How cytoskeleton dynamics mediate cell migration

HFSP Young Investigator Grant Awardees Gaudenz Danuser, Clare Waterman-Storer and colleagues

Cell locomotion represents one of the most critical processes in normal physiology and disease. For instance, during the development of an embryo cells divide into daughter cells over many generations, resulting in millions of cells which differentiate and then must migrate to the right place to organize in tissues and organs with specialized functions. The same migration mechanisms are exploited by cells of the immune system when they chase e.g. a bacterium, catch it, and isolate it from the intruded tissue. The healing of a wound is promoted by yet another class of cells that crawl into the fissure and close it. On the other hand, uncontrolled cell movement is a hallmark of many diseases. A most striking example of abnormal migration is associated with cancer metastasis where genetic modifications initiate increased migration activity of tumor cells. Metastatic cells then migrate out of the tumor, enter blood vessels to travel to new sites of the body, where they crawl into healthy tissue and generate new tumors.
 
The migration machinery of all these cells consists essentially of the actin and microtubule cytoskeletons. Both cytoskeletons dynamically assemble and disassemble and interact mechanically. The actin cytoskeleton acts as the main force generator for cell migration, on the one hand by polymerization and on the other hand by contraction which is mediated by actin-associated molecular motors. It also integrates with transmembrane adhesion complexes that transmit these forces to the adjacent tissue providing the mechanical support for a cell to migrate relative to its surroundings.
 
In their HFSP funded project Danuser and Waterman-Storer at The Scripps Research Institute in La Jolla, CA, collaborate with Inke Näthke in Dundee, UK, on investigating how the dynamics of the actin and microtubule cytoskeleton systems are orchestrated to mediate directed migration. To address this question Danuser and Waterman-Storer have jointly developed novel light microscopical imaging referred to as quantitative fluorescent speckle microscopy (qFSM). qFSM combines high resolution live cell imaging of very low concentrations of fluorescently labeled actin and tubulin subunits incorporated by the cytoskeletons with computational image analysis and statistics to map the kinematics (intracellular movement) and kinetics (turnover) of the two cytoskeleton systems. In their recent paper Ponti et al. in Science exploited this technique to dissect actin cytoskeleton dynamics during cell protrusion, i.e. when a cell initiates forward movement. To their great surprise the authors discovered that this process is mediated not by a homogeneous dendritic network of actin as described in the textbooks but by two materially and molecularly distinct, mechanically weakly coupled, yet spatially overlapping networks. The paper raises more questions for future research than it can answer. Although the analysis provides ample evidence that these networks support different functions in cell migration, their exact roles in promoting protrusion remain unclear. Also, future work will have to explain how the two networks interact chemically and mechanically. Finally, it is very puzzling how a bipartite structure can assemble from a single protein – actin – but selectively bind to different sets of associated proteins which profoundly alter the dynamic behavior and function of the sub-structures. qFSM will continue to be a powerful tool to tackle these issues, in combination with molecular techniques that selectively perturb one of the two networks.
 
In another study published earlier this summer in the Proc. Nat’l Acad. Sci. U.S.A. Vallotton et al. used qFSM to examine the relationship between actin cytoskeleton turnover and contraction. They found that cytoskeleton contraction is consistently accompanied by depolymerization. Their conclusion relied on two pieces of evidence. In a first set of experiments with untreated cells they learned that the cytoskeleton contracts in periodic patterns. Whenever contraction increased, qFSM maps of the kinetic activity indicated an increase in actin disassembly. In a second set of experiments they stimulated contraction by perfusion of the cell with a drug known to activate the actin-associated molecular motor myosin II. Analogously, by qFSM they observed that this perturbation also increased actin depolymerization. These results pose a classic hen-and-egg problem: Is contraction possible because depolymerization reduces the stiffness of the cytoskeleton; or is depolymerization caused by higher motor activity, either via mechanical breakage of actin filaments, or via signaling pathways? Based on the sequence of events in the drug experiments the authors cautiously promote the notion of motor activity triggering the disassembly of the cytoskeleton. Yet, to draw a definitive conclusion better drugs will be required which target the activation of myosin II more specifically than the ones currently available.
 
Both studies indicate the power of qFSM as a means to measure the dynamics of cytoskeleton assemblies in situ. This will allow this research team in the coming years to dissect cytoskeleton activity in many different cell functions and to use cytoskeleton dynamics as a readout of the effects of regulatory signals that control cell behavior via cytoskeleton activity.
 

 
Figure: Analysis of the actin cytoskeleton dynamics at the leading edge of a migrating cell by quantitative Fluorescent Speckle Microscopy. (A) Speckle image where each speckle (bright spot) is an independent fiduciary mark probing the local flow and turnover of the cytoskeleton. (B) By computational modeling of the speckle signal Ponti et al. have extracted complete maps of the turnover, i.e. they can measure in a living cell where the cytoskeleton assembles (red channel) and disassembles (green channel). This calculation can be performed for every frame in a movie indicating the assembly and disassembly dynamics (see supplementary material to the paper in Science). (C) Decomposition of speckles into two classes (red and black) revealed that the cytoskeleton at the leading edge consists of two molecularly and functionally distinct, yet spatially overlapping (see inset) actin networks, referred to as the lamellipodium and lamella.
 
Pubmed link
 

Reference:
Two distinct actin networks drive the protrusion of migrating cells. Ponti A, Machacek M, Gupton SL, Waterman-Storer CM, Danuser G. Science. 2004 Sep 17;305(5691):1782-6