Photocleavable TDP-43 to Control Subcellular Localization

Authors

• JA Shadishm
• JC Lee

Abstract

TAR DNA-binding protein 43 (TDP-43) cytosolic aggregates have been implicated in a host of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. Under healthy conditions, TDP-43 is an ubiquitously expressed RNA-binding protein which is primarily located in the nucleus. The mechanism of TDP-43 pathology, going from a functional nuclear protein to an aggregated cytoplasmic inclusion, remains poorly understood. Understanding this process is critical to developing effective therapeutics. In this work, the subcellular distribution equilibrium of TDP-43 is controlled in a spatiotemporal manner through the genetic incorporation of a photocleavable protein, PhoCl. Upon illumination with cellular compatible violet light (λ = 400 nm), the PhoCl protein undergoes a structural rearrangement to generate disease-related N-terminally truncated TDP-43 variants. The position of the truncation is based on the placement of PhoCl in the TDP-43 sequence. Photocleavable TDP-43 constructs were transfected and expressed in HEK293-T cells. Light exposure generates truncations lacking an N-terminal nuclear localization sequence, shifting TDP-43 equilibrium towards the cytosol. Cells are tracked via timelapse confocal fluorescence microscopy, and the cellular distribution of TDP-43 is quantified by cellular fractionation and western blot. Spatial control over the in cellulo generation of truncations facilitates the probing of biological mechanisms of TDP-43 translocation that are not possible by traditional methods.

Scientific Focus Area: Biomedical Engineering and Biophysics

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