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Researchers at the Indian Institute of Science (IISc) have developed a new technique called Light-Induced Spindle Activation (LISA), which can reversibly activate Aurora A kinase, a key centrosomal enzyme critical for spindle assembly.
The institute said that this technique allows scientists to study centrosomes and spindle assembly in much greater detail, besides opening the door to the possibility of rescuing cell division by artificially manipulating these components and processes when they fail.
Scientists believed that Aurora A is activated through clustering, a process promoted in cells by its evolutionarily conserved scaffold, Cep192.
According to the institute, previous work has shown that Aurora A-coated beads can trigger microtubule nucleation in frog egg extracts; however, Cep192 is required for Aurora A activation in this setting.
“With LISA, researchers can now reversibly induce and study Aurora A clustering using blue light, independently of Cep192, across a range of cell types. LISA can also be applied directly in living cells, whereas the bead-based method is largely restricted to cell extracts,” IISc said.
To develop this method, Sachin Kotak, associate professor at the Department of Microbiology and Cell Biology, and his team identified a novel application for an existing technique called LARIAT, which is typically used to inhibit proteins by inducing their clustering with light.
Prof. Kotak’s team realised that this light-induced clustering could instead be harnessed to activate Aurora A kinase. LARIAT uses the plant proteins CRY2 and CIB1, which can be induced to cluster in response to blue light.
The researchers first engineered human cells to express a fusion protein in which Aurora A kinase was linked to CRY2. When blue light was shone on the fusion protein, CRY2 underwent a conformational change that promoted its interaction with CIB1. This, in turn, drove the clustering of multiple Aurora A assemblies, said Vignesh Olakkal, a PhD student and first author of the study.
“This provides a novel way to trigger and activate an artificial Aurora A kinase complex — Aurora A fused to light-sensitive plant proteins, which is sufficient to induce microtubule nucleation,” he added.
Cells without centrosomes often have defects in spindle assembly, leading to errors in cell division. Prof. Kotak’s team wanted to test whether LISA could overcome this defect. They first chemically eliminated the centrosomes from human cells and then introduced the engineered Aurora A clustering system. When blue light was applied during mitosis, it induced Aurora A clustering and efficiently stimulated microtubule nucleation.
Researchers at the Indian Institute of Science (IISc) have developed a new technique called Light-Induced Spindle Activation (LISA), which can reversibly activate Aurora A kinase, a key centrosomal enzyme critical for spindle assembly.