Actin is a protein within all eukaryotic cells that forms dynamic cables known as microfilaments. These structures are part of the cytoskeleton, a cellular scaffolding that provides structural support, enables cell movement, and facilitates processes like muscle contraction and cell division. Because these functions are so important, there is significant scientific interest in observing how actin behaves inside a cell. Individual actin filaments are only a few nanometers in diameter and are too small to be seen with a standard light microscope.
This physical limitation necessitates specialized visualization strategies. To track actin’s behavior, scientists must attach a detectable marker to it through a process known as labeling. This makes the otherwise invisible actin cytoskeleton visible, allowing researchers to study its architecture and reorganizations. The choice of labeling strategy depends on the biological question, particularly whether the goal is to see a snapshot in time or to watch a process unfold in a living cell.
Methods for Labeling Actin
Labeling techniques fall into two categories: those for fixed (non-living) cells and those for live cells. For fixed specimens, one common method uses a natural toxin called phalloidin. Isolated from the Amanita phalloides mushroom, phalloidin has a high affinity for the filamentous form of actin (F-actin). A fluorescent molecule is attached to the phalloidin, and when this conjugate is introduced to permeabilized cells, it binds to actin filaments, painting the cytoskeleton.
Because phalloidin stabilizes actin filaments and prevents their disassembly, it is unsuitable for studying dynamic processes. It provides a high-quality static image of the actin network at a specific moment. Another method for fixed cells is immunofluorescence, which uses antibodies to detect actin. A primary antibody binds to the actin, and then a fluorescently-tagged secondary antibody is introduced that binds to the primary antibody, amplifying the signal.
Observing actin in living cells requires genetic tagging. This technique involves fusing the gene that codes for actin with a gene for a fluorescent protein, such as Green Fluorescent Protein (GFP). This combined gene is introduced into a cell, which then produces an actin protein that is inherently fluorescent. This fusion protein is incorporated directly into the cell’s cytoskeleton, allowing for the real-time visualization of actin dynamics as they happen.
Visualizing Labeled Actin Dynamics
Labeled actin provides a window into processes like cell motility. Using live-cell imaging of fluorescently tagged actin, researchers can watch as a cell crawls across a surface. This movement is driven by the rapid assembly of actin filaments at the cell’s “leading edge,” pushing the membrane forward in sheet-like structures called lamellipodia and finger-like protrusions known as filopodia. The imaging reveals a dynamic process where new filaments are built at the front while older ones are disassembled toward the rear.
Labeled actin also provides insight into cell division, or cytokinesis. As a cell prepares to divide, a contractile ring composed of actin and myosin motor proteins forms at the cell’s equator. Live imaging shows the ring assembling and then constricting, much like a drawstring on a bag, pinching the cell membrane inward until the cell is cleaved in two. Visualizing the labeled actin in the ring allows for precise measurement of the forces and timing involved.
Beyond motility and division, labeled actin reveals the cytoskeleton’s role in organizing the cell’s interior. For instance, some intracellular pathogens, like the bacterium Listeria monocytogenes, hijack the host cell’s actin network. Labeled actin makes this process directly observable, as these bacteria build actin “comet tails” to propel themselves through the cytoplasm and spread to other cells. Actin filaments also serve as tracks for the transport of organelles and are integral to the structure of cell-to-cell connections.
Instrumentation for Imaging Labeled Actin
Visualizing labeled actin requires fluorescence microscopy. This technology uses a high-energy light source to illuminate the sample at a specific wavelength. This light is absorbed by the fluorescent label on the actin, which then releases the energy as light of a different, longer wavelength. This emitted light is captured by a sensitive camera to form an image.
Standard fluorescence microscopy images can be blurry due to out-of-focus light from above and below the plane being observed. Confocal microscopy solves this problem by using a pinhole aperture in front of the detector, which blocks most of the out-of-focus light. The result is a sharp, high-contrast “optical slice” through the specimen. By capturing a series of these slices at different depths, a computer can reconstruct a detailed three-dimensional image of the actin cytoskeleton.
Super-resolution microscopy is a more recent development that bypasses the traditional diffraction limit of light. Techniques like STED and STORM use sophisticated approaches to achieve much higher resolution. For example, STORM uses photoswitchable dyes that can be turned on and off, allowing the microscope to pinpoint the location of individual molecules before computationally reconstructing a final image. These methods enable scientists to see the fine details of individual actin filaments and their interactions with other proteins.