The long-term goal is to understand the structural and functional mechanisms that control actin cytoskeleton dynamics in health and disease. Malfunction of cytoskeletal components is linked to many diseases, and several bacterial pathogens hijack the cytoskeleton for infection. Here, the focus will be on actin filament nucleation, which determines the time and location for actin polymerization.
Aim 1 will address the molecular mechanism for nucleation, branch formation and debranching by Arp2/3 complex. Arp2/3 complex is activated by nucleation promoting factors (NPFs). Two NPFs are needed for optimal activation. One of the NPFs interacts with subunit ArpC1 of Arp2/3 complex. We will characterize this interaction at the structural and functional levels. New data suggests that interaction of Arp2/3 complex with NPFs promotes binding to the side of the mother filament to form a branch. We will test the role of pre-association of NPFs with actin for binding to the mother filament. In cells, older branches are disassembled by GMF. We will characterize the mechanism of debranching of mammalian Arp2/3 complex by GMF using TIRF microscopy, and establish the role of GMF phosphorylation for this activity.
In Aim 2 will address the molecular mechanism for filament nucleation by Lmod, a muscle-specific nucleator discovered in our lab. We previously characterized the skeletal/cardiac isoform Lmod2. The smooth muscle isoform Lmod1 shares only 37% sequence identity with Lmod2, and is 105-aa longer, which translates into substantial functional differences between the two isoforms. We will investigate the two isoforms in parallel, to understand the structural and functional bases for their muscle-type specific activities.
Aim 3 will address the molecular mechanism for filament nucleation and elongation by Rickettsia Sca2. Sca2 is the only bacterial protein known to promote both, actin nucleation and elongation, mimicking eukaryotic formins to assemble actin comet tails for Rickettsia motility and virulence. We recently discovered that Sca2's functional mimicry of formins is achieved through a novel molecular mechanism. Here we will investigate how the interaction of the N- and C-terminal domains of Sca2 leads to actin nucleation and elongation within a monomeric protein.

Public Health Relevance

This project addresses a major lack of understanding of the mechanisms controlling actin cytoskeleton dynamics in health and disease. A better understanding of these processes, and specifically actin filament nucleation, has both fundamental importance and direct relevance to human health, since malfunctioning of cytoskeletal proteins is the direct cause of many diseases, including cancer metastasis, neurodegenerative disorders and defective immune response.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gindhart, Joseph G
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University of Pennsylvania
Schools of Medicine
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Fowler, Velia M; Dominguez, Roberto (2017) Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles. Biophys J 112:1742-1760
Burke, Thomas A; Harker, Alyssa J; Dominguez, Roberto et al. (2017) The bacterial virulence factors VopL and VopF nucleate actin from the pointed end. J Cell Biol 216:1267-1276
Boczkowska, Malgorzata; Yurtsever, Zeynep; Rebowski, Grzegorz et al. (2017) Crystal Structure of Leiomodin 2 in Complex with Actin: A Structural and Functional Reexamination. Biophys J 113:889-899
Dominguez, Roberto (2016) The WH2 Domain and Actin Nucleation: Necessary but Insufficient. Trends Biochem Sci 41:478-490
Boczkowska, Malgorzata; Rebowski, Grzegorz; Kremneva, Elena et al. (2015) How Leiomodin and Tropomodulin use a common fold for different actin assembly functions. Nat Commun 6:8314
Dominguez, Roberto (2015) Subversive bacteria reveal new tricks in their cytoskeleton-hijacking arsenal. Nat Struct Mol Biol 22:178-9
Rao, Jampani Nageswara; Madasu, Yadaiah; Dominguez, Roberto (2014) Mechanism of actin filament pointed-end capping by tropomodulin. Science 345:463-7
Boczkowska, Malgorzata; Rebowski, Grzegorz; Kast, David J et al. (2014) Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs. Nat Commun 5:3308
Shuman, Henry; Greenberg, Michael J; Zwolak, Adam et al. (2014) A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning. Proc Natl Acad Sci U S A 111:2116-21
Kremneva, Elena; Makkonen, Maarit H; Skwarek-Maruszewska, Aneta et al. (2014) Cofilin-2 controls actin filament length in muscle sarcomeres. Dev Cell 31:215-26

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