polymerization that are quantitatively similar to those observed along the leading edge in live cell experiments. 3221-Pos Board B326 Myosin Motor Dynamics in Filopodia Pavel I. Zhuravlev, Maria Minakova, Yueheng Lan, Garegin A. Papoian. ATP-driven molecular motors are ubiquitously used for various cellular trans- port processes. Myosin motors that walk on actin filaments have been detected in both filopodia and stereocilia. Myosin X plays an important role in filopodial formation and growth, however, the specific mechanisms and cargo of various myosins remain unclear. To address these issues, we have developed a comprehensive stochastic com- putational model of a filopodium, incorporating active transport by motors. Our model indicates that efficient motor mediated transport is hindered by se- questration of G-actin cargo by motors, as well as clogging of the actin fila- ments by multiple motors. Our work suggests potential mechanisms for overcoming these transport bottlenecks. Furthermore, experimentally measured motor concentration profiles along the filopodial tube are difficult to explain. This motivated us to look into the details of this distribution. We created a theoretical model describing the steady-state concentration profile of the motors and compared it with the computer simula- tions. The theoretical results semi-quantitatevly agree with the numerical data. In the stationary state, the model shows a quick saturation of actin filaments with walking motors as a function of distance from the filopodial base. This ad- versely affects the flux of motors and their cargo to the filopodial tip, as space for motors to step into runs out. Thus, the clogging problem is at least as im- portant as the sequestration one, and the cell would probably need to employ special facilities for solving it in order to achieve efficient active transport, and thus longer filopodia. 3222-Pos Board B327 Model of the Role of Actin Crosslinker Proteins during Contractile Ring Assembly in Fission Yeast Nikola Ojkic, Dimitrios Vavylonis. Actin cross linkers play a crucial role in various processes during cell cycle. Contractile ring formation in fission yeast is one of the processes where the role of two actin crosslinkers fimbrin and alpha actinin is important. In fission yeast contractile ring assembles from a broad band of cortical nodes. Fission yeast nodes are macromolecular complexes containing several myosin-II heads and a few formin dimers. The condensation of the broad band of nodes into a ring has been described by a search, capture, pull and release (SCPR) model. In this model, formin nucleates filaments along random directions parallel to the cell membrane. When a growing filament comes in close proximity to a my- osin of a neighboring node, myosin grabs the filament and exerts force that moves nodes toward one other. Using analytical calculations and numerical simulations, we have shown that the SCPR model predicts that nodes coalesce into rings or clumps (i.e. disconnected aggregates), depending on the values of parameters. In experimental images of fission yeast, myosin nodes appear to get stabilized into linear elements towards the late stages of ring assembly, presum- ably due to crosslinkers. This process may assist ring formation by preventing the formation of clumps. To study this possible role of crosslinkers, we incor- porated a short-range aligning force to the SCPR model, representing a short range aligning mechanism. We used simulations and scaling arguments to quantify the resulting dynamics and morphology of myosin aggregates. We de- scribe regions in parameter space in which local alignment prevents clump for- mation. We compare the results to experiments on fission yeast cells in the process of contractile ring formation. 3223-Pos Board B328 Fluctuation-Dissipation Relations in Minimal Models for Active Driving Yair Shokef, Eyal Ben-Isaac, Nir S. Gov. Biological systems exhibit fluctuations driven by active (energy consuming) processes, such as molecular motors in the cytoskeleton and flagella propelling bacteria. It is tempting to treat these fluctuations in the same manner that ther- mal fluctuations are treated in equilibrium systems. Recent experiments have compared these inherent fluctuations with the response to small external pertur- bations. From these measurements, an effective temperature may be defined us- ing the fluctuation-dissipation formalism of equilibrium statistical mechanics. In active systems, this effective temperature is frequency dependent (unlike the thermal case), and is higher than the ambient temperature. Another param- eter that is useful for characterizing deviations from equilibrium of such fluc- tuations is the kurtosis of their distribution function; deviations of the kurtosis from its value for a Gaussian distribution indicate that the system may not be mapped onto an equilibrium ensemble. We address these issues theoretically by examining simple model systems of randomly kicked "particles", with a variable number of kicking "motors". Our generalized particles and motors represent different objects in different real systems, and we make specific choices when comparing to experimental observations. We exactly calculate the non-equilibrium fluctuation-dissipation relations and resulting effective temperatures. We show that their frequency de- pendence is model dependent and generally non-monotonous. We investigate the non-Gaussianity of the velocity distribution functions by a combination of numerical simulations and theoretical estimates. We show that the kurtosis has a non-monotonous dependence on the motor activity. We identify situations in which these two indicators of non-equilibrium behavior contradict. In partic- ular, in the presence of multiple motors, the kurtosis of the distribution recovers its Gaussian value, while the effective temperature may still exhibit strong fre- quency dependence. We compare our results with two recent measurements of oscillations of the red-blood cell membrane, where these two indicators where independently measured. 3224-Pos Board B329 Investigating Function of Nck Adaptor in Tyrosine Kinase Signaling to the Actin Cytoskeleton Sofya Borinskaya, Jon A. Ditlev, Les M. Loew, Bruce J. Mayer. Many extracellular signals are transduced through Receptor Tyrosine Ki- nases. We investigate RTK signaling to the actin cytoskeleton through the Nck adaptor protein. The SH2 domain of Nck binds phosphorylated tyrosine residues of RTK and then SH3 domains of Nck bind and activate N-WASP (Neural Wiskott-Aldrich Syndrome Protein). Activation of N-WASP involves release of the VCA domain (verprolin-homology, central and acidic do- mains). Simultaneous binding of G-actin monomer and actin nucleation fac- tor Arp2/3 to the VCA domain initiates nucleation of a new actin branch. Aggregation of the Nck SH3 domains on plasma membrane in vivo results in the formation of long, dynamic, moderately dense actin comet tails. We uti- lize a variety of techniques to study this artificially induced localized actin polymerization: cell culture and quantitative western blotting, confocal fluo- rescent microscopy and quantitative image analysis, biochemical modeling in the Virtual Cell environment. Our model recapitulates important features of Nck actin comets: length of the comet tails, F-actin concentration within the tails, distance from the head of the aggregate to the maximum F-actin concentration location, and dependence of these parameters on comet veloc- ity and the number of Nck molecules in the membrane cluster. However the model does not account for the fact that Nck binding proteins (WIP, PAK, Abl) can have an effect on actin polymerization. The VCA domain of N- WASP is directly responsible for Arp2/3 activation and can be used in our experimental system to bypass the need for adaptor proteins such as Nck to initiate actin polymerization. Aggregation of VCA domains on the membrane in vivo produced actin ‘‘blobs’’ - short thick structures. Under- standing the differences between the Nck and VCA induced localized actin assemblies will advance our knowledge of RTK signaling to mobilize and direct actin cytoskeleton. 3225-Pos Board B330 Nck Function in Tyrosine Kinase Signaling to the Actin Cytoskeleton Jonathon A. Ditlev, Bruce J. Mayer, Leslie M. Loew. Tyrosine kinase signaling leads to the post-translational modification of pro- teins and their binding partners. These modifications lead to the membrane re- cruitment of signaling proteins, promoting an increase in their local concentration, which results in a cellular response to the phosphorylation of ty- rosine residues. Nck, an SH2/SH3 adaptor protein, functions in tyrosine kinase signaling by linking tyrosine phosphorylation on the membrane with binding partners, such as N-WASp, that function in facilitating actin nucleation and po- lymerization. Recent in vitro studies have demonstrated that N-WASp dimers bind to and activate N-WASp far more efficiently than individual N-WASp molecules. Because Nck has been shown to induce actin comet tail formation in an N-WASp dependent manner, we manipulate this molecular interaction to test the effect of N-WASp dimerization on actin polymerization. To manipulate the pathway, we vary the concentration of Nck within antibody-induced aggre- gates on the cellular surface and then use precise quantitative analysis of Nck density and induced actin polymerization to analyze the system. Using the Vir- tual Cell, we have built a comprehensive, quantitative actin cycle model. With this model we have tested 2 mathematical expressions for dimerized N-WASp activation of Arp2/3, which produce different experimentally testable predic- tions. These results demonstrate a nonlinear relationship between the density of Nck aggregates and actin polymerization and also demonstrate that a qua- dratic-based formula describing dimerized N-WASp activation of Arp2/3 596a Wednesday, March 9, 2011