Optogenetic Protein Fusion for Precise And Low-Impact Actin Cytoskeleton Imaging In Cells

SUMMARY

A light-sensitive version of LifeAct, a peptide used to label actin in cells, to reduce its harmful side effects by controlling its binding through light exposure, improving cell imaging and minimizing cytoskeletal disruptions

The Unmet Need: Innovative solutions that can provide high-quality imaging while minimizing the side effects on cellular dynamics

• The study of the actin cytoskeleton is crucial in cell biology because it plays a significant role in various cellular processes, including cell motility, division, and intracellular transport. Researchers frequently use fluorescent markers to visualize actin structures within cells to understand these processes better. One such widely used marker is LifeAct, a 17-amino acid peptide that binds to actin filaments. Despite its popularity, there is a pressing need for improved imaging techniques, as the current methods can introduce artifacts and interfere with cell physiology. Better imaging tools would not only provide more accurate depictions of the cytoskeleton but also maintain cellular health, thereby improving the reliability of experimental results.

• Studies have shown that LifeAct can alter cytoskeletal dynamics and negatively affect cellular functions by competing with essential proteins like cofilin and myosin for binding sites on actin filaments. This competition can distort the natural behavior of cells, leading to results that do not accurately reflect true biological processes. Current approaches, despite their utility, fail to mitigate these adverse effects, causing potential data misinterpretations.

The Proposed Solution: A modular system combining LifeAct with the photosensitive LOV2 protein allowing precise, temporally-controlled actin visualization and interaction with reduced cellular side effects

• The faculty inventor developed a modified version of LifeAct, a peptide used for labeling the actin cytoskeleton in cells. Traditional LifeAct, consisting of 17 amino acids, has been instrumental in over 1800 studies but has shown to disrupt cytoskeletal dynamics, adversely affecting cell health. To mitigate these issues, researchers have incorporated an optogenetic approach using the LOV2 photosensitive protein. By attaching LifeAct to the C-terminus of LOV2, the peptide remains inactive in the dark, which prevents it from binding to actin and thus, reduces unwanted cytoskeletal alterations. Upon exposure to light, LOV2 unfolds, allowing LifeAct to engage with actin filaments with reduced affinity. This innovation enables the cells to grow unharmed in the dark while facilitating controlled actin labeling during imaging. Additionally, the system holds promise for application in light-patterned actin recruitment and cross-linking.

ADVANTAGES

ADVANTAGES

• Reduces side effects of the imaging reagent by using low affinity LifeAct when cells are grown in the dark

• Allows reversible control of actin binding with light exposure using LOV2

APPLICATIONS

• Cell imaging tool