The Rac-Activating Toxin Cytotoxic Necrotizing Factor 1 Oversees NK Cell-Mediated Activity by Regulating the Actin/Microtubule Interplay

Abstract

The cell cytoskeleton is widely acknowledged as a master for NK cell function. Specifically, actin filaments guide the NK cell binding to target cells, engendering the formation of the so-called immunological synapse, while microtubules direct the killer behavior. All these cytoskeleton-dependent activities are competently governed by the Rho GTPases, a family of regulatory molecules encompassing the three different subfamilies, Rho, Rac, and Cdc42. By using a Rac GTPase-activating bacterial protein toxin from Escherichia coli named cytotoxic necrotizing factor 1 (CNF1), we obtained results supporting the activation of Rac GTPase as a booster for effector cell-binding efficiency, recruitment ability, and, consequently, cytotoxicity. In particular, the augmented killer capacity of CNF1-treated NK cells was associated with the increased expression of certain cell adhesion or activation-associated molecules and the reshaping of the actin and microtubule networks. Importantly, CNF1 counteracted the activity exerted by toxins disrupting the cytoskeletal architecture. Hence, the activation of Rho GTPases, particularly Rac, induced by CNF1, appears to orchestrate a dynamic cross talk between microtubules and actin filaments, leading to a fruitful NK cell activity and polarization state. Our findings suggest that protein toxins might be viewed as modulators of NK cell cytotoxic activity and could possibly be regarded as useful pharmacological tools for certain Rho-linked immune diseases in the near future.

Fas Death Receptor Enhances Endocytic Membrane Traffic Converging into the Golgi Region

“…The small Rho GTPase CDC42 predominantly drives Fasstimulated membrane traffic in T cells, for the following reasons: 1) it promotes the selective traffic of GPI-anchored proteins such as CD59 (Sabharanjak et al, 2002;Mayor and Pagano, 2007), which we found to be rapidly altered after Fas activation (Figures 4 -7); 2) it is activated early after Fas stimulation (Subauste et al, 2000), as confirmed in our experiments (unpublished data); 3) it is selectively inhibited by the semisynthetic compound secramine A (Pelish et al, 2006), which abolishes the alteration of CD59 traffic ( Figures 6 and 7); 4) it is upstream of Rac in promoting filopodia and other surface protrusions, which are blocked by C. difficile toxin B (Malorni et al, 2003), together with the reduction in CD59 traffic alterations ( Figure 6); 5) it is the central regulator of cell polarity (Etienne-Manneville, 2004), and we found that Fas stimulation increases the polarization of membrane traffic toward the Golgi region (Figures 2-7); and 6) when expressed in its constitutively active form, it promotes cell death in Jurkat cells (Chuang et al, 1997), and we found that its inhibition with secramine reduces incipient cell death ( Figure 9C).…”

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

“…The evidence presented clearly demonstrates that CNF1 is able to perturb the physiological actin cytoskeleton organization in myogenic cells, as occurs in other cell types, 20,21 and to dramatically impair myotube formation upon differentiative stimuli. (e-h) Fluorescence microscopy analysis of C2C12 cells co-stained with fluoresceine-conjugated-phalloidin, to define F-actin organization, and with the nuclear dye DAPI.…”

“…In the presence of a high concentration of serum, these cells proliferate as an undifferentiated population, while following serum deprivation they permanently withdraw from the cell cycle, undergo terminal differentiation and fuse into multinucleated myotubes. It is known that CNF1 causes an impressive rearrangement of the actin cytoskeleton in most cell lines studied so far, 20,21 thus representing an extremely powerful tool to study those cellular functions that rely on actin network dynamics. Hence, our investigation started by analyzing the effects of CNF1 on the ultrastructure and actin cytoskeleton organization of C2C12 cells, being the cytoskeleton architecture crucial during the muscle cell differentiation.…”

“…17 Due to its peculiar activating property on Rho GTPases, CNF1 has already contributed to the investigation of relevant cellular processes, such as macropinocytosis, 18 cell motility 19 or NKmediated cytotoxicity. 20 Therefore, in the present study, we have utilized CNF1 as a tool to modulate Rho GTPase activity in the mouse myogenic cell line C2C12, and analyzed how it could influence muscle cell differentiation in relationship to the differential activation of the Rho family members as compared with the physiological process. The aim was to contribute to the definition of the puzzling role played by the Rho GTPases in muscle cells.…”

Defective Chemokine-Directed Lymphocyte Migration and Development in the Absence of Rho Guanosine Diphosphate-Dissociation Inhibitors α and β

“…For example, the chemokine CXCL12 (SDF-1␣) induces Rac activation that mediated microvillar breakdown, leading to T lymphocyte emigration and a fruitful NK cell activity and polarization state (10). CXCL12 also controls efficient up-regulation of the integrin 41-dependent T lymphocyte adhesion (11).…”