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In JoVE (1)
Other Publications (19)
- Microbiology (Reading, England)
- Traffic (Copenhagen, Denmark)
- Current Biology : CB
- Eukaryotic Cell
- Journal of Cell Science
- Toxicological Sciences : an Official Journal of the Society of Toxicology
- The Journal of Cell Biology
- BMC Immunology
- Methods in Molecular Biology (Clifton, N.J.)
- Journal of Immunological Methods
- Journal of Cell Science
- BMC Cell Biology
- Journal of Molecular Biology
- American Journal of Respiratory Cell and Molecular Biology
- Genes & Development
- Journal of Materials Science. Materials in Medicine
- International Journal of Cell Biology
- PloS One
- PloS One
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Articles by David A. Knecht in JoVE
JoVE General
ECIS / टैक्सी के साथ सेलुलर chemotaxis का मापन
1Molecular and Cell Biology, University of Connecticut, 2University of Connecticut
ECIS / टैक्सी प्रणाली एक स्वचालित वास्तविक समय परख है कि सेलुलर chemotaxis उपाय है. इस परख में, कोशिकाओं में agarose की एक परत करने के लिए एक लक्ष्य इलेक्ट्रोड पर पहुंचने के नीचे चलते हैं. सेलुलर आंदोलन एसी चालू 0 से प्रतिरोध की शुरुआत द्वारा मापा जाता है.
Other articles by David A. Knecht on PubMed
Kinetics of Binding, Uptake and Degradation of Live Fluorescent (DsRed) Bacteria by Dictyostelium Discoideum
The kinetics of binding, uptake and degradation of bacteria by vegetative Dictyostelium amoeba using Escherichia coli expressing the recombinant fluorescent protein DsRed have been characterized. There are significant advantages to using DsRed-expressing bacteria for phagocytosis assays. Stable expression of the fluorescent protein, DsRed, provides living bacteria with a bright internal fluorescent signal that is degradable in the phagolysosomal pathway. Unlike assays with chemically labelled bacteria or latex beads, the bacteria are alive and possess a natural, unaltered external surface for receptor interaction. Dictyostelium cells rapidly bind and phagocytose DsRed bacteria. Pulse-chase experiments show that the signal derived from DsRed is degraded with a half-life of approximately 45 min. To distinguish internalized bacteria from those bound to the surface, an assay was developed in which sodium azide was used to release surface-bound particles. Surprisingly, surface particle release appears to be independent of myosin II function. Using this assay it was shown that the uptake of bacteria into cells is extremely rapid. After 1 min incubation, 20% of the signal is derived from internalized bacteria. The proportion of the signal from internalized bacteria increases gradually and reaches 50% at steady state. This assay will be useful in investigations of the molecular machinery of phagocytosis and post-internalization vesicle trafficking.
Visualization of Actin Dynamics During Macropinocytosis and Exocytosis
Macropinocytosis of newly formed resides and exocytosis of post-lysosomes have been visualized using a green fluorescent protein probe that binds specifically to F-actin filaments. F-actin association with macropinocytosis begins as a V-shaped infolding of the membrane. Vesicle enlargement occurs through an inward movement of the proximal point of the V as well as an outward protrusion at the tip of the V to form an elongated invagination. The protrusion eventually closes at its distal margin to become a vesicle and is moved centripetally while recovering its circular shape. The vesicle loses its actin coat within 1 min after internalization. One hour later, post-lysosomal vesicles became weakly surrounded by actin while still cytoplasmic. Some of these vesicles moved to the plasma membrane, docked, and then expelled their contents. Slightly before the vesicle content began to disappear, an increase in F-actin association with the vesicle was observed. This was followed by rapid contraction of the vesicle and then disappearance of the actin signal once the internal content was released. These results show that dynamic changes in actin filament association with the vesicle membrane accompany both endocytosis and exocytosis.
Rho-kinase and Myosin-II Control Phagocytic Cup Formation During CR, but Not FcgammaR, Phagocytosis
Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.
RacB Regulates Cytoskeletal Function in Dictyostelium Spp
Thus far, 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. To investigate the specific function of RacB, we have conditionally expressed wild-type (WT-RacB), dominant negative (N17-RacB), and constitutively activated (V12-RacB) versions of the protein. On induction, cells expressing V12-RacB stopped growing, detached from the surface, and formed numerous spherical surface protrusions while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin-driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin II activity, which distinguishes them from blebs formed by wild-type cells under stress. Finally, we examined the functional consequences of expression of wild-type and mutant RacB. Phagocytosis, endocytosis, and fluid phase efflux rates were reduced in all cell lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin but the consequences of activation appear to be different from other Dictyostelium Rac proteins so far investigated, resulting in different morphological and functional changes in cells.
Cross-linking of Actin Filaments by Myosin II is a Major Contributor to Cortical Integrity and Cell Motility in Restrictive Environments
Cells are frequently required to move in a local environment that physically restricts locomotion, such as during extravasation or metastatic invasion. In order to model these events, we have developed an assay in which vegetative Dictyostelium amoebae undergo chemotaxis under a layer of agarose toward a source of folic acid [Laevsky, G. and Knecht, D. A. (2001). Biotechniques 31, 1140-1149]. As the concentration of agarose is increased from 0.5% to 3% the cells are increasingly inhibited in their ability to move under the agarose. The contribution of myosin II and actin cross-linking proteins to the movement of cells in this restrictive environment has now been examined. Cells lacking myosin II heavy chain (mhcA-) are unable to migrate under agarose overlays of greater than 0.5%, and even at this concentration they move only a short distance from the trough. While attempting to move, the cells become stretched and fragmented due to their inability to retract their uropods. At higher agarose concentrations, the mhcA- cells protrude pseudopods under the agarose, but are unable to pull the cell body underneath. Consistent with a role for myosin II in general cortical stability, GFP-myosin dynamically localizes to the lateral and posterior cortex of cells moving under agarose. Cells lacking the essential light chain of myosin II (mlcE-), have no measurable myosin II motor activity, yet were able to move normally under all agarose concentrations. Mutants lacking either ABP-120 or alpha-actinin were also able to move under agarose at rates similar to wild-type cells. We hypothesize that myosin stabilizes the actin cortex through its cross-linking activity rather than its motor function and this activity is necessary and sufficient for the maintenance of cortical integrity of cells undergoing movement in a restrictive environment. The actin cross-linkers alpha-actinin and ABP-120 do not appear to play as major a role as myosin II in providing this cortical integrity.
Silica-induced Apoptosis in Mouse Alveolar Macrophages is Initiated by Lysosomal Enzyme Activity
Past studies in our laboratory have shown that silica (-quartz) particle exposure of a mouse alveolar macrophage cell line (MH-S) elicits mitochondrial depolarization and caspase 3 and 9 activation, contributing to apoptosis. However, cellular pathways leading to these outcomes have not been extensively investigated. Initial studies revealed that silica exposure elicits lysosomal permeability after 1 h, as evidenced by leakage of FITC-conjugated dextran and acridine orange. We next evaluated a role for the lysosomal acidic compartment in apoptosis. Cells pretreated with the lysosomotropic weak base ammonium chloride, to increase lysosomal pH, showed decreased caspase activation and apoptotic DNA fragmentation. MH-S cells pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, showed decreased caspase 9 and 3 activation as well as a decreased percentage of cells that became apoptotic. DNA fragmentation and caspase 9 and 3 activation were also decreased in cells pretreated with despiramine, an inhibitor of lysosomal acidic sphingomyelinase. Silica pretreated with aluminum lactate (to blunt surface active sites) reduced caspase activation and apoptosis. Although aluminum lactate-treated silica still induced lysosomal permeability (by FITC-dextran leakage), one measure of lysosome integrity and function suggested a reduction in the extent and/or nature of lysosomal injury (by acridine orange retention). A role for reactive oxygen species (ROS) was investigated to explore another pathway for silica-induced apoptosis in addition to lysosomal enzymes; however, no role for ROS was apparent. Thus, following silica exposure, lysosomal injury precedes apoptosis, and the apoptotic signaling pathway includes cathepsin D and acidic sphingomyelinase.
Phosphatidylinositol-4,5-bisphosphate Hydrolysis Directs Actin Remodeling During Phagocytosis
The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.
Metallothionein Mediates Leukocyte Chemotaxis
Metallothionein (MT) is a cysteine-rich, metal-binding protein that can be induced by a variety of agents. Modulation of MT levels has also been shown to alter specific immune functions. We have noticed that the MT genes map close to the chemokines Ccl17 and Cx3cl1. Cysteine motifs that characterize these chemokines are also found in the MT sequence suggesting that MT might also act as a chemotactic factor.
Under-agarose Chemotaxis of Dictyostelium Discoideum
In the vegetative state, Dictyostelium amoebae are chemotactic toward pterins released by bacteria, whereas during multicellular development, they become chemotactic to endogenously produced cAMP. A variety of assays have been used to visualize and quantify chemotactic movement. Under-agarose chemotaxis provides a simple and flexible assay that permits high-resolution imaging and quantification of the motility behavior of individual cells and populations by both transmitted light and fluorescence microscopy. The assay requires cells to deform a solid but flexible matrix; therefore, it also provides a way to measure defects in the ability of mutant cells to move in these restrictive conditions.
Automated Real-time Measurements of Leukocyte Chemotaxis
We have previously described an automated system (ECIS/taxis) for measuring chemotactic movement of Dictyostelium amoebae in a folic acid gradient [Hadjout, N., Laevsky, G., Knecht, D.A. and Lynes, M.A., 2001. Automated real-time measurement of chemotactic cell motility. Biotechniques 31, 1130-1138.]. In the ECIS/taxis system, cells migrate in an under-agarose environment, and their position is monitored by determining the impedance change caused by cells crawling onto the surface of an electrode. In this report, we show that chemotaxis of primary and immortalized leukocytes in response to complement (C5a) could be measured using the ECIS/taxis system. Several modifications to the design of the target electrode were tested, and a linear electrode perpendicular to the direction of movement was found to increase the sensitivity and reliability of the assay. Using the optimized ECIS/taxis assay, the dose response of neutrophils and WBC 265-9C cells was established and compared to the Boyden chamber assay. The ECIS/taxis assay system can be used to compare the movement of different cell types, to assess the effect of complex chemotactic gradients, or to determine the effects of pharmaceuticals on chemotactic motility.
Traction Force Microscopy in Dictyostelium Reveals Distinct Roles for Myosin II Motor and Actin-crosslinking Activity in Polarized Cell Movement
Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA-) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE-) and mhcA- cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.
Actin Binding Domains Direct Actin-binding Proteins to Different Cytoskeletal Locations
Filamin (FLN) and non-muscle alpha-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell.
Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal-targeting-sequence-1-mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase
The peroxisomal targeting sequence 1 (PTS1) is a consensus tripeptide 1 (S/C/A)(K/R/H)(L/M) that is found at the C-terminus of most peroxisomal proteins. However, the only known mammalian protein containing a terminal methionine PTS1 (SKM), human soluble epoxide hydrolase (hsEH), shows both peroxisomal and cytosolic localizations in vivo. Mechanisms regulating the subcellular localization of hsEH thus remain unclear. Here we utilized green fluorescent protein-hsEH fusion constructs to study the peroxisomal targeting of hsEH in transiently and stably transfected Chinese hamster ovary cells. Our results suggest that the peroxisomal import of hsEH is regulated by three factors. First, we show that SKM is required, but not sufficient, for peroxisomal import. Second, by manipulating protein expression levels, we show that SKM mediates peroxisomal import of wild-type hsEH only when expression levels are high. Third, we show that amino acid modifications that decrease subunit oligomerization and presumably enhance accessibility of the SKM motif confer peroxisomal targeting even at low protein expression levels. We conclude that, in hsEH, SKM is a necessary but inefficient and context-dependent PTS1. Peroxisomal import occurs when expression levels are high or when the SKM motif is accessible. These results provide a mechanistic basis for understanding the cell-specific and tissue-specific localization of hsEH in vivo.
The Phagocytosis of Crystalline Silica Particles by Macrophages
Silicosis is a chronic lung disease induced by the inhalation of crystalline silica. Exposure of cultured macrophages to crystalline silica leads to cell death; however, the mechanism of cell-particle interaction, the fate of particles, and the cause of death are unknown. Time-lapse imaging shows that mouse macrophages avidly bind particles that settle onto the cell surface and that cells also extend protrusions to capture distant particles. Using confocal optical sectioning, silica particles were shown to be present within the cytoplasmic volume of live cells. In addition, electron microscopy and elemental analysis showed silica in internal cellular sections. To further examine the phagocytosis process, the kinetics of particle uptake was quantified using an assay in which cells were exposed to ovalbumin (OVA)-coated particles, and an anti-OVA antibody was used to distinguish surface-bound from internalized particles. Fc receptor-mediated uptake of antibody-coated silica particles was nearly complete within 5 minutes. In contrast, no OVA-coated particles were internalized at this time. After 30 minutes, 30% of bound silica was internalized and uptake continued slowly thereafter. OVA-coated latex beads, regardless of surface charge, were internalized at a similarly slow rate. These results demonstrate that macrophages internalize silica and that nonopsonized phagocytosis occurs by a temporally, and possibly mechanistically, distinct pathway from Fc receptor-mediated phagocytosis. Eighty percent of macrophages die within 12 hours of silica exposure. Neither OVA coating nor tetramethylrhodamine isothiocyanate labeling has any effect on cell death. Interestingly, antibody coating dramatically reduces silica toxicity. We hypothesize that the route of particle entry and subsequent phagosome trafficking affects the toxicity of internalized particles.
Tsunami, the Dictyostelium Homolog of the Fused Kinase, is Required for Polarization and Chemotaxis
In a forward genetic screen for chemotaxis mutants in Dictyostelium discoideum, we identified a loss-of-function mutation, designated tsunami, encoding a homolog of the Fused kinase. Cells lacking tsuA function could not effectively perform chemotaxis and were unable to become polarized or correctly orient pseudopods in chemotactic gradients. While tsuA(-) cells were able to couple receptor occupancy to phosphatidylinositol (3,4,5) trisphosphate (PIP3) production and actin polymerization, the PIP3 response was prolonged and basal F-actin levels were increased. Interestingly, TsuA localizes to the microtubule network and puncta mainly found at the cell periphery. Analysis of the gene uncovered a novel C-terminal domain that we designated the Tsunami Homology (TH) domain. Both the kinase domain and the TH domain are required to rescue the phenotypic defects of tsuA(-) cells. While kinase activity is not required for localization to microtubules, the TH domain is essential. Thus, localization of kinase activity to microtubules is critical for TsuA function. We propose that functions in association with the microtubule network may underlie the divergent roles of Fused kinase proteins in different organisms.
Incorporation of Bovine Serum Albumin into Biomimetic Coatings on Titanium with High Loading Efficacy and Its Release Behavior
Bovine serum albumin (BSA) was employed as a model protein to study its loading efficiency into a calcium phosphate (CaP) coating on titanium substrates. It is found that the protein loading efficiency can be adjusted by varying the specific configurations of the coating system such as simulated body fluid (SBF) volume, solution height and container selection for the SBF. A BSA loading efficiency as high as 90% was achieved when the ratio of the substrate surface area to modified SBF (m-SBF) volume was as high as 0.072. The release of BSA from the biomimetic coatings was also investigated in vitro. A sustained release was achieved although a large quantity of BSA was still trapped in the coating after 15 days of immersion in a phosphate buffer solution. A much faster release rate would be expected when the coating is implanted in vivo due to the active involvement of osteoclast cells and enzymes.
Contribution of Filopodia to Cell Migration: A Mechanical Link Between Protrusion and Contraction
Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound by alpha-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.
Cucurbitacin I Inhibits Cell Motility by Indirectly Interfering with Actin Dynamics
Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton.
The Phagocytosis and Toxicity of Amorphous Silica
Inhalation of crystalline silica is known to cause an inflammatory reaction and chronic exposure leads to lung fibrosis and can progress into the disease, silicosis. Cultured macrophages bind crystalline silica particles, phagocytose them, and rapidly undergo apoptotic and necrotic death. The mechanism by which particles are bound and internalized and the reason particles are toxic is unclear. Amorphous silica has been considered to be a less toxic form, but this view is controversial. We compared the uptake and toxicity of amorphous silica to crystalline silica.
