Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization. Infections by enteropathogenic Escherichia coli are an important cause of diarrhea linked to high infant mortality. Such bacteria attach to cells and form actin-rich structures called pedestals, which contain many proteins that play unknown functions during pedestal formation. Here we studied two nearly identical forms (isoforms) of Crk adaptor proteins, CrkII and CrkL, during pedestal formation. Eliminating both isoforms from the cell enhanced pedestal formation, while eliminating only one did not, implying that the isoforms are redundant inhibitors of pedestal formation. We also found that Crk proteins bind the bacterial protein Tir, which binds another adaptor, Nck, to promote actin polymerization in pedestals. We propose that Crk adaptor proteins inhibit actin polymerization by competing with Nck binding to Tir. This work opens the door to investigating how Crk adaptor proteins may participate in numerous actin polymerization pathways.

The Crk adaptor family of proteins comprises the alternatively spliced CrkI and CrkII isoforms, as well as the paralog Crk-like (CrkL) protein, which is encoded by a different gene. Initially thought to be involved in signaling during apoptosis and cell adhesion, this ubiquitously expressed family of proteins is now known to play essential roles in integrating signals from a wide range of stimuli. In this review, we describe the structure and function of the different Crk proteins. We then focus on the emerging roles of Crk adaptors during Enterobacteriaceae pathogenesis, with special emphasis on the important human pathogensSalmonella,Shigella,Yersinia, and enteropathogenicEscherichia coli. Throughout, we remark on opportunities for future research into this intriguing family of proteins.

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

The human neocortex represents a crucial evolutionary advance, the formation of which requires the tight and precise orchestration of both intracellular and extracellular signals. Structures grown in three-dimensional cultures, specifically human-induced pluripotent stem cells (hIPSCs)-derived cerebral organoids (COs), have been fundamental to study the signals that regulate the formation of the cortex, overcoming the limitations of 2D cultures. Amongst these, purinergic signaling driven by extracellular ATP and other nucleotides may encode crucial intercellular communications that govern central nervous system (CNS) development. The ATP that accumulates in the extracellular milieu can interact with both ionotropic P2X and metabotropic P2Y receptors on cells to exert its modulating effects. Although widely studied in different animal models, little is known about the expression and function of this signaling system in the human cortex. Thus, here we analyzed the expression of P2X receptor subunits comprehensively throughout the entire process of CO development, confirming that P2X receptors are functional in ventricular structures of the human cortex. Specifically, we detected the expression of P2X1, P2X4, and P2X6 in CO, showing distinct distributions in Nestin⁺ radial glial cells and/or DCX⁺ newborn neurons. Significantly, we also show how prolonged pharmacological inhibition of P2X activity affects CO development, resulting in smaller organoids with fewer and less well-organized cortical ventricles. Altogether, our findings point to a relevant role of purinergic signaling during the formation of the human cerebral cortex.