Cells contain thousands of protein “micromachines” performing a bewildering number of chemical reactions every second. The challenge for biologists in the 21st century is to integrate information about multiple – or even all – proteins into holistic models for the entire cell. This is a daunting task. The addition of any new component to a system can alter the behavior of the components already there. This phenomenon is especially familiar to biologists studying the cytoskeleton, a complex system of protein filaments that provide the force for cell division and migration, among other things. The building blocks of the cytoskeleton are simple proteins called tubulin and actin that assemble into a remarkable variety of shapes depending on context. While the basic chemistry of this assembly process has been understood in purified systems for decades, how it happens in cells is not well understood. For example, growth of actin filaments is a two-step process: nucleation, or the formation of a new filament, and elongation, or the extension of that existing filament. Both steps happen just fine when actin is present in pure form in a test tube. In cells, however, proteins called profilin and capping protein block these two steps, respectively. Nucleation and elongation can only occur because other proteins overcome these blocks. Thus, a faithful experimental system to study actin assembly as it would occur in a living cell requires – at a minimum – five purified proteins.
One technological advance of great importance is the ability to literally see single molecules (in this case proteins) using advanced fluorescence imaging. In such an experimental system, many details can be captured. In a recent publication in Science, Dr. Dennis Breitsprecher and colleagues in the Goode and Gelles labs, undertook this challenge and directly visualized the effects of key regulatory proteins helping actin proteins nucleate and grow into filaments in the presence of both profilin and capping protein. A previous study from the Goode lab had shown that two proteins, called APC and mDia1, together stimulate the growth of actin into filaments (Okada et al, 2010). In the present study, Breitsprecher and colleagues examined the mechanism by which APC and mDia1 overcome the profilin and capping protein-imposed blocks. To do this, they ‘tagged’ actin, APC and mDia1 with three different fluorescent dyes, each of a different color, and then filmed these molecules (using triple-color TIRF microscopy) in the act of building an actin filament to learn precisely what they are doing.
The authors began by imaging APC and actin (2 colors) at the same time. APC formed discrete spots on the microscope slide, and growing actin filaments emerged from them, suggesting than APC nucleates actin filament formation. As the filament emerged from the APC spot, APC stayed where it was: remaining stably associated at the site of nucleation. Next, the authors added dye-labeled mDia1 to the system, and observed mDia1 molecules staying attached to and ‘riding’ the fast-growing end of actin filament, while protecting it from capping protein.
The most remarkable result came when they visualized all three labeled molecules together (actin, APC, and mDia1). What they saw was that APC and mDia1 first come together in a stable complex even before actin arrives. Then APC recruits multiple actin subunits to initiate the nucleation of a filament. This complex was resistant to the blocks imposed by both profilin and capping protein. As the filament grew from the APC-mDia1 spot, mDia1 separated from APC and stayed bound to the growing end of the filament – protecting it from capping protein while it grows. Thus, even though APC and mDia1 have different activities, participating in different stages of the growth of a filament, they associate together before actin even arrives, likely so that once the actin filament is born, it is immediately protected from capping protein. This mechanism has been compared to a rocket launcher: APC is the launch pad for an actin filament, which is then propelled forward by mDia1.
Rocket launcher mechanism for APC and mDia1 nucleation. Left: Microscopic image of a growing actin filament. APC stays put while mDia1 remains associated with the fast growing end. Right: Model for the rocket launcher mechanism.
The new study provides great detail of the system: for example, the number of APC subunits required to nucleate actin filaments was determined, and the growth rate of actin filaments in the presence and absence of all the other components was measured. Ultimately, all of these data will be required to put together a detailed model of how actin filaments grow inside of real cells: details that would be difficult or impossible to obtain without employing single molecule analysis.
For the future, the authors have set their sights on even more challenging experiments aimed at elucidating the mysterious link between tubulin and actin fibers. APC and mDia1 are implicated in this linkage in living cells, but almost nothing is known about how they physically link and/or communicate information between the two systems. Since APC is mutated in some 80% of colon cancer tumors, understanding its multiple roles is of clinical as well as intellectual importance. This will be an exciting, if challenging, endeavor for the future.
