Design of active transport must be highly intricate: a possible role of myosin and Ena/VASP for G-actin transport in filopodia

Abstract

Recent modeling of filopodia--the actin-based cell organelles employed for sensing and motility--reveals that one of the key limiting factors of filopodial length is diffusional transport of G-actin monomers to the polymerizing barbed ends. We have explored the possibility of active transport of G-actin by myosin motors, which would be an expected biological response to overcome the limitation of a diffusion-based process. We found that in a straightforward implementation of active transport the increase in length was unimpressive, < or = 30%, due to sequestering of G-actin by freely diffusing motors. However, artificially removing motor sequestration reactions led to approximately threefold increases in filopodial length, with the transport being mainly limited by the motors failing to detach from the filaments near the tip, clogging the cooperative conveyer belt dynamics. Making motors sterically transparent led to a qualitative change of the dynamics to a different regime of steady growth without a stationary length. Having identified sequestration and clogging as ubiquitous constraints to motor-driven transport, we devised and tested a speculative means to sidestep these limitations in filopodia by employing cross-linking and putative scaffolding roles of Ena/VASP proteins. We conclude that a naïve design of molecular-motor-based active transport would almost always be inefficient--an intricately organized kinetic scheme, with finely tuned rate constants, is required to achieve high-flux transport.

Figure 1

A schematic representation of the filopodial tip in the model is shown. A bundle of polymerizing actin filaments is enveloped by membrane which affects polymerization rates. Transported G-actin must dissociate before polymerization. Retrograde flow pulls filaments back with constant velocity. Myosin X motors travel the filaments in a directed fashion toward the barbed ends at the filopodial tip. Ena/VASP serves as a scaffold between G-actin monomers and motor molecules, and it is consumed near the tip due to cross-linking of the filaments.

Figure 2

Filopodial stationary length, as a function of model motor-related parameters, is shown. Bulk motor concentration is color-coded. Filament unbinding rate for the motors is equal to 300 s⁻¹ (thick solid lines), 100 s⁻¹ (thin solid lines), and 30 s⁻¹ (dashed lines). As the motor unloading rate k_u essentially defines which fraction of motors carry actin, the length dependence comes mostly from the ratio of motor concentration to unloading rate. For this reason, the latter ratio is used as the variable on x axis.

Figure 3

Only in specific local regions of the parameter space do motors provide an increase in filopodial length. These regions are always characterized by low motor concentrations. High motor concentration area does not lead to stable filopodia as most G-actin required for growth is sequestered.

Figure 4

Comparison between diffusion-limited and linear growth regimes is shown. These are two simulations with different parameters from the Ena/VASP set.

Figure 5

The largest observed stationary length in each set of simulations is shown. In the case of simulations with artificial conditions in which motors do not sequester actin and do not clog the filaments (inasmuch as they are sterically transparent to each other), a linear growth regime was observed that did not reach a stationary length.