important implications for muscular dystrophy and its therapy. Our previous TPA studies, detecting the microsecond dynamics of phosphorescent-labeled actin, showed that both proteins have novel effects on actin flexibility, with utrophin more effective than dystrophin (Prochniewicz et al., 2009, PNAS). We have now compared the effects of the isolated actin-binding domains of dystrophin, ABD1 and ABD2. TPA shows that the enhanced rate of actin rota- tional dynamics is induced primarily by ABD1, while both ABD1 and ABD2 contribute to the restriction in rotational amplitude. Disease-causing point mu- tations in ABD1 decrease the effects on actin’s rotational rate. We propose that this in turn causes the dystrophin-actin complex to be less resilient and thus less able to prevent damage to the muscle cytoskeleton during contraction. Finally, we have attached probes directly to ABD1 in dystrophin and utrophin, to detect changes in structure upon actin binding. High-resolution distance measure- ments, provided by DEER, show that the two lobes (calponin-homology do- mains) within ABD1 undergo a dramatic opening upon actin binding, helping to resolve a previous controversy (Lin et al., 2011, PNAS). Analogous studies with dystrophin show more subtle changes upon actin binding, provid- ing insight into the structural and functional differences in dystrophin and utrophin. 1899-Pos Board B669 Caldesmon Stabilizes Nascent Actin Filaments and Promotes Branching by Arp2/3 Complex Mikkel H. Jensen 1 , Eliza J. Morris 2 , Renjian Huang 3 , Roberto Dominguez 4 , David Weitz 2 , Jeffrey R. Moore 1 , Chih-Lueh Albert Wang 3 . 1 Boston University, Boston, MA, USA, 2 Harvard University, Cambridge, MA, USA, 3 Boston Biomedical Research Institute, Boston, MA, USA, 4 University of Pennsylvania, Philadelphia, PA, USA. Caldesmon is an actin-binding protein found in nearly all vertebrate cells. The heavy caldesmon isoform, which is specific to smooth muscle cells, is known to inhibit actomyosin interactions in vitro in a phosphorylation-dependent man- ner. However, possible roles of caldesmon as a regulator of actin mechanics and turnover are being explored, and the effects of caldesmon on actin dynam- ics and structure are not well understood. We have recently demonstrated that polymerizing actin undergoes an irrevers- ible structural transition (termed ‘‘maturation’’), and that the caldesmon C-ter- minal fragment, H32K, if added during the early stage of actin polymerization, prevents this maturation process (Huang et al., 2010 J Biol Chem 285(1):71- 79). Actin filaments stabilized in this ‘‘nascent’’ state by H32K appear rough under electron microscopy and exhibits attenuated pyrene fluorescence en- hancement compared to normal, mature F-actin, but H32K does not otherwise affect the polymerization kinetics of actin (Collins et al., 2011 Bioarchitechture 1(3):127-133). Both phosphorylated caldesmon and the actin-branching protein complex Arp2/3 are present at the leading edge of motile cells, where actin assembly provides the force needed to protrude the plasma membrane. Here, we study the interaction of H32K-stabilized nascent F-actin with Arp2/3 complex. By di- rect visualization of actin assembly in the presence of Arp2/3 complex using confocal fluorescence microscopy, we show that actin polymerized in the pres- ence of H32K exhibits increased branching activity. We propose that the ob- served change is a result of the structural state of the young actin filament, which is stabilized by the early added H32K. This effect suggests a role of cal- desmon as a regulator of actin structure, in turn dictating the interaction of other actin-binding proteins with actin. 1900-Pos Board B670 Structural and Biochemical Study of the Interaction Between Actin and the Mammalian Formin FMNL3 Morgan E. Thompson 1,2 , Ernest G. Heimsath 2 , Timothy J. Gauvin 2 , Henry N. Higgs 2 , F. Jon Kull 1,2 . 1 Dartmouth College, Hanover, NH, USA, 2 Dartmouth Medical School, Hanover, NH, USA. Formins are a class of proteins that influence the rate of actin filament nucle- ation and elongation. Mammals possess 15 formin isoforms, providing a myriad of possibilities for regulating actin-based structures in cells. The dimeric formin homology 2 (FH2) domain is capable of altering the nucleation rate of new ac- tin filaments and subsequently influences filament elongation via direct interac- tion with the barbed end of an actin filament. The FH2 domain moves processively with the barbed end as the filament elongates. Our goal in this study is to examine FMNL3’s interaction with actin to uncover mechanistic de- tails of formin regulated actin elongation. The FH2 domain of FMNL3 that forms a stable interaction with tetramethylrhodamine-malamide labeled actin (TMR-actin) and has been crystallized and the 3.3 A ˚ resolution structure has been solved. The crystal structure indicates that FMNL3 dimerizes to form a ring around the barbed end of two actin subunits. The formin/actin interface is comprised of multiple interactions that are found in three distinct regions of the FH2 domain. Mutational analysis identified key residues in FMNL3/actin binding as well as those essential for elongation activity. The study of these res- idues has led us to propose a more detailed model for the interaction of the FH2 domain with actin. A previous study with Bni1p, revealed an FH2 dimer con- formation spanning three actin subunits. This structure supports the model of a formin tracking with the growing end of an actin filament by taking steps with the addition of each monomer. The FMNL3/actin complex represents an intermediate step in the formin stepping mechanism in which two actin sub- units are aligned side-by-side surrounded by a symmetric FH2 dimer. This structure reveals mechanistic details of FH2 mediated actin filament elongation via processive capping. 1901-Pos Board B671 Tropomyosin and Caldesmon Enhance the Binding Force of Unphosphory- lated Myosin to Thin Filaments Horia N. Roman 1 , Nedjma B. Zitouni 1 , Apolinary Sobieszek 2 , Anne-Marie Lauzon 1 . 1 McGill University, Montreal, QC, Canada, 2 Institute of Biomedical Aging Research, Innsbruck, Austria. Smooth muscle is unique in its ability to maintain force for long periods of time at low energy consumption. This property, called the latch-state, has been pos- tulated to be a consequence of the dephosphorylation of myosin molecules when attached to the actin thin filament. Alternatively, unphosphorylated (unPHOS) myosin could potentially attach to the thin filaments, contributing to force maintenance. Thus, in this study we verified if unPHOS myosin can bind to actin in presence of tropomyosin and caldesmon and we measured its binding force using the laser trap. Briefly, a microsphere captured in a laser trap was attached to an actin filament. The filament was brought in contact with a pedestal coated with unPHOS myosin. The pedestal was then moved away from the trap at constant velocity. The microsphere followed the pedestal until the force exerted by the trap on the microsphere was sufficient to overcome the binding force of myosin to the actin filament. At this point, the microsphere sprang back into the trap center. The force of unbinding (Funb) was calculated as the product of the trap stiffness and the maximal distance between the trapped microsphere and the trap center. Funb was nor- malized by the number of myosin molecules estimated per actin filament length. Funb of unregulated actin filaments (0.11850.007 pN; mean5SE) was enhanced in presence of tropomyosin (0.1650.008 pN; p<0.05), caldes- mon (0.16950.017 pN; p<0.05) and a mix of both regulatory proteins (0.17450.022 pN; p<0.05). Our data demonstrate that unPHOS myosin binds to regulated actin filaments and that each regulatory protein increases this bind- ing force but that their effect is not cumulative. Thus, the actin regulatory pro- teins might play a major role in the latch-state, by enhancing the binding of unPHOS myosin. 1902-Pos Board B672 Novel Modulation of the S.Cerevsiae Actin-Cyctokeleton by Heterologous Expression of the Short N.Crassa Tropomyosin Seham Ebrahim 1 , Robin Maytum 2 . 1 Queen Mary, University of London, London, United Kingdom, 2 University of Bedfordshire, Luton, United Kingdom. Tropomyosins are actin-regulatory proteins found in eukaryotes from yeast to man. They are best understood in their role in the regulation of muscle contraction in higher eukaryotes. However, only a small subset of the 20 or more isoforms of tropomyosin found in man are involved in muscle regula- tion. Their extensive diversity indicates their importance in regulating the actin cytoskeleton, a role in which their function is only beginning to be understood. Saccharomyces cerrevisiae provides a simple model system for trying to under- stand the functioning of tropomyosins in regulating the actin cytoskeleton. It has two tropomyosins, the larger 5 actin spanning TPM1 and smaller 4 actin spanning TPM2. Knockout of the major TPM1 isoform produces defects in cell division and hence cell size, whilst knockout of the minor TPM2 isoform has no morphological effect. A double knockout is lethal. Previous studies of heterologous expression of other tropomyosins in a DTPM1 background have all produced unremarkable ‘rescue’ of the knockout effects. We have previously reported that Neurospora crassa also has two tropomyo- sins, but in this case the larger is a 4 actin spanning and the shorter an unusual 3 actin spanning tropomyosin. Heterologous expression of this short N. crassa tropomyosin in DTPM1 cells does not produce rescue. Instead we observe 374a Monday, February 27, 2012