Membrane Ruffling Research Articles

Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis

A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunofluorescence and immunohistochemistry. Taking advantage of prostate and pancreatic cancer cell models known to have high basal Rac1-GTP levels, here we have established that this antibody does not recognize Rac1 but rather detects the intermediate filament protein vimentin. Indeed, Rac1-null PC3 prostate cancer cells or cancer models with low levels of Rac1 activation still show a high signal with the anti-Rac1-GTP antibody, which is lost upon silencing of vimentin expression. Moreover, this antibody was unable to detect activated Rac1 in membrane ruffles induced by epidermal growth factor stimulation. These results have profound implications for the study of this key GTPase in cancer, particularly because a large number of cancer cell lines with characteristic mesenchymal features show simultaneous up-regulation of vimentin and high basal Rac1-GTP levels when measured biochemically. This misleading correlation can lead to assumptions about the validity of this antibody and inaccurate conclusions that may affect the development of appropriate therapeutic approaches for targeting the Rac1 pathway.

Secreted gingipains from Porphyromonas gingivalis induce microglia migration through endosomal signaling by protease-activated receptor 2

Much attention has been paid to the connection between periodontitis and Alzheimer's disease (AD). We previously showed that infection of P. gingivalis, one of major periodontal pathogen causing periodontitis, induced migration and inflammatory responses in murine microglia through gingipain-induced activation of protease-activated receptor 2 (PAR2). In this study, we have attempted to clarify effects of secreted gingipains on cell migration of human microglial cell line, cleavage sites of PAR2 by gingipains and subsequent signaling pathways. P. gingivalis culture supernatant induced migration and membrane ruffling, which is necessary for microglia migration, in human microglial cell line HMC3 cells through PAR2. These effects were mainly mediated by gingipains, because cell migration and membrane ruffling were dramatically inhibited by treatment with gingipain inhibitors. Furthermore, pharmacological and genetic inhibition of Src kinase and β-arrestin, which are important for the internalization of G protein-coupled receptors, significantly inhibited P. gingivavlis culture supernatant-induced membrane ruffling in HMC3 cells. After treatment with P. gingivalis culture supernatant in Flag-PAR2-HA transfected HEK293T cells, Flag was removed from the cell surface, and HA was detected in the cytosol, indicating the internalization of PAR2. Furthermore, the phosphorylation level of ERK1/2 increased in PAR2-transfected HEK293T cells after treatment with P. gingivalis culture supernatant. The gingipain inhibitors, Src kinase inhibitor and β-arrestin knockdown suppressed PAR2 internalization and ERK1/2 phosphorylation. These observations suggest that secreted gingipains from P. gingivalis induce Src- and β-arrestin-dependent internalization of PAR2 and further activate the ERK1/2 pathway to promote migration of microglia. PAR2 are activated by the tethered ligands exposed by cleavage of extracellular N-terminal of PAR2. We also estimated potential gingipain cleavage sites in PAR2 and exposed tethered ligands, which are required for PAR2 internalization and membrane ruffling. The identified mechanism in this study might contribute to the retrogression of sporadic AD in patients after infection with P. gingivalis.

Novel function for AP-1B during cell migration.

The epithelial cell-specific clathrin adaptor protein (AP)-1B has a well-established role in polarized sorting of cargos to the basolateral membrane. Here we show that β1 integrin was dependent on AP-1B and its coadaptor, autosomal recessive hypercholesterolemia protein (ARH), for sorting to the basolateral membrane. We further demonstrate an unprecedented role for AP-1B at the basal plasma membrane during collective cell migration of epithelial sheets. During wound healing, expression of AP-1B (and ARH in AP–1B-positive cells) slowed epithelial-cell migration. We show that AP-1B colocalized with β1 integrin in focal adhesions during cell migration using confocal microscopy and total internal reflection fluorescence microscopy on fixed specimens. Further, AP-1B labeling in cell protrusions was distinct from labeling for the endocytic adaptor complex AP-2. Using stochastic optical reconstruction microscopy we identified numerous AP–1B-coated structures at or close to the basal plasma membrane in cell protrusions. In addition, immunoelectron microscopy showed AP-1B in coated pits and vesicles at the plasma membrane during cell migration. Lastly, quantitative real-time reverse transcription PCR analysis of human epithelial-derived cell lines revealed a loss of AP-1B expression in highly migratory metastatic cancer cells suggesting that AP-1B’s novel role at the basal plasma membrane during cell migration might be an anticancer mechanism.

Complement receptor 3 mediates ruffle-like, actin-rich aggregates during phagocytosis of Leishmania infantum metacyclics

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes.

Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL

The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion.

Two Japanese pepper (Zanthoxylum piperitum) fruit-derived compounds attenuate IgE-mediated allergic response in vitro and in vivo via inhibition of mast cell degranulation

Zanthoxylum piperitum (ZP, ‘Japanese pepper’) is a traditional medicine and pepper used in Asian countries such as Japan. Hydroxy-α-sanshool, a pungent-tasting substance contained within ZP, has been reported to slightly suppress immunoglobulin E (IgE)-mediated mast cell degranulation. The current study aims to newly identify anti-allergic compounds derived from ZP. We examine the inhibitory mechanisms behind IgE-mediated mast cell degranulation. By inhibitory effect-guided isolation, we identified degranulation inhibitory compounds derived from ZP fruit: 1-acetoxy-7-hydroxy-3, 7-dimethylocta-2E, 5E-diene (ZP1) and 8-hydroxygeranyl acetate (ZP2). ZP1 and ZP2 inhibited IgE-mediated degranulation and A23187-mediated degranulation in RBL-2H3 mast cells. Our findings suggest the inhibition of degranulation by ZP1 and ZP2 was by inhibition of Lyn phosphorylation, followed by inhibition of intracellular Ca2+ mobilization, protein kinase C alpha phosphorylation, membrane ruffling, and granule-to-plasma membrane fusion. Oral administration of ZP1 or ZP2 attenuated an IgE-mediated passive cutaneous anaphylactic reaction in mice. Histological observation suggests that this effect occurred via inhibition of mast cell degranulation. These findings indicate that ZP1 and ZP2 attenuate allergic reaction via inhibition of IgE-mediated mast cell degranulation.

Cell Derived Matrix Fibulin-1 Associates With Epidermal Growth Factor Receptor to Inhibit Its Activation, Localization and Function in Lung Cancer Calu-1 Cells

Epidermal Growth Factor Receptor (EGFR) is a known promoter of tumor progression and is overexpressed in lung cancers. Growth factor receptors (including EGFR) are known to interact with extracellular matrix (ECM) proteins, which regulate their activation and function. Fibulin-1 (FBLN1) is a major component of the ECM in lung tissue, and its levels are known to be downregulated in non-small cell lung cancers (NSCLC). To test the possible role FBLN1 isoforms could have in regulating EGFR signaling and function in lung cancer, we performed siRNA mediated knockdown of FBLN1C and FBLN1D in NSCLC Calu-1 cells. Their loss significantly increased basal (with serum) and EGF (Epidermal Growth Factor) mediated EGFR activation without affecting net EGFR levels. Overexpression of FBLN1C and FBLN1D also inhibits EGFR activation confirming their regulatory crosstalk. Loss of FBLN1C and FBLN1D promotes EGFR-dependent cell migration, inhibited upon Erlotinib treatment. Mechanistically, both FBLN1 isoforms interact with EGFR, their association not dependent on its activation. Notably, cell-derived matrix (CDM) enriched FBLN1 binds EGFR. Calu-1 cells plated on CDM derived from FBLN1C and FBLN1D knockdown cells show a significant increase in EGF mediated EGFR activation. This promotes cell adhesion and spreading with active EGFR enriched at membrane ruffles. Both adhesion and spreading on CDMs is significantly reduced by Erlotinib treatment. Together, these findings show FBLN1C/1D, as part of the ECM, can bind and regulate EGFR activation and function in NSCLC Calu-1 cells. They further highlight the role tumor ECM composition could have in influencing EGFR dependent lung cancers.

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin.

Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments. In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.

Rac1 Promotes Cell Motility by Controlling Cell Mechanics in Human Glioblastoma.

The failure of existing therapies in treating human glioblastoma (GBM) mostly is due to the ability of GBM to infiltrate into healthy regions of the brain; however, the relationship between cell motility and cell mechanics is not well understood. Here, we used atomic force microscopy (AFM), live-cell imaging, and biochemical tools to study the connection between motility and mechanics in human GBM cells. It was found thatRac1 inactivation by genomic silencing and inhibition with EHT 1864 reduced cell motility, inhibited cell ruffles, and disrupted the dynamics of cytoskeleton organization and cell adhesion. These changes were correlated with abnormal localization of myosin IIa and a rapid suppression of the phosphorylation of Erk1/2. At the same time, AFM measurements of the GBM cells revealed a significant increase in cell elasticity and viscosity following Rac1 inhibition. These results indicate that mechanical properties profoundly affect cell motility and may play an important role in the infiltration of GBM. It is conceivable that the mechanical characters might be used as markers for further surgical and therapeutical interventions.

Epidermal Growth Factor Receptor and Abl2 Kinase Regulate Distinct Steps of Human Papillomavirus 16 Endocytosis.

Human papillomavirus 16 (HPV16), the leading cause of cervical cancer, exploits a novel endocytic pathway during host cell entry. This mechanism shares many requirements with macropinocytosis but differs in the mode of vesicle formation. Previous work indicated a role of the epidermal growth factor receptor (EGFR) in HPV16 endocytosis. However, the functional outcome of EGFR signaling and its downstream targets during HPV16 uptake are not well characterized. Here, we analyzed the functional importance of signal transduction via EGFR and its downstream effectors for endocytosis of HPV16. Our findings indicate two phases of EGFR signaling as follows: a-likely dispensable-transient activation with or shortly after cell binding and signaling required throughout the process of asynchronous internalization of HPV16. Interestingly, EGFR inhibition interfered with virus internalization and strongly reduced the number of endocytic pits, suggesting a role for EGFR signaling in the induction of HPV16 endocytosis. Moreover, we identified the Src-related kinase Abl2 as a novel regulator of virus uptake. Inhibition of Abl2 resulted in an accumulation of misshaped endocytic pits, indicating Abl2's importance for endocytic vesicle maturation. Since Abl2 rather than Src, a regulator of membrane ruffling during macropinocytosis, mediated downstream signaling of EGFR, we propose that the selective effector targeting downstream of EGFR determines whether HPV16 endocytosis or macropinocytosis is induced.IMPORTANCE Human papillomaviruses are small, nonenveloped DNA viruses that infect skin and mucosa. The so-called high-risk HPVs (e.g., HPV16, HPV18, HPV31) have transforming potential and are associated with various anogenital and oropharyngeal tumors. These viruses enter host cells by a novel endocytic pathway with unknown cellular function. To date, it is unclear how endocytic vesicle formation occurs mechanistically. Here, we addressed the role of epidermal growth factor receptor signaling, which has previously been implicated in HPV16 endocytosis and identified the kinase Abl2 as a novel regulator of virus uptake. Since other viruses, such as influenza A virus and lymphocytic choriomeningitis virus, possibly make use of related mechanisms, our findings shed light on fundamental strategies of virus entry and may in turn help to develop new host cell-targeted antiviral strategies.

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1h from day 0 to day 3, every 2h from day 4 to day 7, and every 1h for 24h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

Computational and in vitro Pharmacodynamics Characterization of 1A-116 Rac1 Inhibitor: Relevance of Trp56 in Its Biological Activity.

In the last years, the development of new drugs in oncology has evolved notably. In particular, drug development has shifted from empirical screening of active cytotoxic compounds to molecularly targeted drugs blocking specific biologic pathways that drive cancer progression and metastasis. Using a rational design approach, our group has developed 1A-116 as a promising Rac1 inhibitor, with antitumoral and antimetastatic effects in several types of cancer. Rac1 is over activated in a wide range of tumor types and and it is one of the most studied proteins of the Rho GTPase family. Its role in actin cytoskeleton reorganization has effects on endocytosis, vesicular trafficking, cell cycle progression and cellular migration. In this context, the regulatory activity of Rac1 affects several key processes in the course of the cancer including invasion and metastasis. The purpose of this preclinical study was to focus on the mode of action of 1A-116, conducting an interdisciplinary approach with in silico bioinformatics tools and in vitro assays. Here, we demonstrate that the tryptophan 56 residue is necessary for the inhibitory effects of 1A-116 since this compound interferes with protein-protein interactions (PPI) of Rac1GTPase involving several GEF activators. 1A-116 is also able to inhibit the oncogenic Rac1P29S mutant protein, one of the oncogenic drivers found in sun-exposed melanoma. It also inhibits numerous Rac1-regulated cellular processes such as membrane ruffling and lamellipodia formation. These results deepen our knowledge of 1A-116 inhibition of Rac1 and its biological impact on cancer progression. They also represent a good example of how in silico analyses represent a valuable approach for drug development.

Altered oligomeric states in pathogenic ALS2 variants associated with juvenile motor neuron diseases cause loss of ALS2‐mediated endosomal function

ALS2, the causative gene product for a number of recessive motor neuron diseases, forms a homophilic oligomer and acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5. This oligomerization is crucial for both Rab5 activation and ALS2‐mediated endosome fusion and maturation in cells. Recently, we have shown that pathogenic missense ALS2 variants retaining the Rab5 GEF activity fail to properly localize to endosomes via Rac1‐stimulated macropinocytosis. However, the molecular mechanisms underlying dysregulated distribution of ALS2 variants still unknown. In this study, we clarify the relationship between intracellular localization and oligomeric states of pathogenic ALS2 variants. Upon Rac1 activation, all variants moved from the cytosol to membrane ruffles, but not to macropinosomes and/or endosomes. Further, most wild‐type ALS2 in cultured cells forms tetramers. Importantly, the sizes of an ALS2 complex carrying missense mutations in the N‐terminus of RLD or in‐frame amino acid deletion in the PH domain were shifted toward high molecular weight, whereas the C‐terminal VPS9 domain missense mutant existed as a smaller dimeric or trimeric smaller form. Furthermore, in silico mutagenesis analyses using the RLD protein structure in conjunction with a cycloheximide chase assay in vitro disclosed that these missense mutations led to decrease in the protein stability. These results suggested that disorganized higher structures of ALS2 variants might explain their impaired endosomal localization and the stability, leading to loss of the ALS2 function. Currently, to further clarify the in vivo structure, gel‐filtration analyses of the ALS2 complex in murine tissues, including the brain, spinal cord and liver, are underway.Support or Funding InformationThis work was supported by Japanese Society for Promotion of Science Grants 26290018 and 24650189 and in part by the National Natural Science Foundation of China and JSPS Bilateral Joint Research Project.

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility.

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members.

The metabolic signature of T cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prototypic autoimmune disease manifesting as chronic inflammation of the synovium and leading to acceleration of cardiovascular disease and shortening of life expectancy. The basic defect causing autoimmunity has remained elusive, but recent insights have challenged the notion that autoantigen is the core driver. Emerging data have added metabolic cues involved in the proper maintenance and activation of immune cells as pathogenic regulators. Specifically, studies have unveiled metabolic pathways that enforce T cell fate decisions promoting tissue inflammation; including T cell tissue invasiveness, T cell cytokine release, T cell-dependent macrophage activation and inflammatory T cell death. At the center of the metabolic abnormalities lies the mitochondria, which is consistently underperforming in RA T cells. The mitochondrial defect results at least partially from insufficient DNA repair and leads to lipid droplet accumulation, formation of invasive membrane ruffles, inflammasome activation and pyroptotic T cell death. T cells in patients with RA, even naïve T cells never having been involved in inflammatory lesions, have a unique metabolic signature and the changes in intracellular metabolites drive pathogenic T cell behavior. Recognizing the role of metabolic signals in cell fate decisions opens the possibility for immunomodulation long before the end stage synovial inflammation encountered in clinical practice.

ALS2, the small GTPase Rab17-interacting protein, regulates maturation and sorting of Rab17-associated endosomes

Small GTPase Rab17 has been shown to regulate a wide range of physiological processes including cell migration in tumor cells and dendrite morphogenesis in neurons. However, molecular mechanism underlying Rab17-mediated intracellular trafficking is still unclear. To address this issue, we focused on Rab17-interacting protein ALS2, which was also known as a guanine nucleotide exchange factor (GEF) for Rab5, and investigated how ALS2 contributed to Rab17-associated membrane trafficking in cells. Rab17 was primarily localized to endosomal compartments, particularly to recycling endosomes, which was dependent on Rab11 expression. Upon Rac1 activation, Rab17 along with ALS2 was recruited to membrane ruffles and early endosomes in a Rab5 activity-independent manner. While RABGEF1, another Rab17-interacting Rab5 GEF, functioned as a GEF for Rab17, ALS2 did not possess such catalytic activity but merely interacted with Rab17. Importantly, ALS2 acted downstream of RABGEF1, regulating the maturation of Rab17-residing nascent endosomes to early endosome antigen 1 (EEA1)-positive early endosomes. Further, these Rab17-residing nascent endosomes were arisen via clathrin-independent endocytosis (CIE). Collectively, ALS2 plays a crucial role in the regulation of Rab17-associated endosomal trafficking and maturation, probably through their physical interaction, in cells.

Silencing the Cytoskeleton Protein Iba1 (Ionized Calcium Binding Adapter Protein 1) Interferes with BV2 Microglia Functioning.

Iba1 (ionized calcium binding adapter protein 1) is a cytoskeleton protein specific only for microglia and macrophages, where it acts as an actin-cross linking protein. Although frequently regarded as a marker of activation, its involvement in cell migration, membrane ruffling, phagocytosis or in microglia remodeling during immunological surveillance of the brain suggest that Iba1 is not a simple cytoskeleton protein, but a signaling molecule involved in specific signaling pathways. In this study we investigated if Iba1 could also represent a drug target, and tested the hypothesis that its specific silencing with customized Iba1-siRNA can modulate microglia functioning. The results showed that Iba1-silenced BV2 microglia migrate less due to reduced proliferation and cell adhesion, while their phagocytic activity and P2x7 functioning was significantly increased. Our data are the proof of concept that Iba1 protein is a new microglia target, which opens a new therapeutic avenue for modulating microglia behavior.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro.

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental sampling, pathogen detection and immune signaling. Quantitative assessment of ruffling is important for assessing pathogen responses in macrophages and for analysis of growth factor responses in other cell types but automated and quantitative methods are lacking, and rely on manual and qualitative assessments. Here we present an automated ImageJ macro for quantifying dorsal cell surface protrusions from 3D microscope images. The assay presented here is suitable for high-throughput screening applications to detect drug, pathogen, or growth factor induced changes in cell ruffling by measuring ruffle area and intensity and providing normalized values in an easy to read combined spreadsheet.