Giulia Germena

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Summary Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

Citron kinase controls abscission through RhoA and anillin

In this report, we confirm that the RhoA-binding protein citron kinase (CIT-K) is required for midbody abscission in late cytokinesis, while it has little or no role in early cytokinesis. Moreover, we show that CIT-K, despite being commonly considered a RhoA effector, promotes midbody stability through RhoA and anillin during late cytokinesis.

The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions.

In phagocytes, GTPases of the Rac family control crucial antimicrobial functions. The RacGAP ArhGAP15 negatively modulates Rac activity in leukocytes, but its in vivo role in innate immunity remains largely unknown. Here we show that neutrophils and macrophages derived from mice lacking ArhGAP15 presented higher Rac activity but distinct phenotypes. In macrophages, the loss of ArhGAP15 induced increased cellular elongation and membrane protrusions but did not modify chemotactic responses. Conversely, the lack of ArhGAP15 in neutrophils affected critical Rac-dependent antimicrobial functions, specifically causing enhanced chemotactic responses, straighter directional migration, amplified reactive oxygen species production, increased phagocytosis, and improved bacterial killing. In vivo, in a model of severe abdominal sepsis, these effects contributed to increase neutrophil recruitment to the site of infection, thereby limiting bacterial growth, controlling infection spread, reducing systemic inflammation, and ultimately improving survival in ArhGAP15-null mice. Altogether, these results demonstrate the relevance of ArhGAP15 in the selective regulation of multiple neutrophil functions, suggesting that ArhGAP15 targeting might be beneficial in specific pathologic settings like severe sepsis.

Crossroads of PI3K and Rac pathways

Rac and PI3Ks are intracellular signal transducers able to regulate multiple signaling pathways fundamental for cell behavior. PI3Ks are lipid kinases that produce phosphorylated lipids which, in turn, transduce extracellular cues within the cell, while Rac is a small G protein that impacts on actin organization. Compelling evidence indicates that in multiple circumstances the 2 signaling pathways appear intermingled. For instance, phosphorylated lipids produced by PI3Ks recruit and activate GEF and GAP proteins, key modulators of Rac function. Conversely, PI3Ks interact with activated Rac, leading to Rac signaling amplification. This review summarizes the molecular mechanisms underlying the cross-talk between Rac and PI3K signaling in 2 different processes, cell migration and ROS production.

PI3Ks and small GTPases in neutrophil migration: Two sides of the same coin

Cell migration is a key event in physiological processes such as embryonic development, tissue repair, angiogenesis and immune responses. Alteration of the migration program is an important component in multiple pathologies, including chronic inflammation, autoimmunity and tumor metastasis. Understanding of the precise mechanisms at the basis of cellular migration may lead to the identification of novel therapeutic approach for these diseases. Recent evidences show that the interplay between the lipid kinases phosphatidylinositol 3-kinase (PI3Ks) and small GTPases play a critical role in driving cell migration. In this review we will describe the role of these molecules and the interaction between their signal cascades in leukocyte polarization and amoeboid migration.

Class I Phosphoinositide-3-Kinases and Src Kinases Play a Nonredundant Role in Regulation of Adhesion-Independent and -Dependent Neutrophil Reactive Oxygen Species Generation

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.

Dissection of the Interplay between Class I PI3Ks and Rac Signaling in Phagocytic Functions

Phagocytes, like neutrophils and macrophages, are specialized cells evolved to clear infectious pathogens. This function resides at the core of innate immunity and requires a series of concerted events that lead first to migration to the infected tissue and then to the killing of the invading pathogens. Molecular mechanisms underlying these processes are starting to emerge and point to the interplay between two families of crucial proteins: the PI3K lipid kinases and the Rac GTPases. This review focuses on how these two protein families contribute to migration, phagocytosis, and reactive oxygen species production, as well as their epistatic and feedback relations that finely tune these crucial aspects of the immune response.

Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo

Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest.

Disruption of ArhGAP15 results in hyperactive Rac1, affects the architecture and function of hippocampal inhibitory neurons and causes cognitive deficits

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

Mutation in the CD45 Inhibitory Wedge Modulates Integrin Activation and Leukocyte Recruitment during Inflammation

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. Src family kinases (SFKs) activation is required for integrin and chemokine signaling as well as immune cell function. The receptor-like protein tyrosine phosphatase CD45 positively regulates chemoattractant signaling acting on SFK activity. To further investigate the role of CD45 in neutrophil recruitment and function, we analyzed transgenic mice carrying a single point mutation (CD45E613R), which constitutively activates CD45. By using intravital microscopy experiments, we demonstrated that different steps of the leukocyte recruitment cascade were affected in CD45E613R mutant mice. The rolling velocity of CD45E613R mutant neutrophils was decreased compared with wild-type neutrophils that subsequently resulted in an increased number of adherent cells. The analysis of β2 integrins LFA-1 and macrophage-1 Ag (Mac-1) showed that in CD45E613R mutant neutrophils LFA-1 adhesiveness was impaired, and avidity was enhanced, whereas Mac-1 adhesiveness was increased. Because of the increased Mac-1 adhesiveness, neutrophil crawling was impaired in CD45E613R mutant compared with wild-type neutrophils. In an Escherichia coli lung infection model, CD45E613R mice displayed a decreased neutrophil recruitment into the alveolar compartment, which resulted in an increased number of CFUs in the lung. Our data demonstrate that the CD45E613R mutation modulates integrin activation and leukocyte recruitment during inflammation.