Biomedical subjects

J Condeelis

Publications and source records attributed to J Condeelis.

At least 19 recordsLinked to original sources

PubMed2004

Cellular localization of activated N-WASP using a conformation-sensitive antibody.

The main regulators of Arp2/3 activity appear to be N-WASP and the other members of the Scar/WAVE family of proteins. We show here that after EGF stimulation, N-WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N-WASP within 1 min after EGF stimulation. The timing of N-WASP recruitment mirrors the timing of barbed-end formation at the leading edge. To determine the cellular activation of N-WASP after EGF stimulation, we made a conformation-sensitive antibody (CSA) against the CRIB domain of N-WASP that is predicted to recognize N-WASP in its open, active conformation, but not in its closed, inactive conformation. The ability of CSA to detect only active N-WASP was demonstrated by in vitro experiments using immunoprecipitation of active N-WASP from EGF-stimulated cells and Cdc42 activation of N-WASP activity. In cell staining experiments, N-WASP is maximally accessible to CSA 40 sec after EGF stimulation and this activated N-WASP is in the nucleation zone. These results indicate that active N-WASP is present at the leading edge of lamellipods, an unexpected finding given its reported involvement in filopod formation. This work establishes the feasibility of using antibodies directed against specific conformations or epitopes with changing accessibilities as a window on the status and localization of activity.

Actin Cytoskeleton↗

PubMed2003

The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism.

The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 A. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.

Actins↗

PubMed2001

The physiological significance of beta -actin mRNA localization in determining cell polarity and directional motility.

beta-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3' untranslated region, the "zip code." Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3' untranslated region leads to delocalization of beta-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after beta-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of beta-actin protein. These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Actins↗

PubMed2001

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension.

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.

Actin-Related Protein 2↗

PubMed2001

EGF-stimulated lamellipod extension in adenocarcinoma cells.

The extension of lamellipodia has been triggered by the application of epidermal growth factor (EGF). We have used an atomic force microscope (AFM) to investigate this lamellipodial extension. During extension we could detect an increase in height from about 500 nm for the stable lamellipodium to typical values of 600-800 nm for the extending lamellipodium. The AFM was also used to determine the mechanical properties of the lamellipodium where we found a decrease of the elastic modulus by a factor of 1.4 at the same location within the same cell. Both findings are consistent with the cortical expansion hypothesis, suggesting that severing of actin filaments, leading to a swelling of the cytoskeleton, generates the protrusive force during lamellipodial extension.

Adenocarcinoma↗

PubMed2001

How is actin polymerization nucleated in vivo?

Actin polymerization in vivo is dependent on free barbed ends that act as nuclei. Free barbed ends can arise in vivo by nucleation from the Arp2/3 complex, uncapping of barbed ends on pre-existing filaments or severing of filaments by cofilin. There is evidence that each mechanism operates in cells. However, different cell types use different combinations of these processes to generate barbed ends during stimulated cell motility. Here, I describe recent attempts to define the relative contributions of these three mechanisms to actin nucleation in vivo. The rapid increase in the number of barbed ends during stimulation is not due to any single mechanism. Cooperation between capping proteins, cofilin and the Arp2/3 complex is necessary for the development of protrusive force at the leading edge of the cell: uncapping and cofilin severing contributing barbed ends, whereas activity of the Arp2/3 complex is necessary, but not sufficient, for lamellipod extension. These results highlight the need for new methods that enable the direct observation of actin nucleation and so define precisely the relative contributions of the three processes to stimulated cell motility.

Actin-Related Protein 2↗

PubMed2001

Filamins as integrators of cell mechanics and signalling.

Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Cell Membrane↗

PubMed2001

Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways.

BACKGROUND: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, incluing the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulated diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis; however, a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. MATERIALS AND METHODS: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. RESULTS: Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the co-localization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. CONCLUSIONS: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Adenocarcinoma↗

PubMed1999

Mycobacterial infection of macrophages results in membrane-permeable phagosomes.

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.

Animals↗

PubMed1999

Correlation of beta-actin messenger RNA localization with metastatic potential in rat adenocarcinoma cell lines.

The actin cytoskeleton is involved in the motility of tumor cells. It has been shown in several cell types that beta-actin mRNA is localized in the protrusions of cells in which actin is actively polymerized, and the ability to localize mRNA is correlated with the efficiency of motility. In this context, we studied the distribution of beta-actin mRNA in two different tumor cell lines and correlated it with their metastatic potential. The two cell lines used were the highly metastatic MTLn3 cells and nonmetastatic MTC cells. Nonmetastatic MTC cells have two different pools of beta-actin mRNA (perinuclear and at the leading edge), whereas highly metastatic MTLn3 cells have only a perinuclear distribution of beta-actin mRNA. These differences in mRNA localization are correlated with profound differences in the polarity and plasticity of cell motility of these cells in culture and the histopathology of primary breast tumors derived from these cells. In particular, MTLn3 cells are unpolarized by all cell shape and motility criteria in culture and in their histopathological organization in primary tumors. By comparison, MTC cells are polarized in all identical measurements. These results suggest that the increased plasticity of cell locomotion and the invasiveness of MTLn3 cells result from the failure of metastatic cells to localize beta-actin mRNA properly, causing them to be less polarized and therefore more flexible in their direction of motility. Thus, differences in the polarized organization of cells in the primary tumor that are correlated with beta-actin mRNA localization may have prognostic value in predicting metastatic potential.

Actins↗

PubMed1998

The effect of F-actin on the binding and hydrolysis of guanine nucleotide by Dictyostelium elongation factor 1A.

Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF1A, formerly EF1alpha), a major actin-binding protein. The basal mechanism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are required to prevent protein aggregation and loss of enzymatic activity. The dissociation constants (Kd) for GDP and GTP are 2.5 microM and 0.6 microM, respectively, and the kcat of GTP hydrolysis is 1.0 x 10(-3) s-1. When eEF1A binds to F-actin, there is a 7-fold decrease in the affinity for guanine nucleotide and an increase of 35% in the rate of GTP hydrolysis. Based upon our results and the relevant cellular concentrations, the predominant form of cellular eEF1A is calculated to be GTP.eEF1A.F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP.eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.

Actins↗

PubMed1996

EGF stimulates lamellipod extension in metastatic mammary adenocarcinoma cells by an actin-dependent mechanism.

Changes in lamellipod extension and chemotaxis in response to EGF were analysed for MTLn3 cells (a metastatic cell line derived from the 13762NF rat mammary adenocarcinoma). Addition of EGF produced a cessation of ruffling followed by extension of hyaline lamellipods containing increased amounts of F-actin at the growing edge. A non-metastatic cell line (MTC) derived from the same tumor did not show such responses. Lamellipod extension was maximal within 5 min, followed by retraction and resumption of ruffling. Maximal area increases due to lamellipod extension occurred at about 5 nM EGF. Chemotactic and chemokinetic responses, measured using a microchemotaxis chamber, were also greatest at 5 nM. Cytochalasin D inhibited EGF-stimulated responses including lamellipod extension, increases in F-actin in lamellipods, and chemotaxis. Nocodazole affected chemotaxis at higher concentrations but not EGF-induced lamellipod extension. We conclude that polymerization of F-actin at the leading edges of lamellipods is necessary for extension of lamellipods and chemotaxis of MTLn3 cells in response to EGF. The motility and chemotaxis responses of this metastatic cell line have strong similarities to those seen in well-characterized chemotactic cells such as Dictyostelium and neutrophils.

Actins↗

PubMed1996

pH, EF-1alpha and the cytoskeleton.

One of the unexpected cellular components found interacting with the cytoskeleton is elongation factor 1 alpha (EF-1alpha). How this interaction is regulated is not clear, but pH may be a potent regulator. Interestingly, pH also regulates the amount of protein translation occurring in many cell systems. In this paper, the authors suggest that sequestration of EF-1alpha in the cytoskeleton may play a key role in regulating the spatial distribution of macromolecular assembly in a way that is dependent on cytoplasmic pH.

Journal Article↗

PubMed1996

F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction.

The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin-binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F-actin, we have studied the effect of F-actin on the ability of EF-1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa-tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF-1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus.

Actins↗

PubMed1996

Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends.

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.

Actin Cytoskeleton↗

PubMed1996

Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

Actins↗

PubMed1996

Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma.

Overexpression of elongation factor-1 alpha (EF1 alpha) mRNA has been correlated with increased metastatic potential in mammary adenocarcinoma; however, this relationship was not explored at the level of protein expression. As EF1 alpha has been shown in other cell types to be a component of the actin cytoskeleton, a likely effector in metastasis, the actin binding activity of EF1 alpha from metastatic and nonmetastatic rat breast tumors and cell lines was investigated. We have shown that EF1 alpha protein is overexpressed in metastatic compared to nonmetastatic cells and whole tumors. Similarly to other EF1 alpha s, both types of tumor EF1 alpha bind to F-actin, but EF1 alpha from metastatic cells has a reduced affinity for actin. In addition, there is a high correlation between the intracellular distribution of filamentous actin and EF1 alpha in those cytoskeletal structures thought to be important for supporting the cellular motility required for metastasis. Following stimulation with EGF, there is a parallel increase in the amount of F-actin and EF1 alpha associated with the cytoskeleton. The response to EGF can be blocked with cytochalasin D indicating that the binding of EF1 alpha to the cytoskeleton is mediated by F-actin. We propose that a weakened association of EF1 alpha with actin may be related to the metastatic process via an altered organization of the actin cytoskeleton and the differential translation of mRNAs associated with the cytoskeleton.

Actins↗

PubMed1995

pH regulation of the F-actin binding properties of Dictyostelium elongation factor 1 alpha.

ABP50, an F-actin bundling protein from Dictyostelium, is also the protein synthesis co-factor, elongation factor 1 alpha (EF1 alpha). Concomitant with cAMP stimulation in Dictyostelium is a cytoplasmic alkalinization (Aerts, R. J., DeWit, R. J. W., and Van Lookeren Campagne, M. M. (1987) FEBS Lett. 220, 366-370) and a redistribution of EF1 alpha (Dharmawardhane, S., Demma, M., Yang, F., and Condeelis, J. (1991) Cell Motil. Cytoskel. 20, 279-288). In addition, others have shown a correlation between intracellular pH and the level of protein synthesis in Dictyostelium (Aerts, R. J., Durston, A. J., and Moolenaar, W. H. (1985) Cell 43, 653-657). The present study investigates the relationship between pH and the F-actin binding properties of EF1 alpha. We found that increasing pH over the physiological range 6.2-7.8 causes a loss of EF1 alpha-mediated F-actin bundling and single filament binding, with corresponding increases in the amount of free EF1 alpha in vitro. Similar results also were obtained by cell fractionation and confocal immunofluorescence microscopy. The EF1 alpha binding constant (Kd) for F-actin is increased from 0.2 microM to > 2.2 microM over the same pH range. In addition, EF1 alpha-induced actin bundle formation is freely reversible by changes in pH. Thus, pH may be a potent modulator of cytoarchitecture in Dictyostelium and may also influence mRNA translation rates by modifying the interactions between the protein synthetic machinery and the actin cytoskeleton.

Actins↗