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Stossel, Thomas

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Stossel

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Stossel, Thomas
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    Documentation and Localization of Force-mediated Filamin A Domain Perturbations in Moving Cells
    (2014) Nakamura, Fumihiko; Song, Mia; Hartwig, John; Stossel, Thomas
    Endogenously and externally generated mechanical forces influence diverse cellular activities, a phenomenon defined as mechanotransduction. Deformation of protein domains by application of stress, previously documented to alter macromolecular interactions in vitro, could mediate these effects. We engineered a photon-emitting system responsive to unfolding of two repeat domains of the actin filament (F-actin) crosslinker protein filamin A (FLNA) that binds multiple partners involved in cell signaling reactions and validated the system using F-actin networks subjected to myosin-based contraction. Expressed in cultured cells, the sensor-containing FLNA construct reproducibly reported FLNA domain unfolding strikingly localized to dynamic, actively protruding, leading cell edges. The unfolding signal depends upon coherence of F-actin-FLNA networks and is enhanced by stimulating cell contractility. The results establish protein domain distortion as a bona fide mechanism for mechanotransduction in vivo.
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    From the Bench to the Field in Low-Cost Diagnostics: Two Case Studies
    (Wiley-Blackwell, 2015) Kumar, Ashok Ashwin; Hennek, Jonathan; Smith, Barbara; Kumar, Shailendra; Beattie, Patrick Daniel; Jain, Sidhartha; Rolland, Jason P.; Stossel, Thomas; Chunda-Liyoka, Catherine; Whitesides, George
    Despite the growth of research in universities on point-of-care (POC) diagnostics for global health, most devices never leave the laboratory. The processes that move diagnostic technology from the laboratory to the field—the processes intended to evaluate operation and performance under realistic conditions—are more complicated than they might seem. Two case studies illustrate this process: the development of a paper-based device to measure liver function, and the development of a device to identify sickle cell disease based on aqueous multiphase systems (AMPS) and differences in the densities of normal and sickled cells. Details of developing these devices provide strategies for forming partnerships, prototyping devices, designing studies, and evaluating POC diagnostics. Technical and procedural lessons drawn from these experiences may be useful to those designing diagnostic tests for developing countries, and more generally, technologies for use in resource-limited environments.
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    Mechanical Perturbation of Filamin A Immunoglobulin Repeats 20-21 Reveals Potential Non-equilibrium Mechanochemical Partner Binding Function
    (Nature Publishing Group, 2013) Chen, Hu; Chandrasekar, Saranya; Sheetz, Michael P.; Stossel, Thomas; Nakamura, Fumihiko; Yan, Jie
    The actin crosslinking protein filamin A (FLNa) mediates mechanotransduction, a conversion of mechanical forces into cellular biochemical signals to regulate cell growth and survival. To provide more quantitative insight into this process, we report results using magnetic tweezers that relate mechanical force to conformational changes of FLNa immunoglobulin-like repeats (IgFLNa) 20–21, previously identified as a mechanosensing domain. We determined the force magnitudes required to unfold previously identified structural organizations of the β-strands in the two domains: IgFLNa 20 unfolds at ~15 pN and IgFLNa 21 unfolding requires significantly larger forces. Unfolded domain IgFLNa 20 can exist in two different conformational states, which lead to different refolding kinetics of the IgFLNa 20 and imply a significant impact on the reformation of the domain pair at reduced force values. We discuss the relevance of the findings to force bearing and mechanosensing functions of FLNa.
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    Plasma Gelsolin Depletion and Circulating Actin in Sepsis—A Pilot Study
    (Public Library of Science, 2008) Lee, Po-Shun; Patel, Sanjay R.; Christiani, David; Bajwa, Ednan; Stossel, Thomas; Waxman, Aaron
    Background: Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in septic patients and animals. We hypothesized that the extent of pGSN reduction correlates with outcomes of septic patients and that circulating actin is a manifestation of sepsis. Methodology/Principal Findings: We assayed pGSN in plasma samples from non-surgical septic patients identified from a pre-existing database which prospectively enrolled patients admitted to adult intensive care units at an academic hospital. We identified 21 non-surgical septic patients for the study. Actinemia was detected in 17 of the 21 patients, suggesting actin released into circulation from injured tissues is a manifestation of sepsis. Furthermore, we documented the depletion of pGSN in human clinical sepsis, and that the survivors had significantly higher pGSN levels than the non-survivors (163±47 mg/L vs. 89±48 mg/L, p = 0.01). pGSN levels were more strongly predictive of 28-day mortality than APACHE III scores. For every quartile reduction in pGSN, the odds of death increased 3.4-fold. Conclusion: We conclude that circulating actin and pGSN deficiency are associated with early sepsis. The degree of pGSN deficiency correlates with sepsis mortality. Reversing pGSN deficiency may be an effective treatment for sepsis.
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    Decreased Levels of the Gelsolin Plasma Isoform in Patients with Rheumatoid Arthritis
    (BioMed Central, 2008) Osborn, Teresia M.; Verdrengh, Margareta; Stossel, Thomas; Tarkowski, Andrej; Bokarewa, Maria
    Introduction Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of \(200 \pm 50\) mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis.Methods Circulating and intra-articular levels of plasma gelsolin were measured in 78 patients with rheumatoid arthritis using a functional (pyrene-actin nucleation) assay and compared with 62 age- and gender-matched healthy controls.Results Circulating plasma gelsolin levels were significantly lower in patients with rheumatoid arthritis compared with healthy controls (\(141 \pm 32\) versus \(196 \pm 40\) mg/L, P = 0.0002). The patients' intra-articular plasma gelsolin levels were significantly lower than in the paired plasma samples (\(94 \pm 24\) versus \(141 \pm 32\) mg/L, P = 0.0001). Actin was detected in the synovial fluids of all but four of the patients, and immunoprecipitation experiments identified gelsolin-actin complexes.Conclusions The plasma isoform of gelsolin is decreased in the plasma of patients with rheumatoid arthritis compared with healthy controls. The reduced plasma concentrations in combination with the presence of actin and gelsolin-actin complexes in synovial fluids suggest a local consumption of this potentially anti-inflammatory protein in the inflamed joint.
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    Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations
    (Public Library of Science, 2009) Heikkinen, Outi; Pentikäinen, Olli T.; Kasza, Karen E.; Kupiainen, Olga; Permi, Perttu; Kilpeläinen, Ilkka; Ylänne, Jari; Nakamura, Fumihiko; Osborn, Teresia; Weitz, David; Hartwig, John; Stossel, Thomas
    Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/principal findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D \(\beta-strands\) of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress. Conclusions/significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.