Profilin as a part of the musculo-skeletal system that motors our cells around. But how can we tell?
Our cells have structural elements called the cytoskeleton. The term is a misnomer, since the cytoskeleton comprises the muscles of the cell as well as its rigid supports. There are three types of rigid element- actin filaments, intermediate filaments, and microtubules. Intermediate filaments are the stable, relatively inert part of the equation, making up structures like keratins that shape our skin, hair, and nails. Actin and microtubules, however are highly dynamic and contribute to amoeboid motion, developmental cell motions, neural extensions, and all kinds of other shape changes cells perform. Microtubules are bigger and stiffer, (25 nm diameter, hundreds of times stiffer than actin filaments), and participate in big, discrete processes like separating the chromosomes at division, and forming the core of cilia that wave from the outside of the cell.
Actin (6 nm diameter) is more pervasive all over the cell, and is what provides the main motive force of ameboid motions and cell shape change. Indeed, our muscles are mostly composed of great quantities of actin along with interdigitated filaments of its corresponding motor protein (myosin) in orderly, almost crystalline, arrays. Both myosin and actin create motion in two ways- by their own polymerization / depolymerization, and also by way of motors that can move along their lengths.
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| Images of cells showing fluorescence labeling of skeletal components. Microtubules are shown in green, and DNA in blue. Panel C shows a neuronal growth cone with actin labeled in red. Note how microtubules and actin cooperate, with actin in the lead, pushing out the cell edges by force of its own polymerization. Panel A shows resting cells, with the microtubule organizing center in red. E shows a yeast cell with microtubules spanning its length. G shows a dividing cell at M phase, where microtubules organize the separation of chromosomes, after the microtubule organizing center has itself first divided into two. |
A recent paper discussed new tools in the quest to visualize profilin, one of the many accessory proteins involved in managing the cytoskeleton. The most basic role of profilin is to bind to monomers of actin helping them recharge (that is, exchange their ADP for a new ATP). There is a lot of profilin in the cell, and it mostly sits around complexed with actin, preventing it from spontaneously polymerizing. But then if a signal comes in, profilin has binding sites for formin proteins, which tend to be the main instigators of cell shape change and actin polymerization, and can orchestrate the handoff of actin from profilin to growing actin filaments.
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| The overall actin cycle. Actin monomers are constantly coming on and off of filaments. ATP-charged actin is held in reserve in complex with profilin (dark shapes). Then formins or other accessory proteins can encourage addition to a filament, at one end, called the barbed end. While in filaments, actin gradually hydrolyzes its ATP, forming ADP. Actin with ADP is prone to dissociation, which may be encouraged or discouraged by various other accessory proteins. The resulting actin monomers are then re-bound by profilin and the cycle begins again. |
But how can we see all this? Making proteins fluorescent has been now for decades the amazingly effective way to vizualize them. And one can do that either live, or dead. For the latter, the cell is chemically embalmed and permeabilized, then treated with antibodies that bind to the protein(s) of interest. Then a second set of antibodies are applied that bind to the first set, and are labeled with some fluorescent tag, and voila- images of where your protein of interest is, or was. But much more compelling is to see all this in living, working, and moving cells. To do that, the protein of interest is mutated to add an intrinsically fluorescent tag, such as green fluorescent protein. But profilin is so small, and so packed with critical binding sites, that there is little room for a fluorescent tag protein that is, in fact, almost twice as large as profilin itself.
What to do? These researchers attached a little tail to one end of the protein, off which they then added their tag, in this case a protein called mApple, chosen for its nice red fluorescence spectrum that doesn't interfere with the other greens and blues typically used in these experiments. The paper is mostly then a laborious verification that this new form of profilin fully functions in cells as the wild type does, engages in all the same interactions, (as far as known), and thus consitutes a wonderful new tool for the field.
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| An atomic structure of profilin bound to actin. Profilin is a very small protein with many important interactions. That makes altering it very tricky. How to create a fluorescent form, or squeeze in some other tag? Profilin binds to actin, to microtubules, to formins and other proteins with PLP (poly-proline) domains, and to phosphoinositide 4,5-bisphosphate (PIP2), which is not even shown here. |
It turns out that profilin binds to microtubules as well as to actin. And so do formins. As shown above in the image of a neural growth cone, though the composition of actin and microtubules and their size and other characteristics are very different, they cooperate extensively, thus must have mechanisms of crosstalk. Not much is known, unfortunately, about how this works- while a good bit is known individually how each of the actin and microtubule systems work, how they work together is poorly understood. But one thing these researchers show is that profilin, along with its abundance all over the cell, is also concentrated at the microtubule organizing center. Indeed, some mutations that cause the disease ALS occur right in these regions of profilin that bind microtubules. So something important is going on, and hopefully this new tool will speed work towards greater understanding of how the cytoskeletons operate.
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| Profilin imaged in a live cell, with other tagged molecules. At left, profilin occurs all over the cell in its role as actin buffer and storage partner. But note a couple of dots on each side. Next is shown the same cell labeled on alpha tubulin, the major component of microtubules. Next is show DNA, which is condensed, as this cell is undergoing division. Last is shown the merged images, with DNA in blue, tubulin in green, and profilin in red/orange. The dots turn out to be the microtubule organizing centers that run the spindle which is orchestrating chromosome segregation. |
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