This project started over 35 years ago to isolate, characterize and define cell functions for molecules controlling actin filament (F-actin) remodeling responsible for mammalian cell motility and phagocytosis. In the 1970s, we discovered two proteins contributing to cytoplasmic """"""""gel-sol transformations,"""""""" filamin A (FLNa), which gels F-actin and gelsolin, which regulates this gelation by reversible F-actin severing. Although filamin A and gelsolin are clearly important for cell motility, and their deficiency or mutation lead to human diseases, with time these proteins have become imbedded within hundreds of others contributing to actin remodeling, including filamin and gelsolin isoforms. We have therefore increasingly focused on the structure and function of filamin A. During the current grant cycle, our ability to generate full-length, truncated and domain-swapped FLNa proteins helped clarify how FLNa orients to specifically and potently cross-link F-actin. It also facilitated the discovery of calcium regulation of F-actin binding by FLNa through calmodulin and of unsuspected F-actin binding regions in FLNa subunits. We demonstrated that F-actin binding by FLNa slows F-actin turnover in vitro and in vivo. We also identified that FLNa subunit flexibility, conferred in part by a surprisingly small hinge sequence, enables pre-stress to cause FLNa-F-actin gels to achieve high stiffness values associated with living substrate-attached cells. We also showed that myosin II filaments within FLNa-F-actin gels impose pre- stress, introducing a unique biomimetic machine. The multi-domain contributions of FLNa to F-actin mechanics and dynamics pale in complexity before the scores and probably hundreds of binding partners interacting with and functionally influenced by FLNa, including receptors, signaling intermediates, enzymes and transcription factors. We believe that only atomic structural information on FLNa and its interfaces with binding partners to inform point mutant reagents will enable us to sort out its cellular functions with confidence and think about designing inhibitors to treat diseases. We therefore now collaborate with structural biologists to solve FLNa atomic structures and prove concept that point mutations selectively impair specific FLNa functions, leaving most others intact. We also showed how FLNa's substructure accommodates both its F-actin cross-linking and partner binding and suggested how domain unfolding might regulate partner binding. We propose to build on this strategy, solving additional FLNa substructures as well as complexes with selected representative partners: CXCR4, CFTR and FilGAP, a Rac GAP we discovered that determines cell polarity. We will use point mutants to study the regulation of FilGAP in vivo and also investigate its role in neutrophil oxidase activity as well as obtain additional evidence for FLNa domain unfolding in partner binding regulation.

Public Health Relevance

This research concerns certain machinery parts in living cells that control how cells move, change shape, multiply and undertake many other functions. When these parts are missing or altered by genetic mutations, depending on the abnormality, mild or severe diseases occur. The research plan is to define the chemistry of one of these parts in sufficient detail to enable future development of drugs to ameliorate such diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL019429-36
Application #
8082744
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Eu, Jerry Pc
Project Start
1979-06-01
Project End
2012-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
36
Fiscal Year
2011
Total Cost
$713,654
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Song, Mia; He, Qianjing; Berk, Benjamin-Andreas et al. (2016) An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu). Biochem Biophys Res Commun 469:659-64
Gómez-Moutón, Concepción; Fischer, Thierry; Peregil, Rosa M et al. (2015) Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors. Blood 125:1116-25
Yang, Zhiping; Chiou, Terry Ting-Yu; Stossel, Thomas P et al. (2015) Plasma gelsolin improves lung host defense against pneumonia by enhancing macrophage NOS3 function. Am J Physiol Lung Cell Mol Physiol 309:L11-6
Nakamura, Fumihiko; Song, Mia; Hartwig, John H et al. (2014) Documentation and localization of force-mediated filamin A domain perturbations in moving cells. Nat Commun 5:4656
Zhou, Xiaohui; Massol, Ramiro H; Nakamura, Fumihiko et al. (2014) Remodeling of the intestinal brush border underlies adhesion and virulence of an enteric pathogen. MBio 5:
Xu, Tianyou; Lannon, Herbert; Wolf, Sébastein et al. (2013) Domain-domain interactions in filamin A (16-23) impose a hierarchy of unfolding forces. Biophys J 104:2022-30
Sun, Chunxiang; Forster, Carol; Nakamura, Fumihiko et al. (2013) Filamin-A regulates neutrophil uropod retraction through RhoA during chemotaxis. PLoS One 8:e79009
Ehrlicher, A J; Nakamura, F; Hartwig, J H et al. (2011) Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A. Nature 478:260-3
Nakamura, Fumihiko; Stossel, Thomas P; Hartwig, John H (2011) The filamins: organizers of cell structure and function. Cell Adh Migr 5:160-9
Kasza, K E; Broedersz, C P; Koenderink, G H et al. (2010) Actin filament length tunes elasticity of flexibly cross-linked actin networks. Biophys J 99:1091-100

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