Alexa Fluor® Dyes Shed Light on a New Actin Assembly Factor
What cellular factors regulate the assembly and function of actin structures? Actin filaments are the basic building blocks for a wide array of cytoskeletal features. Several classes of proteins have been identified that direct the assembly of these building blocks into macromolecular domains, including Arp2/3, which serves as a template for branched filament formation, and Spire, which nucleates unbranched filament assembly. Given the wealth of genome data available to researchers, database searches are turning up proteins whose sequence homology to these known actin nucleators suggests potentially related functions.
In their current report, Zuchero and colleagues investigate the actin assembly role of JMY, a protein previously known as a transcriptional activator but with sequence homology to the WASp Homology 2 (WH2) domain, an Arp2/3-activating sequence. They observed that JMY is capable of accelerating actin assembly in a dose-dependent fashion, either in concert with Arp 2/3 or on its own, as visualized in several cell types by labeling with Alexa Fluor® phalloidin conjugates (Figure 1). This acceleration was shown to be a consequence of nucleation of new actin filaments rather than enhanced elongation. Supporting this, sequence analysis revealed homology between JMY and Spire, and kinetic assays demonstrated the two proteins possess nearly identical actin nucleation activities. The authors observed that JMY is largely localized in the nucleus of relatively nonmotile cells, but is found more prominently at the leading edge of growth regions in highly motile cells such as neutrophils, suggesting a role in cell motility.
Other experiments showed significantly reduced rates of wound healing in cells when JMY expression was suppressed by RNAi knockdown. Given the known transcriptional function of JMY combined with its actin-nucleating activity demonstrated here, impaired healing could be attributed to either decreased motility or altered transcription, or some combination of the two. The authors present the clearly defined transcriptional and cytoskeletal roles of JMY as evidence that it represents a new class of actin assembly factor.
Figure 1. JMY nucleates actin filaments and activates the Arp2/3 complex. Expression of haemagglutinin (HA)-tagged JMY (top panels; revealed by indirect immunofluorescence of HA, green) in U2OS cells induces the formation of filamentous actin structures (revealed with Alexa Fluor® 568 Phalloidin, red). These elongated actin structures colocalize with JMY and are not seen in untransfected cells (bottom panels). Nuclei were revealed with DAPI (blue). Scale bar, 10 μm. Reprinted by permission from Macmillan Publishers Ltd: Nature Cell Biology 11:451(2009).