Date Published: November 9, 2009
Publisher: Public Library of Science
Author(s): Vedakumar Tatavarty, Eun-Ji Kim, Vladimir Rodionov, Ji Yu, Robert Alan Arkowitz. http://doi.org/10.1371/journal.pone.0007724
Abstract: Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.
Partial Text: The majority of the excitatory synapses in central nervous systems are formed onto dendritic spines. Morphologically, dendritic spines appear to be micrometer-sized membrane protrusion from the neuronal dendrites; functionally they serve as compartments for post-synaptic molecules. They come in a variety of shapes , most commonly as one of the following: filopodia-like, stubby, mushroom-shaped and cup-shaped. The shape and the size of a spine is determined by the underlying actin cytoskeleton , as spines contain a high concentration of filamentous (F-) actin molecules and are mostly devoid of microtubules. In recent years, advanced live cell imaging techniques have revealed that the spines are remarkably dynamic, changing size and shape in a matter of minutes -. These morphological changes are widely believed to affect functional properties of the individual synapses and by extension the neuronal network, and therefore are directly linked to brain’s cognitive functions, such as memory and learning. A large body of evidence now exists to support this proposition. For example, many studies have demonstrated changes in spine morphology following electrophysiologically induced long-term potentiation (LTP) or long-term depression (LTD) . Furthermore, a dynamic F-actin cytoskeleton is required for establishing LTP and LTD -. Finally, recent studies in culture showed that the direct application of stimuli to individual spines resulted in an enlargement of the spine and this enlargement required actin , . Therefore understanding the actin cytoskeleton is of central importance to the studies of synaptic and neuronal function.
In this paper we demonstrated a single-molecule imaging assay for analyzing the kinematic dynamics of F-actin in dendritic spines. The method is general enough so that it should be useful for other small cellular compartments. Studying actin kinematics provides clues to the underlying cytoskeletal organization. We argue that at least three mechanisms can give rise to kinematic movements. First, elongation of an F-actin filament from the barbed end, when reaching resistive barrier, will generate force to push filament back. Movement of this type is along the orientation of the filament and towards the pointed end. Secondly, nucleation and/or growth of other filaments could run into and push the filament under observation, causing it to move. In this case, the movement does not need to follow the direction of the filament. Finally, force generating molecular motors, such as myosin, could also produce movements of F-actin. In our case, jasplakinolide stabilized F-actin shows no sign of kinematic movements, which seems to suggest the first two mechanisms. Although a better test of the effects of motor proteins is to chemically inhibit motor activities, unfortunately, the drug commonly used for this purpose, blebbistatin, is a fluorescent molecule itself and thus strongly interfere with single-molecule detection. Therefore we were unable to perform this test. On the other hand, the existence of many randomly moving molecules cannot be accounted for by the first mechanism. It is likely that the second mechanism plays an important role in spines.