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Updated: May 27, 2026
Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells
Published on: February 22, 2020
A targeted siRNA screen identifies regulators of Cdc42 activity at the natural killer cell immunological synapse
Leo M Carlin1, Rachel Evans, Hanna Milewicz
1Richard Dimbleby Department of Cancer Research, King's College London, London SE1 1UL, UK.
Abstract:
Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.
Insights
Natural killer (NK) cell function relies on cytoskeletal changes. This study reveals oscillating Cdc42 activity in NK cells, crucial for targeting cancer cells and requiring specific signaling pathways for proper immune response.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Natural killer (NK) cells are vital for eliminating tumor and virally infected cells.
- Effective NK cell responses depend on cytoskeletal rearrangements, coordinated by Rho GTPase Cdc42.
- Oscillatory Rho GTPase activity, observed in plant cells, was previously uncharacterized in mammalian cells.
Purpose of the Study:
- To investigate whether Cdc42 activity oscillates in NK cells during target cell interaction.
- To identify key regulators of Cdc42 activity and oscillation within the NK cell immunological synapse.
- To understand the role of Cdc42 oscillations in NK cell cytotoxic function.
Main Methods:
- Utilized fluorescence lifetime imaging with a Cdc42 biosensor in live NK cells interacting with target cells.
- Employed protein-protein interaction networks and structural databases to identify candidate regulators.
- Conducted a targeted short interfering RNA (siRNA) screen to assess the function of candidate proteins.
Main Results:
- Observed oscillating Cdc42 activity in NK cells forming immunological synapses, following an initial activation.
- Identified RhoGEF6 and RhoGEF7 as essential guanine nucleotide exchange factors for NK cell Cdc42 activation.
- Demonstrated that the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) are required for Cdc42 oscillation periodicity and cytotoxic vesicle polarization.
Conclusions:
- Cdc42 activity oscillates in human NK cells during target cell engagement, a novel finding in mammalian cells.
- Specific signaling molecules, including RhoGEF6/7, Akt, and PI3K, regulate NK cell Cdc42 oscillations and cytotoxic function.
- Findings highlight the importance of monitoring innate immune function, particularly NK cell activity, during PI3K-targeted cancer therapies.
