Science Spotlight

Pavarotti’s (actin) debut

From the Parkhurst lab, Basic Sciences Division

The inner cortex of eukaryotic cells is largely composed of the protein actin, organized in a filamentous network that forms the cytoskeleton. The actin cytoskeleton plays major roles in cellular functions ranging from providing basic cellular architecture to wound repair. To perform these functions, the actin cytoskeleton associates with an array of specialized protein complexes. In wound repair, cytoskeleton remodeling is driven by the accumulation of actin and myosin in cells around the edge of the wound. As actin and myosin, surround the wound and contract, the rupture is healed. The formation of actin-myosin (actomyosin) rings is dependent on the function of several GTP-hydrolyzing enzymes (GTPases). 

Using the fruit fly Drosophila melanogaster embryo as a model of cellular wound repair, the Parkhurst lab (Basic Sciences Division) previously uncovered the role of Rho GTPases in regulating actomyosin ring formation during repair. GTPases function by cycling between GTP- and GDP-bound forms. This process often requires other proteins, such as GTPase activating proteins (GAPs) and the opposing guanine nucleotide exchange factors (GEFs). The researchers found that the protein Pebble (Pbl), a Rho GEF, and the protein Tumbleweed (Tum), a Rho GAP, are recruited to wounds and regulate the spatiotemporal dynamics of the actomyosin ring formation. The study was led by Dr. Mitsutoshi Nakamura, a postdoc in the Parkhurst lab. “While Pebble and Tumbleweed are also required to form the actomyosin ring during cytokinesis, their spatial localization during cell wound repair suggested that they work independently in this context.” Dr. Nakamura explained. “Since Tumbleweed forms a complex with Pavarotti during cytokinesis, it made us wonder what role, if any, Pavarotti might be playing during cell wound repair.” He added.

Dr. Nakamura and colleagues were surprised to find that the while the Pav, Tum and Pbl work together during cytokinesis, they have separate roles during cell wound repair. During cytokinesis, Tum forms a heterotetramer with Pavarotti (Pav) and together they create the centralspindlin complex. Pav is a member of a kinesin-like microtubule (MT)-dependent molecular motor family of proteins. Kinesins are eukaryotic motor proteins that move along microtubule filaments, powered by ATP hydrolysis. In a new study, published in Journal of Cell Biology, Nakamura et al., found that Pavarotti and Tumbleweed function independently of their canonical complex during cellular wound repair and oogenesis.

The researchers took advantage of an established model for wound repair that uses Drosophila embryos visualized in real time using 4D confocal imaging. Using this model, they found that Pbl, Tum, and Pav exhibit distinct localization patterns in cell wound repair. In addition, loss of function analysis with Tum and Pav knockdowns indicated that Pav and Tum are still recruited to wounds in the absence of the other, contrary to what is seen in cytokinesis. Pav and Tum knockdown mutant embryos also exhibited a distinct wound repair phenotype. For example, Pav mutants showed a narrow actin ring, delayed actin accumulation around the wound edge, and a slower closure rate compared with control. In contrast, Tum mutants had a wider actin ring, wound overexpansion, and a slightly faster closure rate.

A graphical abstract shows the different roles of Pav and the centralspindlin complex in cytokinesis, oogenesis, and cell wound repair. Pav and Tum, as the centralspindlin complex, exhibit MT-dependent functions during cytokinesis. Pav, working independently of Tum, binds to actin during cell wound repair. Both Centralspindlin complex–dependent and –independent functions of Pav are required for normal oogenesis.
A graphical abstract shows the different roles of Pav and the centralspindlin complex in cytokinesis, oogenesis, and cell wound repair. Pav and Tum, as the centralspindlin complex, exhibit MT-dependent functions during cytokinesis. Pav, working independently of Tum, binds to actin during cell wound repair. Both Centralspindlin complex–dependent and –independent functions of Pav are required for normal oogenesis. Courtesy of Mitsutoshi Nakamura

Another surprising finding of the study is that Pav localization to wounds is dependent on actin. This was surprising because while microtubule binding by Pav is well-established, its binding to actin was not. The researchers found that Pav binds directly to actin both in vitro and in vivo. Using in vitro bundling assays and purified Pav protein fragments, the authors mapped actin binding to the head domain of Pav. Finally, the authors established that the actin binding activity of Pav is required for both wound repair and oogenesis by studying a Pav mutant that is unable to bundle actin. 

In summary, the study provides new insights into the biological function of Pavarotti, as well as the processes of cell wound repair and oogenesis. The study also raises several questions. “What are the molecular mechanisms underlying Pavarotti’s regulation of actin dynamics during cell wound repair and oogenesis?” Dr. Nakamura wonders. He also would like to figure out how cells choose between Pavarotti’s actin- or microtubule-dependent functions in different contexts, or if there are other kinesins that can bind to actin. Future research by Dr. Nakamura and his colleagues may find answers to these questions. 

UW/Fred Hutch Cancer Consortium Member Susan Parkhurst contributed to this work.

This work was supported by National Institutes of Health.

Nakamura M, Verboon JM, Prentiss CL, Parkhurst SM. 2020. The kinesin-like protein Pavarotti functions noncanonically to regulate actin dynamics. Journal of Cell Biology doi: 10.1083/jcb.201912117