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AVI file - 4312K, Time-lapse video-recording of a representative JVM3 cell expressing GFP-CCR7 distribution and migrating in a CCL19 gradient (source of CCL19 towards the bottom).

AVI file - 503K, Time-lapse video-recording of a representative JVM3 cells exposed to a CCL19 gradient (source of CCL19 towards the bottom) over a period of 5 min.

AVI file - 1553K, Time-lapse video-recording of a representative JVM3 cell expressing GFP-actin and migrating in a CCL19 gradient (source of CCL19 towards the bottom).

PDF file - 57K, Accession numbers and amplified context sequences of the genes analyzed by quantitative RT-PCR.

PDF file - 54K, Additional information regarding CCR7 internalization.

AVI file - 1602K, Supplemental Movies 6-8 shows Time-lapse video-recording of zoomed sections of the lamellipodium of a representative JVM3 cell expressing GFP-CIP4 and migrating in a CCL19 gradient. The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

AVI file - 1134K, Time-lapse video-recording of a representative CIP4 knocked-down JVM3 cells exposed to a CCL19 gradient (source of CCL19 towards the bottom) over a period of 12 min.

PDF file - 93K, CIP4 is dispensable for CCR7 internalization (S1), CIP4 knock-down impairs the polarized morphology of JVM3 cells (S2).

AVI file - 3816K, Time-lapse video-recording of a representative JVM3 cell expressing GFP-CIP4 and migrating over a period of 37 min in a CCL19 gradient (source of CCL19 towards the bottom). The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

AVI file - 2015K, Supplemental Movies 6-8 shows Time-lapse video-recording of zoomed sections of the lamellipodium of a representative JVM3 cell expressing GFP-CIP4 and migrating in a CCL19 gradient. The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

AVI file - 2141K, Supplemental Movies 6-8 shows Time-lapse video-recording of zoomed sections of the lamellipodium of a representative JVM3 cell expressing GFP-CIP4 and migrating in a CCL19 gradient. The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

Background: Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P 2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. Methods: For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. Results: We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P 2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P 2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P 2 and the cation channel TRPML (transient receptor potential mucolipin) 1 and TPC (two pore segment channel) 2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. Conclusions: These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P 2 control granule integrity both in megakaryocytes and platelets.

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

Clathrin-mediated endocytosis is a paradigm of a cellular processes that is orchestrated by phosphoinositides, where the plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in the formation of clathrin-coated structures. Among the early endocytic proteins that reach at the plasma membrane, the F-BAR domain only protein 1 and 2 complex (FCHo1/2) is thought to orchestrate the early stages but, the exact mechanism triggering its nucleation on membranes is not well understood. By combining bottom-up synthetic approaches on in vitro and cellular membranes, we show the molecular dynamics of FCHo2 self-assembly on membranes. Our results indicate that PI(4,5)P2 guide the recruitment of FCHo2 at specific regions of the membrane, where it self-assembles into ring-like shape protein patches flanked by membrane invaginations. Importantly, we show that binding of FCHo2 on cellular membranes promotes the enrichment of PI(4,5)P2 at the boundary of specific cargo receptors, such as the epidermal growth factor receptor (EGFR). Thus, our results provide a mechanistic framework that could explain the orchestration of early PI(4,5)P2-interacting proteins recruitment at endocytic sites.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3‐kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell–cell junctions by increasing Rab11‐dependent VE‐cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.

The emergence of Digital Twins in manufacturing enables in-depth integration of the physical and informational worlds. Meanwhile, the concept of Reconfigurable Manufacturing Systems exhibits new opportunities and new constraints in the way the manufacturing systems are designed and managed. Among others, the notion of modularity is a prerequisite for both physical components and their informational counterparts. To provide the benefits of Digital Twin to Reconfigurable Manufacturing Systems, this paper introduces a modular design of the Digital Twin counterpart of the components. A prototype system is developed to demonstrate the modular design’s benefits, with an application on a reconfigurable learning factory.

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.

Phosphoinositides (PIPs) are lipid messengers with different functions according to their localization. After their local production by the action of lipid kinases or phosphatases, PIPs regulate various biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, or gene expression through binding of their phosphorylated inositol head group with different protein domains such as PH, PX, and FYVE. It is well known that PIPs regulate the activity of small GTPases by interacting with and activating Guanyl-nucleotide Exchange Factor (GEF) proteins through specific domains such as the ones mentioned above. However, most of the in vitro assays to assess the activation of GTPases focus on the GTPase only and neglect the fact that co-activators, such as membranes and protein activators, have a significant effect in vivo. Herein, we describe not only the classical protein-lipid overlay and liposome sedimentation methods but also an assay we have developed, which contains three partners: a liposome which composition reproduces the membrane of the target of the GTPase, the recombinant specific DH-(PIP affinity) GEF domain, and the recombinant GTPase to be tested by different PIPs. This assay allows us to clearly quantify the GTPase activation.

Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, β, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2β), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.

International audience; Membrane shape is a key feature of many cellular processes, including cell differentiation, division, migration, and trafficking. The development of nanostructured surfaces allowing for the in situ manipulation of membranes in living cells is crucial to understand these processes, but this requires complicated and limited-access technologies. Here, we investigate the self-organization of cellular membranes by using a customizable and benchtop method allowing one to engineer 1D SiO2 nanopillar arrays of defined sizes and shapes on high-performance glass compatible with advanced microscopies. As a result of this original combination, we provide a mapping of the morphology-induced modulation of the cell membrane mechanics, dynamics and steady-state organization of key protein complexes implicated in cellular trafficking and signal transduction.

Our knowledge on the role of the different lipid messengers produced by phosphoinositide 3-kinases (PI3Ks) in normal and cancer cells as well as in platelets during arterial thrombosis has greatly expanded these last 15 years. PI3Ks are a family of lipid kinases that catalyze the phosphorylation of the D3 position of the inositol ring of phosphoinositides to produce phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol-3,4,5 trisphosphate (PtdIns(3,4,5)P3). These D3-phosphoinositides act as intracellular messengers recruiting effector proteins involved in the control of diverse cellular functions including survival, proliferation, migration, membrane trafficking, and cytoskeleton dynamics. The current idea is that the different isoforms of PI3Ks produce specific pools of lipids that regulate in time and space, at the membrane/cytosol interface, the formation of appropriate functional protein complexes. Dysregulation of PI3K-dependent pathways is directly involved in the etiology of several pathologies including cancers where the PI3K/AKT/mTORC1 axis is frequently aberrantly activated. Moreover, PtdIns(3,4,5)P3 production has been shown to play an essential role in platelet functions, particularly in the formation of a stable platelet thrombus at high shear rate. Therefore, PI3Ks are attractive therapeutic targets in the treatment of cancer and arterial thrombosis. In this review, we will discuss the role of the different lipid products of PI3K isoforms in the context of cancer and thrombosis and the development of selective PI3Ks inhibitors in the treatment of these diseases.

Membrane shape is a key feature of many cellular processes, including cell differentiation, division, migration, and trafficking. The development of nanostructured surfaces allowing for the in situ manipulation of membranes in living cells is crucial to understand these processes, but this requires complicated and limited-access technologies. Here, we investigate the self-organization of cellular membranes by using a customizable and bench top method allowing to engineer 1D SiO2 nanopillar arrays of defined sizes and shapes on high-performance glass compatible with advanced microscopies. As a result of this original combination, we provide a mapping of the morphology-induced modulation of the cell membrane mechanics, dynamics and steady-state organization of key protein complexes implicated in cellular trafficking and signal transduction.

By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field.

UNLABELLED Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Forster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP2 and PIP3 molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110β deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110β and the more subtle role of p110α in the production of PIP3 molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP2 during stimulation, opening new perspectives in phosphoinositide signaling in platelets.

Within the glycerophospholipid family, phosphoinositides, which are minor components of eukaryotic cell membranes, play a critical role as spatiotemporal organizers of cell dynamics. By specifically interacting with proteins, they coordinate the formation and the organization of multiprotein complexes involved in cell signaling, intracellular trafficking and cytoskeleton rearrangement. The highly precise spatiotemporal dynamics of phosphoinositides-regulated mechanisms is ensured by kinases and phosphatases that specifically produce, hydrolyze and control the interconversion of these lipids. The direct implication of these enzymes in human pathologies such as genetic diseases, cancer or infectious pathologies, and the recent arrival of inhibitors targeting some phosphoinositide kinases in clinic, illustrate the mandatory functions of these fascinating lipids.

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein-Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities. In addition, PLIF is suitable for screening inhibitors of protein- phosphoinositide interaction, allowing identification of potential pharmacological compounds. © 2017 by John WileySons, Inc.

Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein-Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities. In addition, PLIF is suitable for screening inhibitors of protein- phosphoinositide interaction, allowing identification of potential pharmacological compounds. © 2017 by John Wiley & Sons, Inc.

The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1–UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1–UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation. Gavriilidis et al. show that MTM1, which is mutated in X-linked centronuclear myopathy, and UBQLN2 recognize misfolded desmin and vimentin and trigger their degradation to clear misfolded intermediated filaments prior to aggregate formation.

Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors.

Autophagy is a cellular process whereby cytoplasmic substrates are targeted for degradation in the lysosome via the membrane structures autophagosomes. This process is initiated by specific phosphoinositides, PtdIns3P and PtdIns5P, which play a key role in autophagy by recruiting effectors such as Atg18/WIPI2. Therefore, quantifying those lipids is important to better understand the assembly of the complex autophagic machinery. Herein, we describe in detail methods to quantify PtdIns3P and PtdIns5P by specific mass assays feasible in most laboratories.

Phospholipid-enriched membranes such as the plasma membrane can serve as direct regulators of kinase signaling. Pak1 is involved in growth factor signaling at the plasma membrane, and its dysregulation is implicated in cancer. Pak1 adopts an autoinhibited conformation that is relieved upon binding to membrane-bound Rho GTPases Rac1 or Cdc42, but whether lipids also regulate Pak1 in vivo is unknown. We show here that phosphoinositides, particularly PIP(2), potentiate Rho-GTPase-mediated Pak1 activity. A positively charged region of Pak1 binds to phosphoinositide-containing membranes, and this interaction is essential for membrane recruitment and activation of Pak1 in response to extracellular signals. Our results highlight an active role for lipids as allosteric regulators of Pak1 and suggest that Pak1 is a "coincidence detector" whose activation depends on GTPases present in phosphoinositide-rich membranes. These findings expand the role of phosphoinositides in kinase signaling and suggest how altered phosphoinositide metabolism may upregulate Pak1 activity in cancer cells.

Within the glycerophospholipid family, phosphoinositides, which are minor components of eukaryotic cell membranes, play a critical role as spatiotemporal organizers of cell dynamics. By specifically interacting with proteins, they coordinate the formation and the organization of multiprotein complexes involved in cell signaling, intracellular trafficking and cytoskeleton rearrangement. The highly precise spatiotemporal dynamics of phosphoinositides-regulated mechanisms is ensured by kinases and phosphatases that specifically produce, hydrolyze and control the interconversion of these lipids. The direct implication of these enzymes in human pathologies such as genetic diseases, cancer or infectious pathologies, and the recent arrival of inhibitors targeting some phosphoinositide kinases in clinic, illustrate the mandatory functions of these fascinating lipids.

Cyclophilin A is involved in many cellular processes, including protein folding and intracellular transports. Because cyclophilin A has been shown to interact with HIV-1 gag proteins and to enhance the viral infectivity, nonimmunosuppressive cyclophilin A ligands may represent a new class of therapeutic agents against HIV. Here, we report a virtual screening using structure- and pharmacophore-based design to identify original nonpeptidic cyclophilin ligands. Following a lead identification of compounds 1 [1-(3-benzyloxypyridin-2-yl)-3-(3-chlorophenyl)urea] and 2 [1-(3-benzyloxypyridin-2-yl)-3-(3-trifluoromethylphenyl)urea] (IC(50) = 0.3 microM), a series of molecules were synthesized from a diarylurea scaffold and evaluated for their in vitro ability to inhibit the cis-trans isomerase activity of cyclophilin A. Molecular modifications provided several more potent compounds, in particular analogues 4d and 4i with IC(50) of 14 and 20 nM, respectively. Then, we evaluated the effect of analogues 1 and 2 on HIV virion infectivity in both immortalized and primary cells. Both 1 and 2 reduced virion infectivity in the replication-defective one-round infection assay, but only 1 impaired wild-type HIV infection in human peripheral blood mononuclear cells.

International audience; Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

PtdIns5P is a lipid messenger acting as a stress-response mediator in the nucleus, and known to maintain cell activation through traffic alterations upon bacterial infection. Here, we show that PtdIns5P regulates actin dynamics and invasion via recruitment and activation of the exchange factor Tiam1 and Rac1. Restricted Rac1 activation results from the binding of Tiam1 DH-PH domains to PtdIns5P. Using an assay that mimics Rac1 membrane anchoring by using Rac1-His and liposomes containing Ni(2+)-NTA modified lipids, we demonstrate that intrinsic Tiam1 DH-PH activity increases when Rac1 is anchored in a PtdIns5P-enriched environment. This pathway appears to be general since it is valid in different pathophysiological models: receptor tyrosine kinase activation, bacterial phosphatase IpgD expression and the invasive NPM-ALK(+) lymphomas. The discovery that PtdIns5P could be a keystone of GTPases and cytoskeleton spatiotemporal regulation opens important research avenues towards unravelling new strategies counteracting cell invasion.

Protein kinases are among the most important drug targets; however the structural conservation of the ATP-binding pocket of kinases can lead to promiscuous inhibition of additional unintended kinase targets. Allosteric inhibitors that target less conserved regions of protein kinases represent an alternative approach that may provide more selective kinase inhibition. In this report, protocols are provided for the screening and identification of Pak1 inhibitors acting via an allosteric mechanism.

Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.

International audience; Rho GTPases act as molecular switches central in cellular processes such as cytoskeleton dynamics, migration, cell proliferation, growth or survival. Their activation is tightly regulated downstream of cell surface receptors by Guanine nucleotide Exchange Factors (GEFs), that are responsible for the specificity, the accuracy, and the spatial restriction of Rho GTPases response to extracellular cues. Because there is about four time more RhoGEFs that Rho GTPases, and GEFs do not always show a strict specificity for GTPases, it is clear that their regulation depends on specific interactions with the subcellular environment. RhoGEFs bear a peculiar structure, highly conserved though evolution, consisting of a DH-PH tandem, the DH (Dbl homology) domain being responsible for the exchange activity. The function of the PH (Pleckstrin homology) domain known to bind phosphoinositides, however, remains elusive, and reports are in many cases rather confusing. This review summarizes data on the regulation of RhoGEFs activity through interaction of the PH-associated DH domain with phosphoinositides which are considered as critical players in the spatial organization of major signaling pathways.