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1.  Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs 
PLoS ONE  2009;4(6):e5961.
Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.
Methodology/Principal Findings
We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.
Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.
PMCID: PMC2694364  PMID: 19536333
2.  Actin polymerization drives polar growth in Arabidopsis root hair cells 
Plant Signaling & Behavior  2014;9:e29401.
In plants, the actin cytoskeleton is a prime regulator of cell polarity, growth, and cytoplasmic streaming. Tip growth, as observed in root hairs, caulonema, and pollen tubes, is governed by many factors, including calcium gradients, exocytosis and endocytosis, reactive oxygen species, and the cytoskeleton. Several studies indicate that the polymerization of G-actin into F-actin also contributes to tip growth. The structure and function of F-actin within the apical dome is variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in the elongating apices of tip-growing cells suggests that this cytoskeletal array is tightly regulated. We recently reported that sublethal concentrations of fluorescently labeled cytochalasin could be used to visualize the distribution of microfilament plus ends using fluorescence microscopy, and found that the tip region of the growing root hair cells of a legume plant exhibits a clear response to the nodulation factors secreted by Rhizobium.1 In this current work, we expanded our analysis using confocal microscopy and demonstrated the existence of highly dynamic fluorescent foci along Arabidopsis root hair cells. Furthermore, we show that the strongest fluorescence signal accumulates in the tip dome of the growing root hair and seems to be in close proximity to the apical plasma membrane. Based on these findings, we propose that actin polymerization within the dome of growing root hair cells regulates polar growth.
PMCID: PMC4203500  PMID: 25763621
Root hairs; actin polymerization sites; fluorescently labeled cytochalasin D; Arabidopsis
3.  The Circular F-Actin Bundles Provide a Track for Turnaround and Bidirectional Movement of Mitochondria in Arabidopsis Root Hair 
PLoS ONE  2014;9(3):e91501.
The movement of organelles in root hairs primarily occurs along the actin cytoskeleton. Circulation and “reverse fountain” cytoplasmic streaming constitute the typical forms by which most organelles (such as mitochondria and the Golgi apparatus) in plant root hair cells engage in bidirectional movement. However, there remains a lack of in-depth research regarding the relationship between the distribution of the actin cytoskeleton and turnaround organelle movement in plant root hair cells.
In this paper, Arabidopsis seedlings that had been stably transformed with a GFP-ABD2-GFP (green fluorescent protein-actin-binding domain 2-green fluorescent protein) construct were utilized to study the distribution of bundles of filamentous (F)-actin and the directed motion of mitochondria along these bundles in root hairs. Observations with a confocal laser scanning microscope revealed that there were widespread circular F-actin bundles in the epidermal cells and root hairs of Arabidopsis roots. In root hairs, these circular bundles primarily start at the sub-apical region, which is the location where the turnaround movement of organelles occurs. MitoTracker probes were used to label mitochondria, and the dynamic observation of root hair cells with a confocal laser scanning microscope indicated that turnaround mitochondrial movement occurred along circular F-actin bundles.
Relevant experimental results demonstrated that the circular F-actin bundles provide a track for the turnaround and bidirectional movement of mitochondria.
PMCID: PMC3953408  PMID: 24626218
4.  A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division 
eLife  null;4:e08828.
Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.
eLife digest
Mitochondria are structures within cells that provide the energy to power many biological processes that are essential for complex life. These structures are also highly dynamic and go through cycles of fission (in which a single mitochondrion splits in two) and fusion (in which two mitochondria merge into one). These processes both maintain the correct number of mitochondria in a cell and remove damaged ones, and defects in either can result in many diseases.
Previous research had shown that mitochondria are in close contact with another cellular structure called the endoplasmic reticulum. The points of contact mark the sites where mitochondria undergo fission, as small tubes of the endoplasmic reticulum wrap around, and then constrict, to split a mitochondrion.
Other recent work revealed that a protein called INF2 is anchored on the endoplasmic reticulum where it promotes mitochondrial constriction. This protein builds actin subunits into long filaments that provide the force for constriction. However, it was not clear how INF2 became active, and whether there are proteins on mitochondria that interact with INF2 or actin.
Manor, Bartholomew et al. have now used a combination of microscopy-based methods and biochemical analysis to discover that a mitochondrial protein called Spire1C performs all of these roles. Spire1C is found on the outer membrane of mitochondria; it interacts with INF2 to drive the formation of actin filaments that constrict mitochondria. These results suggest that Spire1C bridges the endoplasmic reticulum with the network of actin filaments. Further experiments then showed that increasing Spire1C levels in cells resulted in the mitochondria becoming fragmented due to increased constriction. On the other hand, depleting Spire1C had the opposite effect and caused mitochondria to become unusually elongated. Following on from this work, the next challenge is to see if Spire1C is used differently or similarly in the different processes that involve mitochondrial fission.
PMCID: PMC4574297  PMID: 26305500
actin; mitochondria; endoplasmic reticulum; cytoskeleton; membranes; organelles; human; mouse
5.  Ectopic Expression of an Activated RAC in Arabidopsis Disrupts Membrane CyclingD⃞V⃞ 
Molecular Biology of the Cell  2005;16(4):1913-1927.
Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active AtRAC10 fused to the green fluorescent protein. The activated AtRAC10 induced deformation of root hairs and leaf epidermal cells and was primarily localized in Triton X-100–insoluble fractions of the plasma membrane. Actin cytoskeleton reorganization was revealed by creating double transgenic plants expressing activated AtRAC10 and the actin marker YFP-Talin. Plants were further analyzed by membrane staining with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) under different treatments, including the protein trafficking inhibitor brefeldin A or the actin-depolymeryzing agents latrunculin-B (Lat-B) and cytochalasin-D (CD). After drug treatments, activated AtRAC10 did not accumulate in brefeldin A compartments, but rather reduced their number and colocalized with FM4-64–labeled membranes in large intracellular vesicles. Furthermore, endocytosis was compromised in root hairs of activated AtRAC10 transgenic plants. FM4-64 was endocytosed in nontransgenic root hairs treated with the actin-stabilizing drug jasplakinolide. These findings suggest complex regulation of membrane cycling by plant RACs.
PMCID: PMC1073671  PMID: 15703216
6.  Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin 
Molecular Biology of the Cell  2007;18(8):3002-3014.
The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of subunits through actin filament barbed ends. Collectively, these results and our understanding of other actin-binding proteins implicate CAP1 as a central player in regulating the pool of unpolymerized ATP–actin.
PMCID: PMC1949352  PMID: 17538023
7.  The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns 
PLoS Pathogens  2013;9(4):e1003290.
Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.
Author Summary
The cytoskeleton is a dynamic platform for sensing and responding to a diverse array of biotic and abiotic stresses. The nature and timing of the changes in actin organization range from excessive bundling, to massive depolymerization, to new filament assembly, depending on the particular signal and the responding cell type. Here, we use the Arabidopsis–Pseudomonas pathosystem to dissect pathogen-derived cues that elicit changes in the plant host-cell cytoskeleton. Overall, we provide the first evidence that the actin cytoskeleton rearranges in response to a phytopathogenic bacterium and we quantified the temporal response of epidermal cells to Pseudomonas syringae pv. tomato DC3000 strains and susceptible Arabidopsis mutants, using a robust set of tools for measuring changes in actin organization. An immediate but transient increase in actin filament abundance was associated with pattern-triggered immunity. This response could be mimicked with microbe-associated molecular pattern peptide treatments. Second, we observed a late increase in actin filament bundling that appears to be part of effector-triggered susceptibility. We dissected the initial steps involved in the host-cell signaling pathway and demonstrated that FLS2, BAK1, and BIK1 were required for the actin response. Collectively, these findings demonstrate that rapid changes in host-cell cytoskeleton organization occur in response to receptor-mediated signaling during plant innate immunity.
PMCID: PMC3616984  PMID: 23593000
8.  A Mutation in MRH2 Kinesin Enhances the Root Hair Tip Growth Defect Caused by Constitutively Activated ROP2 Small GTPase in Arabidopsis 
PLoS ONE  2007;2(10):e1074.
Root hair tip growth provides a unique model system for the study of plant cell polarity. Transgenic plants expressing constitutively active (CA) forms of ROP (Rho-of-plants) GTPases have been shown to cause the disruption of root hair polarity likely as a result of the alteration of actin filaments (AF) and microtubules (MT) organization. Towards understanding the mechanism by which ROP controls the cytoskeletal organization during root hair tip growth, we have screened for CA-rop2 suppressors or enhancers using CA1-1, a transgenic line that expresses CA-rop2 and shows only mild disruption of tip growth. Here, we report the characterization of a CA-rop2 enhancer (cae1-1 CA1-1) that exhibits bulbous root hairs. The cae1-1 mutation on its own caused a waving and branching root hair phenotype. CAE1 encodes the root hair growth-related, ARM domain-containing kinesin-like protein MRH2 (and thus cae1-1 was renamed to mrh2-3). Cortical MT displayed fragmentation and random orientation in mrh2 root hairs. Consistently, the MT-stabilizing drug taxol could partially rescue the wavy root hair phenotype of mrh2-3, and the MT-depolymerizing drug Oryzalin slightly enhanced the root hair tip growth defect in CA1-1. Interestingly, the addition of the actin-depolymerizing drug Latrunculin B further enhanced the Oryzalin effect. This indicates that the cross-talk of MT and AF organization is important for the mrh2-3 CA1-1 phenotype. Although we did not observe an apparent effect of the MRH2 mutation in AF organization, we found that mrh2-3 root hair growth was more sensitive to Latrunculin B. Moreover, an ARM domain-containing MRH2 fragment could bind to the polymerized actin in vitro. Therefore, our genetic analyses, together with cell biological and pharmacological evidence, suggest that the plant-specific kinesin-related protein MRH2 is an important component that controls MT organization and is likely involved in the ROP2 GTPase-controlled coordination of AF and MT during polarized growth of root hairs.
PMCID: PMC2031828  PMID: 17957256
9.  Actin Turnover-Mediated Gravity Response in Maize Root Apices 
Plant Signaling & Behavior  2006;1(2):52-58.
The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap.
PMCID: PMC2633879  PMID: 19521476
actin cytoskeleton; gravisensing; graviresponding; root cap
10.  Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling 
The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. Here, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (≤ 10 μM) of the actin depolymerizing drugs, latrunculin B (Lat B) or cytochalasin B (CB), enhanced both formyl peptide receptor and Ca2+ ionophore stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP) inhibited primary granule exocytosis measured as myeloperoxidase release, but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly prior to primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodelling was highly polarized when cells were primed with CB, however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis was blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor, NSC23766, also inhibited actin remodelling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not Ca2+ ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.
PMCID: PMC2878813  PMID: 18799653 CAMSID: cams777
Rac GTPase; actin; latrunculin; cytochalasin; jasplakinolide; NSC23766
11.  Unveiling Interactions among Mitochondria, Caspase-Like Proteases, and the Actin Cytoskeleton during Plant Programmed Cell Death (PCD) 
PLoS ONE  2013;8(3):e57110.
Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD). PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B), a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA) to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1) activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs). The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK) or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.
PMCID: PMC3590192  PMID: 23483897
12.  Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin 
PLoS Biology  2011;9(9):e1001161.
A novel microfluidic approach allows the analysis of the dynamics of individual actin filaments, revealing both their local ADP/ADP-Pi-actin composition and that Pi release is a random mechanism.
The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi) that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds) and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.
Author Summary
Actin proteins assemble into microfilaments that control cell shape and movement by polymerizing or depolymerizing. These actin monomers can bind ATP or ADP molecules. The incorporation of an ATP-actin monomer into a growing filament results in rapid cleavage of ATP into ADP and inorganic phosphate (Pi), followed by a slower release of Pi. As a consequence, actin filaments are composed mainly of ADP- and ADP-Pi-actin subunits, which have different depolymerization kinetics and mechanical properties, and can be targeted specifically by regulatory proteins that affect filament function. Hence, the understanding of many cellular processes requires a knowledge of the ADP/ADP-Pi composition of actin filaments at a molecular scale. This has so far remained elusive because traditional studies rely on measuring an average over many filaments in solution. To address this issue, we developed a microfluidics setup to monitor individual filaments with light microscopy while rapidly changing their chemical environment. We find that depolymerization accelerates progressively and corresponds to an exponential ADP-Pi-actin profile in the filament, meaning that each subunit releases its Pi with the same rate. Our method also provides novel insight into the function of profilin, a protein important for regulation of actin dynamics in cells, thus demonstrating the method's potential in the functional analysis of actin regulators.
PMCID: PMC3181223  PMID: 21980262
13.  Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy 
Journal of Virology  2014;88(14):7904-7914.
Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells.
IMPORTANCE HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.
PMCID: PMC4097803  PMID: 24789789
14.  Distribution of G-actin is Related to Root Hair Growth of Wheat 
Annals of Botany  2006;98(1):49-55.
• Background and Aims Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth.
• Methods G-actin was labelled by fluorescein isothiocyanate–DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate–phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy.
• Key Results Thick F-actin bundles did not extend into a region of approx. 5–10 µm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G-actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs.
• Conclusions These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips.
PMCID: PMC2803535  PMID: 16675602
G-actin; F-actin; root hairs; plant tip-growth; Ca2+; BAPTA-AM; Triticum aestivum; wheat
15.  Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton 
PLoS Biology  2013;11(11):e1001720.
Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton.
Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.
Author Summary
Cilia are microscopic cell surface hair-like protrusions that can act as antennae to mediate cell signaling. Mutations disrupting ciliogenesis can cause many developmental anomalies associated with syndromes known as “ciliopathies.” Some developmental defects, such as limb polydactyly, arise from disruption of cilia-transduced sonic hedgehog signaling, while other defects, such as aberrant patterning of hair cells in the inner ear, arise from disrupted Wnt signaling resulting in modulation of planar cell polarity (PCP)—a process whereby cells are polarized and aligned. While ciliopathy phenotypes would suggest that cilia are involved in modulating PCP, the mechanistic link between cilia and PCP has been elusive. Our study using a mouse model carrying a mutation in Wdpcp, a gene required for both ciliogenesis and PCP, suggest that Wdpcp modulation of PCP involves interactions with the actin cytoskeleton separate from its function in ciliogenesis. We observe Wdpcp localization in cilia, where it is required for recruitment of proteins essential for ciliogenesis. Wdpcp interacts with Sept2, and is also found in actin filaments, where it regulates actin dynamics essential for PCP. Together, these findings show that PCP regulation by Wdpcp is distinct from its function in ciliogenesis and involves direct modulation of the actin cytoskeleton.
PMCID: PMC3841097  PMID: 24302887
16.  Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites 
eLife  null;4:e11553.
While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.
eLife digest
Inside cells, structures called mitochondria supply the energy needed to carry out the processes that sustain life. Mitochondria constantly divide (a process known as fission) or fuse together, which helps to keep them in good working condition and well distributed around the cell. Several neurological disorders, including Parkinson’s disease and Alzheimer’s, are associated with problems that affect mitochondrial fission.
Many different molecules work together to help mitochondria divide, including a protein called Drp1. A number of Drp1 molecules can associate with each other to form an “oligomer” in the shape of a ring around a mitochondrion. The ring then constricts to split the mitochondrion in two.
It is often assumed that Drp1 molecules are recruited to the mitochondria immediately before fission and then form the oligomer ring. However, by using microscopy to track the movement of fluorescently labeled Drp1 molecules in human cells, Ji, Hatch et al. now suggest that Drp1 is continuously binding to and releasing from mitochondria, regardless of the need for fission. The experiments showed that when bound to surface of the mitochondrion, Drp1 switches between assembling and disassembling the oligomer ring. This process of Drp1 assembly and oligomerization on mitochondria is called maturation.
Specific signals for fission can push Drp1 toward maturation, which then leads to fission. Ji, Hatch et al. found that one such signal is the assembly of filaments of a protein called actin. Preventing actin filaments from forming reduced the amount of Drp1 that accumulated at mitochondria, and resulted in the mitochondria dividing less frequently. Further biochemical experiments also revealed that actin interacts directly with Drp1 and stimulates Drp1 activity, helping the ring to organize and assist mitochondrial fission.
The formation of actin filaments is not the only mechanism that can recruit Drp1 to mitochondria. Future work should investigate whether other mechanisms work with actin to recruit Drp1. As with actin filaments, other signals might be predicted to influence the balance of maturation and disassembly of Drp1 oligomers.
PMCID: PMC4755738  PMID: 26609810
formin; myosin; INF2; ionomycin; Mff; Human
17.  Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface 
The Journal of Cell Biology  1995;130(2):345-354.
Using fluorescent membrane potential sensing dyes to stain budding yeast, mitochondria are resolved as tubular organelles aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microscopy reveals region-specific, directed mitochondrial movement during polarized yeast cell growth and mitotic cell division. Mitochondria in the central region of the mother cell move linearly towards the bud, traverse the bud neck, and progress towards the bud tip at an average velocity of 49 +/- 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly less movement. Yeast strains containing temperature sensitive lethal mutations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term shift to semi-permissive temperatures. Thus, the actin cytoskeleton is important for normal mitochondrial movement during inheritance. To determine the possible role of known myosin genes in yeast mitochondrial motility, we investigated mitochondrial inheritance in myo1, myo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondrial spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine motor activity on isolated yeast mitochondria. Rhodamine-phalloidin labeled yeast actin filaments bind to immobilized yeast mitochondria, as well as unilamellar, right- side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 microM), we observed F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 microM-2 mM), bound filaments are released from mitochondria and mitochondrial outer membranes. The maximum velocity of mitochondria- driven microfilament sliding (23 +/- 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extracts, is sensitive to protease treatment, and displays an ATP concentration dependence similar to that of members of the myosin family of actin-based motors. This is the first demonstration of an actin-based motor activity in a defined organelle population.
PMCID: PMC2199926  PMID: 7615636
18.  Actin-Dynamics in Plant Cells: The Function of Actin Perturbing Substances Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins 
This chapter will give an overview of the most common F-actin perturbing substances, that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement or when apoptosis has to be induced. These substances can be divided into two major subclasses – F-actin stabilizing and polymerizing substances like jasplakinolide, chondramides and F-actin severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane permeable F-actin stabilizing and polymerizing agent, which may even have anti-cancer activities. Cytochalasins, derived from fungi show an F-actin severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges, however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin.
For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin- and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given.
PMCID: PMC4869834  PMID: 26498789
Actin filaments; Chondramides; Cytochalasins; Depsipeptide; Jasplakinolide; Latrunculin; Phalloidin; Phallotoxin; Swinholide
19.  High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A 
The Journal of Cell Biology  1997;137(2):399-416.
We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.
LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.
PMCID: PMC2139767  PMID: 9128251
Neuroscience  2005;138(1):17-24.
Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca2+ imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca2+ accumulation to 53±7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca2+ signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca2+ influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca2+-dependent physiological and pathological processes.
PMCID: PMC2927977  PMID: 16359816
actin filament; AMPA; NMDA; latrunculin; channel; receptor
21.  AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana  
Journal of Experimental Botany  2012;64(2):585-597.
Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin–microtubule cross-talk.
PMCID: PMC3542049  PMID: 23202131
Actin; Arabidopsis; At5g25500; LatB; microtubules; SMIFH2; VAEM
22.  Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins, Mmm1p and Mdm10p  
The Journal of Cell Biology  1998;141(6):1371-1381.
Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.
PMCID: PMC2132797  PMID: 9628893
23.  Myosin XIK of Arabidopsis thaliana Accumulates at the Root Hair Tip and Is Required for Fast Root Hair Growth 
PLoS ONE  2013;8(10):e76745.
Myosin motor proteins are thought to carry out important functions in the establishment and maintenance of cell polarity by moving cellular components such as organelles, vesicles, or protein complexes along the actin cytoskeleton. In Arabidopsis thaliana, disruption of the myosin XIK gene leads to reduced elongation of the highly polar root hairs, suggesting that the encoded motor protein is involved in this cell growth. Detailed live-cell observations in this study revealed that xik root hairs elongated more slowly and stopped growth sooner than those in wild type. Overall cellular organization including the actin cytoskeleton appeared normal, but actin filament dynamics were reduced in the mutant. Accumulation of RabA4b-containing vesicles, on the other hand, was not significantly different from wild type. A functional YFP-XIK fusion protein that could complement the mutant phenotype accumulated at the tip of growing root hairs in an actin-dependent manner. The distribution of YFP-XIK at the tip, however, did not match that of the ER or several tip-enriched markers including CFP-RabA4b. We conclude that the myosin XIK is required for normal actin dynamics and plays a role in the subapical region of growing root hairs to facilitate optimal growth.
PMCID: PMC3792037  PMID: 24116145
24.  Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse 
eLife  null;5:e14850.
T cell receptor (TCR) engagement opens Ca2+ release-activated Ca2+ (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca2+ influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca2+-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca2+ as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca2+ influx may modulate TCR signaling.
eLife digest
An effective immune response requires the immune system to rapidly recognize and respond to foreign invaders. Immune cells known as T cells recognize infection through a protein on their surface known as the T cell receptor. The T cell receptor binds to foreign proteins displayed on the surface of other cells. This interaction initiates a chain of events, including the opening of calcium channels embedded in the T cell membrane known as CRAC channels, which allows calcium ions to flow into the cell. These events ultimately lead to the activation of the T cell, enabling it to mount an immune response against the foreign invader.
As part of the activation process, the T cell spreads over the surface of the cell that is displaying foreign proteins to form an extensive interface known as an immune synapse. The movement of the T cell's internal skeleton (the cytoskeleton) is crucial for the formation and function of the synapse. Actin filaments, a key component of the cytoskeleton, flow from the edge of the synapse toward the center; these rearrangements of the actin cytoskeleton help to transport clusters of T cell receptors to the center of the synapse and enable the T cell receptors to transmit signals that lead to the T cell being activated. It is not entirely clear how the binding of T cell receptors to foreign proteins drives the actin rearrangements, but there is indirect evidence suggesting that calcium ions may be involved.
Hartzell et al. have now investigated the interactions between calcium and the actin cytoskeleton at the immune synapse in human T cells. T cells were placed on glass so that they formed immune synapse-like connections with the surface, and actin movements at the synapse were visualized using a specialized type of fluorescence microscopy. When calcium ions were prevented from entering the T cell, the movement of actin stopped almost entirely. Thus, the flow of calcium ions into the T cell through CRAC channels is essential for driving the actin movements that underlie immune synapse development and T cell activation.
In further experiments, Hartzell et al. tracked the movements of CRAC channels and actin at the synapse and found that actin filaments create a constricting “corral” that concentrates CRAC channels in the center of the synapse. Thus, by driving cytoskeleton movement, calcium ions also help to organize calcium channels at the immune synapse. Future work will focus on identifying the actin remodeling proteins that enable calcium ions to control this process.
PMCID: PMC4956410  PMID: 27440222
immunological synapse; endoplasmic reticulum; T lymphocyte; actin dynamics; calcium release-activated calcium (CRAC) channels; calcium; Human
25.  Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection 
Increased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.
Methods and Findings
Here, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.
These results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.
Author Summary
An important clinical characteristic of dengue hemorrhagic fever/dengue shock syndrome is increased vascular permeability. Actin cytoskeleton is a significant element of endothelial barrier function regulation. In vitro study showed that dengue virus infection could induce redistributions of actin cytoskeleton. It is not precisely clear the roles of actin and the mechanisms of its reorganization during the infection. Using immunochemical assays, drug inhibition assays and protein interaction profiling methods, we aimed to identify the ways in which dengue virus serotype 2 interacts with actin cytoskeleton. The study showed that dynamic treadmilling of actin is necessary for dengue virus entry, production and release, while small GTPase Rac1 also plays multiple roles during these processes. In addition, we demonstrated the association of viral E protein with actin, indicating a direct effect of viral protein on the structural modifications of actin cytoskeleton. Our results provide evidence for the participation of Rac1 signaling pathways in viral protein-induced actin reorganizations, which may be a mechanism involved in the etiology of dengue hemorrhagic fever.
PMCID: PMC2930870  PMID: 20824170

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