Membrane Ruffling Research Articles

Septins regulate junctional integrity of endothelial monolayers.

Junctional integrity of endothelial monolayers is crucial to control movement of molecules and cells across the endothelium. Examining the structure and dynamics of cell junctions in endothelial monolayers, we discovered a role for septins. Contacts between adjacent endothelial cells were dynamic, with protrusions extending above or below neighboring cells. Vascular endothelial cadherin (VE-cadherin) was present at cell junctions, with a membrane-associated layer of F-actin. Septins localized at cell-junction membranes, in patterns distinct from VE-cadherin and F-actin. Septins assumed curved and scallop-shaped patterns at junctions, especially in regions of positive membrane curvature associated with actin-rich membrane protrusions. Depletion of septins led to disrupted morphology of VE-cadherin junctions and increased expression of VE-cadherin. In videos, septin-depleted cells displayed remodeling at cell junctions; regions with VE-cadherin were broader, and areas with membrane ruffling were wider. Septin depletion and junction disruption led to functional loss of junctional integrity, revealed by decreased transendothelial electric resistance and increased transmigration of immune cells. We conclude that septins, as cytoskeletal elements associated with the plasma membrane, are important for cell junctions and junctional integrity of endothelial monolayers, functioning at regions of positive curvature in support of actin-rich protrusions to promote cadherin-based cell junctions.

Abstract 100: High-throughput Screening of FDA-approved Drugs Identifies Novel Inhibitors of Macropinocytosis

Aims: Macropinocytosis has been implicated in atherosclerosis, cancer, allergic disorders, and other pathologies. Unfortunately, most currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. The goal of the present study was to perform a high-throughput screen (HTS) of FDA-approved drugs to identify new, clinically relevant inhibitors of macropinocytosis. Methods: In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC 50 values, and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis, clathrin- and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to investigate the mechanisms by which selected compounds inhibit macropinocytosis. Results: The HTS campaign identified 14 compounds that at ~10 μM concentration inhibit >95% of macropinocytotic solute internalization. Our results demonstrated that three lead compounds, namely imipramine, phenoxybenzamine, and vinblastine, potently inhibit (IC 50 ≤ 130 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Mechanistically, we found that imipramine inhibits translocation of ADP-ribosylation factor 6 to the plasma membrane and prevents membrane ruffle formation, a critical early step leading to macropinocytosis. Imipramine inhibited macropinocytosis in multiple cell types, including cancer cells, dendritic cells, and macrophages. Finally, incubation of macrophages with imipramine inhibited nLDL macropinocytosis and foam cell formation in vitro . Innovation and Conclusion: The identified macropinocytosis inhibitors may prove useful as new pharmacological tools to more fully discern the role of macropinocytosis in pathological processes and as therapeutic agents in various disorders involving macropinocytosis.

Rab34 regulates adhesion, migration, and invasion of breast cancer cells.

The small GTPase Rab34 regulates spatial distribution of the lysosomes, secretion, and macropinocytosis. In this study, we found that Rab34 is over-expressed in aggressive breast cancer cells, implying a potential role of Rab34 in breast cancer. Silencing Rab34 by shRNA inhibits cell migration, invasion, and adhesion of breast cancer cells. Rab34 specifically binds to the cytoplasmic tail of integrin β3, and depletion of Rab34 promotes the degradation of integrin β3. Interestingly, EGF induces the translocation of Rab34 to the membrane ruffle, which is greatly enhanced by the expression of Src kinase. Accordingly, Rab34 is tyrosine phosphorylated by Src at Y247 residue. A mutant mimicking phosphorylated form of Rab34 (Rab34Y247D) promotes cell migration and invasion. Importantly, the tyrosine phosphorylation of Rab34 is inhibited in cells in suspension, and increased with the cells re-adhesion. In addition, Rab34Y247D promotes cell adhesion, and enhances integrin β3 endocytosis and recycling. The results uncover a role of Rab34 in migration and invasion of breast cancer cells and its involvement in cancer metastasis, and provide a novel mechanism of tyrosine phosphorylation of Rab34 in regulating cell migration, invasion, and adhesion through modulating the endocytosis, stability, and recycling of integrin β3.

Immunolocalization of Hsc70 at Bacterially‐Generated Actin‐Rich Structures

The actin cytoskeleton is commonly hijacked by bacterial pathogens. Listeria commandeers this filament system during their internalization, and for actin‐rich comet tail formation that enable the microbes to move within the infected cells as well as transfer from cell to cell. Enteropathogenic Escherichia coli (EPEC), an extracellular microbe, controls this cytoskeletal system when they generate E. coli pedestals as part of their firm docking to the plasma membrane of their host's cells and as they “surf” atop the infected cells. Finally, Salmonella exploit the host actin cytoskeleton to enter eukaryotic cells by forcing the generation of actin‐based membrane ruffling. We recently identified Hsc70 in a proteomics screen of EPEC pedestal components. This protein is normally involved with chaperoning functions within the cell. Despite there being a lack of evidence for an association of Hsc70 and actin, due to our proteomics identification of Hsc70 at EPEC pedestals we hypothesized that Hsc70 could be a common target of bacterial pathogens that control the actin cytoskeleton during their disease processes. To test this hypothesis we immunolocalized Hsc70 during Listeria, EPEC and Salmonella infections and found it concentrated at all actin structures generated by the microbes, demonstrating that this protein is a universal component for bacterial actin‐based structures.Support or Funding InformationFunding provided through NSERC. (Grant No. 355316 to J. A. G.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterizing the adult Hematopoietic Stem cell (HSC) niche in a zebrafish model for fetal bone marrow

Hematopoietic stem and progenitor cells (HSPCs) originate from the dorsal aorta and migrate to the intermediate niche, the fetal liver, where they undergo rapid expansion before migrating to the prospective adult niche, the bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish embryos can be used to directly visualize HSPC‐niche cell interactions unperturbed because they develop externally and are transparent. Importantly, their HSC ontogeny is highly similar to mammals. The zebrafish kidney is equivalent to the adult bone marrow niche and gets colonized from 4 to 5 days post fertilization (dpf). To track HSPCs during this dynamic process, we used our previously characterized HSPC‐specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation that these cells mark a highly pure population of long‐term engrafting hematopoietic stem cells. Using a tissue clearing technique that allows visualization of deeper tissues, we detected ~100 HSPCs in the kidney marrow in 5 dpf embryos. These cell numbers could be manipulated through drug treatments and gene knockdowns. For live‐imaging of the kidney marrow, we applied lightsheet microscopy that allows rapid image acquisition with high spatio‐temporal resolution, providing a highly dynamic view of the entire developing niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found that a similar cellular structure forms within the kidney marrow niche. To resolve the ultrastructure of the HSPC and its surrounding cells in context of the entire niche, we performed Correlative Light and Electron Microscopy. For this, the embryos imaged on lightsheet were fixed and stained for EM. MicroCT was used to orient and trim the embedded sample to isolate the kidney niche and serial blockface scanning EM to capture high resolution serial sections. By matching anatomical markers such as blood vessels observed in light and EM 3D data sets, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was identified lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This is suggestive of distinct anatomical niche sites within the marrow that may support different functions, such as proliferation versus quiescence. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche.Support or Funding InformationASH, NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insights into calcium-sensing receptor trafficking and biased signalling by studies of calcium homeostasis.

The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel β-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the Gq/11 pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders.

Downregulation of lumican accelerates lung cancer cell invasion through p120 catenin

The overexpression of lumican has been found in lung cancer cells; however, the functional role of lumican in lung cancer cells remains unclear. In this study, we found lumican functioned as a tubulin-binding protein and the depletion of lumican by transfection with its specific shRNA increased lung cancer cell invasion. Such alterations led to morphological changes and actin cytoskeleton remodeling, including the induction of membrane ruffling or protrusion and stress fiber formation, correlated with the increased activities of Rac and Rho. The downregulation of lumican was also implicated in macrophage-conditioned media (maCM)-induced cell invasion. Immunofluorescence images and immunoprecipitation assays revealed the co-localization of p120-catenin (p120ctn) and lumican. Reduction in the levels of p120ctn induced membrane ruffling and the activation of the Rho family, which accelerated cell invasion. Our data indicated that lumican is associated with microtubule-modulated p120ctn signaling, providing important insights into lung cancer progression.

Bradykinin mediates the association of collecting duct cells to form migratory colonies, through B2 receptor activation

It is known that bradykinin (BK) B2 receptor (B2R) is expressed in the collecting duct (CD) cells of the newborn rat kidney, but little is known about its role during early postnatal life. Therefore, we hypothesize that BK could participate in the mechanisms that mediate CD formation during the postnatal renal development. Performing primary cultures, combined with biochemical, immunocytochemical, and time-lapse analysis, we studied the role of BK in CD cell behavior isolated from renal papilla of neonatal rats. A reverse relationship was observed between B2R expression and the degree of CD epithelial cell sheet maturation. BK stimulation induced CD cell association upon B2R activation. The lack of B2R expression in cells showing mature adherens junctions suggested that BK is mostly involved in early adhesive events, thus favoring the initial formation of CD during development. Time-lapse analysis revealed that BK induced a high protrusive activity of CD cells, denoted by ruffle formation and lamellipodia extension. PI3K was involved in the BK-induced CD cell-cell association and the acquisition of the migratory phenotype since, when inhibited, membrane ruffles, and filopodia between cells diminished. Results indicate that the actions of BK mediated by PI3K activation were due to the downstream Akt and Rac pathways. This study, performed with CD cells that were not genetically manipulated, provides new experimental evidence supporting a novel role of BK in rat renal CD organization. As B2R blockade results in abnormal tubular differentiation, our results contribute to better understanding the etiology of human congenital renal malformation and diseases.

A phosphoglycolate phosphatase/AUM-dependent link between triacylglycerol turnover and epidermal growth factor signaling

Mammalian phosphoglycolate phosphatase (PGP, also known as AUM or glycerol-3-phosphate phosphatase) is a small molecule-directed phosphatase important for metabolite repair and lipid metabolism. Although PGP was first characterized as an enzyme involved in epidermal growth factor (EGF) signaling, PGP protein substrates have remained elusive. Here we show that PGP depletion facilitates fatty acid flux through the intracellular triacylglycerol/fatty acid cycle, and that phosphatidylinositol-4,5-bisphosphate (PIP2), produced in a side branch of this cycle, is critical for the impact of PGP activity on EGF-induced signaling. Loss of endogenous PGP expression amplified both EGF-induced EGF receptor autophosphorylation and Src-dependent tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1). Furthermore, EGF enhanced the formation of circular dorsal ruffles in PGP-depleted cells via Src/PLCγ1/protein kinase C (PKC)-dependent signaling to the cytoskeleton. Inhibition of adipose triglyceride lipase normalized the increased PIP2 content, reduced EGF-dependent PLCγ1 hyperphosphorylation, and decreased the elevated dorsal ruffle formation of PGP-depleted cells. Our data explain how PGP exerts control over EGF-induced cellular protein tyrosine phosphorylation, and reveal an unexpected influence of triacylglycerol turnover on growth factor signaling.

Carboxy-terminal domain of Cas differentially modulates c-Jun expression, DNA synthesis, and membrane ruffling induced by insulin, EGF, and IGF-1

ABSTRACTp130 Crk-associated substrate (Cas) is an adaptor protein associating with many other signaling proteins and regulates a various biological processes including cell adhesion, migration, and growth factor stimulation. However, the exact functional role of Cas in growth factor signaling pathway was poorly understood. Here we investigated the role of Cas and its domains in the effects of insulin, EGF, and IGF-1 on c-Jun gene expression, DNA synthesis, cytoskeletal reorganization. We found that microinjection of anti-Cas antibody and C-terminal domain of Cas (Cas-CT) specifically inhibited EGF-induced, but not insulin- or IGF-1-induced, c-Jun expression. Cell cycle progression and cytoskeleton reorganization induced by insulin and EGF, but not by IGF-1, were inhibited by microinjected anti-Cas and Cas-CT. In contrast, microinjection of the substate domain (Cas-SD) of Cas did not have any inhibitory effects. These results revealed that the Cas-CT is differentially implicated in insulin and EGF-mediated, but not IGF-1-mediated, c-Jun expression, DNA synthesis and membrane ruffling.

The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent

Angiogenin (ANG), a member of the RNase superfamily (also known as RNase 5) has neurotrophic, neuroprotective and angiogenic activities. Recently it has also been shown to be important in stem cell homeostasis. Mutations in ANG are associated with neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal dementia (FTD). ANG is a secreted protein which is taken up by cells and translocated to the nucleus. However, the import pathway/s through which ANG is taken up is/are still largely unclear. We have characterised the uptake of ANG in neuronal, astrocytic and microglial cell lines as well as primary neurons and astrocytes using pharmacological agents as well as dominant negative dynamin and Rab5 to perturb uptake and intracellular trafficking. We find that uptake of ANG is largely clathrin/dynamin independent and microtubule depolymerisation has a marginal effect. Perturbation of membrane ruffling and macropinocytosis significantly inhibited ANG uptake suggesting an uptake mechanism similar to RNase A. Our findings shed light on why mutations which do not overtly affect RNase activity but cause impaired localization are associated with neurodegenerative disease.

Accelerated invagination of vacuoles as a stress response in chronically heat-stressed yeasts

When exposed to sublethal high temperatures, budding yeast cells can survive for a period of time; however, a sufficient amount of ubiquitin is necessary for this survival. To understand the nature of the stress, we examined the morphological changes in yeast cells, focusing on the vacuoles. Changes in vacuolar morphology were notable, and ruffled vacuolar membranes, accelerated invaginations of vacuolar membranes, and vesicle-like formations were observed. These changes occurred in the absence of Atg1, Atg9 or Ivy1 but appeared to require endosomal sorting proteins, such as Vps23, Vps24 or Pep12. Furthermore, the serial sections of the vacuoles analysed using an electron microscopic analysis revealed that spherical invaginated structures were linked together in a vacuole. Because degradation of cell surface proteins is induced from heat stress, fusion of endosomal and vacuolar membranes might occur frequently in heat-stressed cells, and yeast cells might be able to cope with a rapid increase in vacuolar surface area by such invaginations.

The SH3 Domain of Unconventional Myosin IB from E. histolytica Interacts With a GEF (EhFP10) and Regulates Phagocytosis by Affecting Actin Bundling

Unconventional myosins form the largest class of myosin Superfamily. Unlike conventional two-headed myosins, they are involved in organelle movement, vesicle trafficking, phagocytosis, pinocytosis, membrane ruffling and the formation of lamellipodia. Several isoforms of class I myosins differing in structure and function have been reported in lower as well as higher eukaryotes. E.histolytica has only two myosins. The sole unconventional EhMyosin IB is a long-tailed myosin I with a c-terminal SH3. In the present work, we have determined the crystal structure of the Sh3 domain of EhMyosin IB, in native and PEG-bound form. Structural studies of PEG-bound EhMySh3 domain revealed characteristics of its probable ligand. We have identified a GTPase exchange factor, EhFP10 with APC basic domain to be one of the binding partner of EhMySH3 and validated it through SPR studies and co-crystallization of the target peptide with the EhMySH3 domain. EhMyosin IB co-localizes with EhFP10 during pinocytosis and phagocytosis. Interaction with EhMySh3 was observed to inhibit the actin bundling activity by APC domain at high EhMySH3: APC ratios while as the concentration of APC increases, it was able to overcome the inhibition. Our findings reveal a new mode of regulation of endocytic processes in E. histolytica, a highly motile gastric pathogen.

The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution.

Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system-1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane-bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system-1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time-lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper-replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation.

Burkholderia cenocepacia Induces Macropinocytosis to Enter Macrophages.

Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.

Renitence vacuoles facilitate protection against phagolysosomal damage in activated macrophages.

As professional phagocytes, macrophages are susceptible to endolysosomal membrane damage inflicted by the pathogens and noxious particles they ingest. Whether macrophages have mechanisms for limiting such damage is not well understood. Previously, we reported a phenomenon, termed “inducible renitence,” in which lipopolysaccharide (LPS) activation of macrophages protected their endolysosomes against damage initiated by the phagocytosis of silica beads. To gain mechanistic insight into the process, we analyzed the kinetics of renitence and morphological features of LPS-activated versus resting macrophages following silica bead–mediated injury. We discovered novel vacuolar structures that form in LPS-activated but not resting macrophages following silica bead phagocytosis. Because of their correlation with renitence and damage-resistant nature, we termed these structures “renitence vacuoles” (RVs). RVs formed coincident with silica bead uptake in a process associated with membrane ruffling and macropinocytosis. However, unlike normal macropinosomes (MPs), which shrink within 20 min of formation, RVs persisted around bead-containing phagosomes. RVs fused with lysosomes, whereas associated phagosomes typically did not. These findings are consistent with a model in which RVs, as persistent MPs, prevent fusion between damaged phagosomes and intact lysosomes and thereby preserve endolysosomal integrity.

Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells.

IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.

Neuropathic pain inhibitor, RAP-103, is a potent inhibitor of microglial CCL1/CCR8

Chemokine signaling is important in neuropathic pain, with microglial cells expressing chemokine (C-C motif) receptor CCR2, CCR5 and CCR8, all playing key roles. In the previous report (Padi et al., 2012), oral administration of a short peptide, RAP-103, for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rodents. As for the mechanism of the inhibiting effect of RAP-103, it was speculated to be due to dual blockade of CCR2 and CCR5. We report here that RAP-103 exhibits stronger antagonism for CCR8 (half maximal inhibitory concentration [IC50] 7.7 fM) compared to CCR5 (IC50 < 100 pM) in chemotaxis using primary cultured mouse microglia. In addition, RAP-103 at a concentration of 0.1 pM completely inhibits membrane ruffling and phagocytosis induced by chemokine (C-C motif) ligand 1 (CCL1), an agonist for CCR8. It has been shown that CCL1/CCR8 signaling is important in tactile allodynia induced by nerve ligation. Therefore, CCR8, among other chemokine receptors such as CCR2/CCR5, could be the most potent target for RAP-103. Inhibitory effects of RAP-103 on plural chemokine receptors may play important roles for broad clinical use in neuropathic pain treatment.

Cobl-like promotes actin filament formation and dendritic branching using only a single WH2 domain.

Local actin filament formation powers the development of the signal-receiving arbor of neurons that underlies neuronal network formation. Yet, little is known about the molecules that drive these processes and may functionally connect them to the transient calcium pulses observed in restricted areas in the forming dendritic arbor. Here we demonstrate that Cordon-Bleu (Cobl)-like, an uncharacterized protein suggested to represent a very distantly related, evolutionary ancestor of the actin nucleator Cobl, despite having only a single G-actin-binding Wiskott-Aldrich syndrome protein Homology 2 (WH2) domain, massively promoted the formation of F-actin-rich membrane ruffles of COS-7 cells and of dendritic branches of neurons. Cobl-like hereby integrates WH2 domain functions with those of the F-actin-binding protein Abp1. Cobl-like-mediated dendritic branching is dependent on Abp1 as well as on Ca2+/calmodulin (CaM) signaling and CaM association. Calcium signaling leads to a promotion of complex formation with Cobl-like's cofactor Abp1. Thus, Ca2+/CaM control of actin dynamics seems to be a much more broadly used principle in cell biology than previously thought.

Cell membrane dynamics induction using optogenetic tools

Structures arising from actin-based cell membrane movements, including ruffles, lamellipodia, and filopodia, play important roles in a broad spectrum of cellular functions, such as cell motility, axon guidance in neurons, wound healing, and micropinocytosis. Previous studies investigating these cell membrane dynamics often relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies did not allow the modulation of protein activity at specific regions of cells, tissues, and organs in animals with high spatial and temporal precision. Recently, optogenetic tools for inducing cell membrane dynamics have been developed which address several disadvantages of previous techniques. In a recent study, we developed a powerful optogenetic tool, called the Magnet system, to change cell membrane dynamics through Tiam1 and PIP3 signal transductions with high spatial and temporal resolution. In this review, we summarize recent advances in optogenetic tools that allow us to induce actin-regulated cell membrane dynamics and unique membrane ruffles that we discovered using our Magnet system.