Time-lapse fluorescence (left) and differential interference contrast microscopy (right) images showing the protrusion of filopodia in a neuroblastoma cell overexpressing Rif and fluorescently labeled actin.
Cells interact with their surroundings through protrusions known as filopodia. These protrusions dynamically extend and retract through the assembly and disassembly of microfilaments—bundles of actin proteins—beneath the membrane surface. The whole formation process is managed by a network of proteins that are regulated by the protein Cdc42.
Recent studies suggest that Rif, another protein belonging to the same family as Cdc42, also participates in the formation of filopodia, although some ambiguity remains. “The data on Rif were generated using fixed cells,” explains Sohail Ahmed from the A*STAR Institute of Medical Biology. “However, its role in the formation of filopodia had not been investigated in living cells, and we felt this was essential.”
To address this question, Ahmed and his co-workers used imaging techniques that enabled them to monitor cell behavior in real-time. They began by expressing Rif and fluorescently labeled actin in cells derived from mouse brain tumors. The fluorescent label allowed the researchers to directly visualize the formation of actin-rich filopodia in the presence of Rif.
Ahmed’s team has studied Cdc42 extensively, and their work has uncovered a number of factors acting downstream of this protein. The researchers were able to selectively eliminate the expression of several of these proteins without impairing Rif-induced filopodia formation, suggesting that Rif acts via an independent signaling pathway.
Two proteins known as mDia1 and mDia2 facilitate the assembly of individual actin monomers into filaments, and previous studies have indicated that mDia2 in particular may partner with both Cdc42 and Rif to facilitate the formation of filopodia. Ahmed and his co-workers monitored Rif’s association with both proteins using a technique called Förster resonance energy transfer (FRET), which provides a sensitive visual readout of the interactions between two fluorescently labeled proteins.
Although both mDia1 and mDia2 contribute to Rif-mediated filopodia formation, the researchers were surprised to find no evidence for direct Rif–mDia2 interaction within these protrusions. Instead, the FRET experiments revealed an unexpected binding between Rif and mDia1. The result demonstrates how techniques like FRET and live-cell imaging can change the current understanding of how cells function.
Ahmed hypothesizes that Rif and Cdc42 may each modulate actin dynamics in different cellular processes. For example, Rif is believed to contribute to the formation of signal-receiving dendritic spines in neurons. To better resolve its function, his team is now looking to uncover Rif’s partners. “Ultimately, we would like to know all of the proteins involved in forming and regulating filopodia,” says Ahmed.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Medical Biology.
