamycin.

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