Computer-assisted tracking of actin filament motility.

In vitro motility assays, in which fluorescently labeled actin filaments are propelled by myosin molecules adhered to a glass coverslip, require that actin filament velocity be determined. We have developed a computer-assisted filament tracking system that reduced the analysis time, minimized investigator bias, and provided greater accuracy in locating actin filaments in video images. The tracking routine successfully tracked filaments under experimental conditions where filament density, size, and extent of photobleaching varied dramatically. Videotaped images of actin filament motility were digitized and processed to enhance filament image contrast relative to background. Once processed, filament images were cross correlated between frames and a filament path was determined. The changes in filament centroid or center position between video frames were then used to calculate filament velocity. The tracking routine performance was evaluated and the sources of noise that contributed to errors in velocity were identified and quantified. Errors originated in algorithms for filament centroid determination and in the choice of sampling interval between video frames. With knowledge of these error sources, the investigator can maximize the accuracy of the velocity calculation through access to user-definable computer program parameters.     

Theoretical modeling of aging effects in microtubule dynamics

The microtubule (MT) network, an important part of the cytoskeleton, is constantly remodeled by alternating phases of growth and shrinkage of individual filaments. Plus-end tracking proteins (+TIPs) interact with the MT and in many cases alter its dynamics. Although it is established that some +TIPs modify MT dynamics by increasing rescues, the plus-end tracking mechanism is still under debate. We present a model for MT dynamics in which a rescue factor is dynamically added to the filament during growth. As a consequence, the filament shows aging behavior that should be experimentally accessible and thus allow one to exclude some hypothesized models regarding the inclusion of rescue factors at the MT plus end. This result is not limited to +TIPs and can be extended to any kind of mechanism shifting the parameters of dynamic instability. Additionally, we show that the cell geometry has a strong influence on the quantitative results.     

VASP is a processive actin polymerase that requires monomeric actin for barbed end association

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (K(d) = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (K(off) = 0.69 s(-1)) polymerases that deliver multiple actin monomers per barbed end-binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.     

Nematode sperm motility: nonpolar filament polymerization mediated by end-tracking motors

In nematode sperm cell motility, major sperm protein (MSP) filament assembly results in dynamic membrane protrusions in a manner that closely resembles actin-based motility in other eukaryotic cells. Paradoxically, whereas actin-based motility is driven by addition of ATP-bound actin subunits onto actin filament plus-ends located at the cell membrane, MSP dimers assemble from solution into nonpolar filaments that lack a nucleotide binding site. Thus, filament polarity and on-filament ATP hydrolysis, although essential for actin-based motility, appear to be unnecessary for membrane protrusions by MSP. As a potential resolution to this paradox, we propose a model for MSP filament assembly and force generation by MSP filament end-tracking proteins. In this model, ATP hydrolysis drives affinity-modulated, processive interactions between membrane-associated proteins and elongating filament ends. However, in contrast to the "actoclampin" model for actin filament end-tracking motors, ATP activates the tracking protein (or a soluble cofactor) rather than the MSP subunits themselves (in contrast to activation of actin subunits by ATP binding). The MSP end-tracking model predicts properties that are consistent with several key observations of MSP-based motility, including persistent membrane attachment, polymerization of filament ends at the membrane with depolymerization of free-filament ends away from the membrane, as well as a saturating dependence of polymerization rate on the concentration of non-MSP soluble cytoplasmic components.     

Properties of intermediate filament networks assembled from keratin 8 and 18 in the presence of mg(2+)

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg²⁺). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg²⁺ at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg²⁺-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.     

In vitro sliding of actin filaments labelled with single quantum dots

We recently refined the in vitro motility assay for studies of actomyosin function to achieve rectified myosin induced sliding of actin filaments. This paves the way, both for detailed functional studies of actomyosin and for nanotechnological applications. In the latter applications it would be desirable to use actin filaments for transportation of cargoes (e.g., enzymes) between different predetermined locations on a chip. We here describe how single quantum dot labelling of isolated actin filaments simultaneously provides handles for cargo attachment and bright and photostable fluorescence labels facilitating cargo detection and filament tracking. Labelling was achieved with preserved actomyosin function using streptavidin-coated CdSe quantum dots (Qdots). These nanocrystals have several unique physical properties and the present work describes their first use for functional studies of isolated proteins outside the cell. The results, in addition to the nanotechnology developments, open for new types of in vitro assays of isolated biomolecules.     

Thin filament length regulation in striated muscle sarcomeres: pointed-end dynamics go beyond a nebulin ruler

The actin (thin) filaments in striated muscle are highly regulated and precisely specified in length to optimally overlap with the myosin (thick) filaments for efficient myofibril contraction. Here, we review and critically discuss recent evidence for how thin filament lengths are controlled in vertebrate skeletal, vertebrate cardiac, and invertebrate (arthropod) sarcomeres. Regulation of actin polymerization dynamics at the slow-growing (pointed) ends by the capping protein tropomodulin provides a unified explanation for how thin filament lengths are physiologically optimized in all three muscle types. Nebulin, a large protein thought to specify thin filament lengths in vertebrate skeletal muscle through a ruler mechanism, may not control pointed-end actin dynamics directly, but instead may stabilize a large core region of the thin filament. We suggest that this stabilizing function for nebulin modifies the lengths primarily specified by pointed-end actin dynamics to generate uniform filament lengths in vertebrate skeletal muscle. We suggest that nebulette, a small homolog of nebulin, may stabilize a correspondingly shorter core region and allow individual thin filament lengths to vary according to working sarcomere lengths in vertebrate cardiac muscle. We present a unified model for thin filament length regulation where these two mechanisms cooperate to tailor thin filament lengths for specific contractile environments in diverse muscles.     

Emergence and maintenance of variable-length actin filaments in a limiting pool of building blocks

Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the length of single actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different lengths using the same set of molecular building blocks. Here, we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth-rate modulation by actin-binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneity. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning filamentous actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.     

Multiple-particle tracking measurements of heterogeneities in solutions of actin filaments and actin bundles

One of the central functions of actin cytoskeleton is to provide the mechanical support required for the establishment and maintenance of cell morphology. The mechanical properties of actin filament assemblies are a consequence of both the available polymer concentration and the actin regulatory proteins that direct the formation of higher order structures. By monitoring the displacement of well-dispersed microspheres via fluorescence microscopy, we probe the degree of spatial heterogeneity of F-actin gels and networks in vitro. We compare the distribution of the time-dependent mean-square displacement (MSD) of polystyrene microspheres imbedded in low- and high-concentration F-actin solutions, in the presence and absence of the F-actin-bundling protein fascin. The MSD distribution of a 2. 6-microM F-actin solution is symmetric and its standard deviation is similar to that of a homogeneous solution of glycerol of similar zero-shear viscosity. However, increasing actin concentration renders the MSD distribution wide and asymmetric, an effect enhanced by fascin. Quantitative changes in the shape of the MSD distribution correlate qualitatively with the presence of large heterogeneities in F-actin solutions produced by increased filament concentration and the presence of actin bundles, as detected by confocal microscopy. Multiple-particle tracking offers a new, quantitative method to characterize the organization of biopolymers in solution.     

Myosin V passing over Arp2/3 junctions: branching ratio calculated from the elastic lever arm model

Myosin V is a two-headed processive motor protein that walks in a hand-over-hand fashion along actin filaments. When it encounters a filament branch, formed by the Arp2/3 complex, it can either stay on the straight mother filament, or switch to the daughter filament. We study both probabilities using the elastic lever arm model for myosin V. We calculate the shapes and bending energies of all relevant configurations in which the trail head is bound to the actin filament before Arp2/3 and the lead head is bound either to the mother or to the daughter filament. Based on the assumption that the probability for a head to bind to a certain actin subunit is proportional to the Boltzmann factor obtained from the elastic energy, we calculate the mother/daughter filament branching ratio. Our model predicts a value of 27% for the daughter and 73% for the mother filament. This result is in good agreement with recent experimental data.     

Microheterogeneity controls the rate of gelation of actin filament networks

Rapid sol-gel transitions of the actin cytoskeleton are required for many key cellular processes, including cell spreading and cell locomotion. Actin monomers assemble into semiflexible polymers that rapidly intertwine into a network, a process that in vitro takes approximately 1 min for an actin concentration of 1 mg/ml. The same actin filament network, however, takes approximately 1 h to exhibit a steady-state elasticity. We hypothesize that the slow gelation of F-actin is due to the slow establishment of a homogeneous meshwork. Using a novel method, time-resolved multiple particle tracking, which monitors the range of thermally excited displacements of microspheres imbedded in the network, we show that the increase in elasticity in a polymerizing solution of actin parallels the progressive decline of the network microheterogeneity. The rates of gelation and network homogenization slightly decrease with actin concentration and in the presence of the F-actin cross-linking proteins alpha-actinin and fascin, whereas the rate of actin polymerization increases dramatically with actin concentration. Our measurements show that the slow spatial homogenization of the actin filament network, not actin polymerization or the formation of polymer overlaps, is the rate-limiting step in the establishment of an elastic actin network and suggest that a new activity of F-actin binding proteins may be required for the rapid formation of a homogeneous stiff gel.     

Fluctuating elastic filaments under distributed loads

Filaments under distributed loads are common in biological systems. In this paper, we study the thermo-mechanical properties of an extensible thermally fluctuating elastic filament under distributed forces. The ground state of the filament is solved first, followed by an investigation of the thermal fluctuations around the ground state. We first consider a special case where the tangential component of the distributed force tau is uniform along the filament. For the force-extension relation in this case, we show that the filament is equivalent to one under end-to-end applied force F = tauL0/2 where L0 is the length of the filament. To study the thermal fluctuations under more general distributed loadings, the filament is first discretized into segments, and its energy is approximated up to quadratic order. Then the partition function of the discretized filament, or chain, is evaluated using multi-dimensional Gaussian integrals, from which free energy and other properties of the filament are derived. We show that a filament under distributed loads suffers larger thermal fluctuations than one with the end loads of the same magnitude. We also show that our results for a discretized filament agree with continuum theory for a continuous rod. Finally, we give some applications of our ideas to the stretching and fluctuation of DNA in non-uniform microfluidic channels.     

Numerical simulations of odorant detection by biologically inspired sensor arrays

The antennules of many marine crustaceans enable them to rapidly locate sources of odorant in turbulent environmental flows and may provide biological inspiration for engineered plume sampling systems. A substantial gap in knowledge concerns how the physical interaction between a sensing device and the chemical filaments forming a turbulent plume affects odorant detection and filters the information content of the plume. We modeled biological arrays of chemosensory hairs as infinite arrays of odorant flux-detecting cylinders and simulated the fluid flow around and odorant flux into the hair-like sensors as they intercepted a single odorant filament. As array geometry and sampling kinematics were varied, we quantified distortion of the flux time series relative to the spatial shape of the original odorant filament as well as flux metrics that may be important to both organisms and engineered systems attempting to measure plume structure and/or identify chemical composition. The most important predictor of signal distortion is the ratio of sensor diameter to odorant filament width. Achieving high peak properties (e.g. sharpness) of the flux time series and maximizing the total number of odorant molecules detected appear to be mutually exclusive design goals. Sensor arrays inspired specifically by the spiny lobster Panulirus argus and mantis shrimp Gonodactylaceus falcatus introduce little signal distortion but these species' neural systems may not be able to resolve plume structure at the level of individual filaments via temporal properties of the odorant flux. Current chemical sensors are similarly constrained. Our results suggest either that the spatial distribution of flux across the aesthetasc array is utilized by P. argus and G. falcatus, or that such high spatiotemporal resolution is unnecessary for effective plume tracking.     

Filament-Filament Switching Can Be Regulated by Separation Between Filaments Together with Cargo Motor Number

How intracellular transport controls the probability that cargos switch at intersections between filaments is not well understood. In one hypothesis some motors on the cargo attach to one filament while others attach to the intersecting filament, and the ensuing tug-of-war determines which filament is chosen. We investigate this hypothesis using 3D computer simulations, and discover that switching at intersections increases with the number of motors on the cargo, but is not strongly dependent on motor number when the filaments touch. Thus, simply controlling the number of active motors on the cargo cannot account for in vivo observations that found reduced switching with increasing motor number, suggesting additional mechanisms of regulation. We use simulations to show that one possible way to regulate switching is by simultaneously adjusting the separation between planes containing the crossing filaments and the total number of active motors on the cargo. Heretofore, the effect of filament-filament separation on switching has been unexplored. We find that the switching probability decreases with increasing filament separation. This effect is particularly strong for cargos with only a modest number of motors. As the filament separation increases past the maximum head-to-head distance of the motor, individual motors walking along a filament will be unable to reach the intersecting filament. Thus, any switching requires that other motors on the cargo attach to the intersecting filament and haul the cargo along it, while motor(s) engaged on the original filament detach. Further, if the filament separation is large enough, the cargo can have difficulty proceeding along the initial filament because the engaged motors can walk underneath the intersecting filament, but the cargo itself cannot fit between the filaments. Thus, the cargo either detaches entirely from the original filament, or must dip to the side of the initial filament and then pass below the crossing filament.     

Diffusion rate limitations in actin-based propulsion of hard and deformable particles

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.     

Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

Actin and myosin function in acanthamoeba

We have studied the functions of contractile proteins in Acanthamoeba by a combination of structural, biochemical and physiological approaches. We used electron microscopy and image processing to determine the three-dimensional structure of actin and the orientation of the molecule in the actin filament. We measured the rate constants for actin filament elongation and re-evaluated the effect of MgCl2 on the filament nucleation process. In Acanthamoeba actin polymerization is regulated, at least in part, by profilin, which binds to actin monomers, and by capping protein, which both nucleates polymerization and blocks monomer addition at the 'barbed' end of the filament. To test for physiological functions of myosin-II, we produced a monoclonal antibody that inhibits the actin-activated ATPase. When microinjected into living cells, this active-site-specific antibody inhibits amoeboid locomotion. We expect that similar experiments can be used to test for the physiological functions of the other components of the Acanthamoeba contractile system.     

Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.     

SWAP-70 identifies a transitional subset of actin filaments in motile cells

Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.