The Fire Inside: Eukaryotic Locomotion

The GTP-based Rho/Rac system of actin regulation runs in unseen waves of activation.

One of the amazing capabilities of eukaryotic cells, inherited in part from their archaeal parents, is free movement and phagocytosis. These cells have an internal cytoskeleton, plus methods to anchor to a substrate, (via focal adhesions), which allows them to manipulate their membrane, their shape, and their locomotion. The cytoskeleton is composed of two main types of fibers, actin and microtubules. Microtubules are much larger than actin and organize major trackways of organelle movement around the cell (including the movement of chromosomes in mitosis), and also form the core of cilia and flagella. But it is actin that does most of the work of moving cells around, with dynamic networks that generate the forces behind spiky to ruffly protrusions, that power things like the adventuresome pathfinding of neurons as they extend their axons into distant locations.

Schematic of the actin cytoskeleton of a typical eukaryotic cell.

Actin is an ATPase all by itself. ATP promotes its stability, and also its polymerization into filaments. So, cell edges can grow just by adding actin to filament ends. Actin cross-linking proteins also exist, that create the meshwork that supports extended filopodia. But obviously, actin all by itself is not a regulated solution to cell movement. There is an ornately complex system of control, not nearly understood, that revolves around GTPase and binding proteins. These proteins (mainly RhoA, Rac1, and Cdc42, though there are twenty related family members in all in humans) have knife-edge regulation, being on when binding GTP, and off after they cleave off the phosphate and are left binding GDP (the typical, default, state). Yet other proteins regulate these regulators- GTPase exchange factors (GEFs) encourage release of GDP and binding of GTP, while GTPase activating proteins (GAPs) encourage the cleavage of GTP to GDP. The GTP binding proteins interact (depending on their GTP status) with a variety of effector proteins. One example is a family of formins, which chaperone the polymerization of actin. At the head of the pathway, signals coming from external or internal conditions regulate the GTPases, creating (in extremely simplified terms) a pathway that gets the cell to respond by moving toward things it wants, and away from things it does not want. 

This is a very brief post, just touching on one experiment done on this system. Exploring its full complexity is way beyond my current expertise, though we may return to aspects of this fascinating biological pathway periodically in the future. An important paper in the field hooked up fluorescent dyes to one of the effector protein domains that binds only GTP/active RhoA. They tethered this to the (inside) membrane of their cultured cells, and took movies of what the cell looked like, using a microscopy method that looks at very thin sections- only the membrane, essentially not the rest of the cell. RhoA, though graced with a small lipid tail, is typically cytoplasmic when inactive, and travels to the membrane when activated. They were shocked to find that in resting cells, without much locomotion going on, there were recurring waves of activation of RhoA that swept hither and yon across the cell membranes. 

Four examples of RhoA getting bound in its active state in a wave-like way, over 7 1/2 minutes in a resting cell. GEF-H1 is ARHGEF2, one of the regulators that can turn RhoA on. The first three panels have ARHGEF2 versions that are operational, but the fourth (bottom right) is of a cell with an anti-RNA to ARHGEF2, turning its expression level down. In this cell, the waves of RhoA activation and recruitment to the membrane are substantially dampened.

These pulses were made even more intense if the cells were treated with nocodazole, which disrupts microtubules, destabilizes the cytoskeleton, and makes the actin regulatory / structure system work harder. They found that myosin (the motor protein that moves cargoes over actin filaments) was also rapidly relocalized, mirroring some of what happened with RhoA. They also found that ARHGEF2 contained two RhoA binding domains, (one binding active RhoA, one binding inactive RhoA), enabling it to feedback-amplify the positive activation of RhoA, thereby explaining some of the extremely dynamic activity seen here. 

And they also found that the arrival of negative regulators such as ARHGAP35 was delayed by a couple of seconds vs the activation of RhoA, providing the time window needed to see wave formation out of a mechanism of positive feedback followed by squelching by a negative regulator. Lastly, they found that these dynamics were significantly different if the cells were grown on stiffer vs softer substrates. Stiffer substrates allowed the formation of stronger surface attachments, concentrating RhoA and myosin at these adhesion locations. 

These researchers are clearly only scratching the surface of this system, as there are endless complexities left to investigate. The upshot of this one set of observations is that neurons are not the only excitable cells. With a bit of molecular / experimental magic, heretofore unseen intracellular dynamics can be visualized to show that eukaryotic cells have an exquisitely regulated internal excitation system that is part of what drives their shape-shifting capabilities, including processes like phagocytosis and neuronal growth / path-finding. 

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