Protrusion Velocity Research Articles

Actin filament branching and protrusion velocity in a simple 1D model of a motile cell

We formulate and analyse a 1D model for the spatial distribution of actin density at the leading edge of a motile cell. The model incorporates nucleation, capping, growth and decay of actin filaments, as well as retrograde flow of the actin meshwork and known parameter values based on the literature. Using a simplified geometry, and reasonable assumptions about the biochemical processes, we derive PDEs for the density of actin filaments and their tips. Analytic travelling wave solutions are used to predict how the speed of the cell depends on rates of nucleation, capping, polymerization and membrane resistance. Analysis and simulations agree with experimental profiles for measured actin distributions. Extended versions of the model are studied numerically. We find that our model produces stable travelling wave solutions with reasonable cell speeds. Increasing the rate of nucleation of filaments (by the actin related protein Arp2/3) or the rate of actin polymerization leads to faster cell speed, whereas increasing the rate of capping or the membrane resistance reduces cell speed. We consider several variants of nucleation (spontaneous, tip, and side branching) and find best agreement with experimentally measured spatial profiles of filament and tip density in the side branching case.

New Partners for Ena/VASP

The Ena/VASP family proteins are important regulators of the cytoskeleton that are recruited to sites of actin remodeling during cell adhesion, polarization, and migration. Within locations such as focal adhesions and lamellipodia, Ena/VASP proteins alter local actin dynamics through their interactions with actin and profilin, as well as through their associations with proteins that bind to their proline-rich protein interaction domain, EVH1. Less well understood is how Ena/VASP proteins locally alter actin dynamics in response to specific cell signals. Two related adaptor proteins have now been identified as Ena/VASP binding partners that could provide the link between localized membrane-based signaling events and cytoskeletal remodeling. Krause et al. have determined that mammalian Lamellipodin (Lpd), whose C. elegans ortholog, Mig-10, controls neuronal migration, binds to Ena/VASP proteins. Lpd and Ena/VASP proteins colocalized at the tips of lamellipodia in a variety of mammalian cell types and were isolated in a protein complex from primary cortical neurons. However, Lpd localization did not depend on the presence of Ena/VASP. Rather, Lpd directed Ena/VASP protein localization, a recruitment dependent on the EVH1-binding motifs within Lpd. A Ras-association (RA) and lipid-binding PH domain in Lpd were required for targeting to the leading edge of lamellipodia. The PH domain specifically associated with PI(3,4)P2, a phosphoinositide involved in cell polarization. Overexpression of Lpd also increased lamellipodia protrusion velocity in fibroblasts, an effect that required Ena/VASP expression. Knockdown of Lpd by RNA interference reduced actin polymerization in lamellipodia and impaired lamellipodia formation. Similar in Lpd, Rap1-interacting adaptor molecule (RIAM) was identified by Lafuente et al. as another Ena/VASP interacting protein. Both RIAM and Lpd exhibit similar protein domain structure, comprising a new family of adaptor proteins called MRL (Mig-10/RIAM/Lpd). In activated T cells, RIAM associated with activated Rap1, a Ras superfamily member that localizes to the plasma membrane to control cell adhesion and spreading. RIAM was also required for activated Rap1 to localize to the plasma membrane and stimulate integrin-mediated adhesion in T cells. RIAM was distributed throughout the leading edge of lamellipodia, and its overexpression induced cell adhesion. Knockdown of RIAM expression by RNA interference reduced the cellular content of polymerized F actin and adhesion. Although RIAM and Lpd are both found in the leading edge of lamellipodia, Lpd did not bind to activated Rap1 and did not localize to focal adhesions, which suggests that the two adaptors may couple Ena/VASP to distinct signaling pathways within similar subcellular locations. The two adaptor proteins may integrate signals from both phosphoinositides and Ras superfamily proteins and direct actin through association with Ena/VASP. M. Krause, J. D. Leslie, M. Stewart, E. M. Lafuente, F. Valderrama, R. Jagannathan, G. A. Strasser, D. A. Rubinson, H. Liu, M. Way, M. B. Yaffe, V. A. Boussiotis, F. B. Gertler, Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Devel. Cell 7 , 571-583 (2004). [Online Journal] E. M. Lafuente, A. A. F. L. van Puijenbroek, M. Krause, C. V. Carman, G. J. Freeman, A. Berezovskaya, E. Constantine, T. A. Springer, F. B. Gertler, V. A. Boussiotis, RIAM, an Ena/VASP and profilin ligand, interacts with RAP1-GTP and mediates Rap1-induced adhesion. Devel. Cell 7 , 585-595 (2004). [Online Journal]

Lamellipodin, an Ena/VASP Ligand, Is Implicated in the Regulation of Lamellipodial Dynamics

Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P 2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP.

Regulation of Actin Dynamics in Rapidly Moving Cells:A Quantitative Analysis

We develop a mathematical model that describes key details of actin dynamics in protrusion associated with cell motility. The model is based on the dendritic-nucleation hypothesis for lamellipodial protrusion in nonmuscle cells such as keratocytes. We consider a set of partial differential equations for diffusion and reactions of sequestered actin complexes, nucleation, and growth by polymerization of barbed ends of actin filaments, as well as capping and depolymerization of the filaments. The mechanical aspect of protrusion is based on an elastic polymerization ratchet mechanism. An output of the model is a relationship between the protrusion velocity and the number of filament barbed ends pushing the membrane. Significantly, this relationship has a local maximum: too many barbed ends deplete the available monomer pool, too few are insufficient to generate protrusive force, so motility is stalled at either extreme. Our results suggest that to achieve rapid motility, some tuning of parameters affecting actin dynamics must be operating in the cell.

The physiological significance of beta -actin mRNA localization in determining cell polarity and directional motility.

beta-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3' untranslated region, the "zip code." Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3' untranslated region leads to delocalization of beta-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after beta-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of beta-actin protein. These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Quantifying Lamella Dynamics of Cultured Cells by SACED, a New Computer-Assisted Motion Analysis

Formation of lamellipodia and the retraction of ruffles are essential activities during motility and migration of eukaryotic cells. We have developed a computer-assisted stroboscopic method for the continuous observation of cell dynamics (stroboscopic analysis of cell dynamics, SACED) that allows one to analyze changes in lamellipodia protrusion and ruffle retraction with high resolution in space and time. To demonstrate the potential of this method we analyzed keratinocytes in culture, unstimulated or stimulated with epidermal growth factor (EGF), which is known to induce cell motility and migration. Keratinocytes stimulated with EGF exhibited a 2.6-fold increase in their migration velocity, which coincided with enhanced ruffle retraction velocity (144%) and increased ruffle frequency (135%) compared to control cells. We also recorded an enhanced frequency of lamellipodia (135%), whereas the velocity of lamellipodia protrusion remained unchanged. These results on ruffle and lamellipodia dynamics in epidermal cells show that SACED is at least equal to established methods in terms of accuracy. SACED is, however, advantageous concerning resolution in time and therefore allows one to analyze the activity of lamellipodia and ruffles in as yet unknown detail. Moreover, SACED offers two opportunities that render this technique superior to established methods: First, several parameters relevant to cell motility can be analyzed simultaneously. Second, a large number of cells can conveniently be examined, which facilitates the compilation of statistically significant data.

Numerical simulation of flying phenomena of the running tape on rotating drum

This paper analyzes the running mechanism of flexible and thin tape above rotating protrusion through a numerical simulation. The scope of analysis is confined to the phenomena of elastohydrodynamic lubrication between the rotating drum with a protrusion and the running tape. This model is based on the modified Reynolds equation and the equation of plate considering the effect of geometric nonlinearity and geometry of protrusion. Finite element method of Bubnov-Galerkin type is adopted as a numerical simulation technique to solve the above two coupled nonlinear equations. In numerical simulation, the influences of tape tension and protrusion velocity are evaluated in simple model. In complex models, the reciprocal action of two protrusions is simulated.