A light way for nuclear cell biologists

Abstract

The nucleus is a very complex organelle present in eukaryotic cells. Having the crucial task to safeguard, organize and manage the genetic information, it must tightly control its molecular constituents, its shape and its internal architecture at any given time. Despite our vast knowledge of nuclear cell biology, much is yet to be unravelled. For instance, only recently we came to appreciate the existence of a dynamic nuclear cytoskeleton made of actin filaments that regulates processes such as gene expression, DNA repair and nuclear expansion. This suggests further exciting discoveries ahead of us. Modern cell biologists embrace a new methodology relying on precise perturbations of cellular processes that require a reversible, highly spatially confinable, rapid, inexpensive and tunEable external stimulus: light. In this review, we discuss how optogenetics, the state-of-the-art technology that uses genetically encoded light-sensitive proteins to steer biological processes, can be adopted to specifically investigate nuclear cell biology.

Keywords: dynamics; nucleus; optogenetics; photosensors; protein engineering.

Graphical abstract

Fig. 1.

Overview of optogenetic strategies to control a protein of interest. Shown are schematic illustrations of selected optogenetic strategies. Since some photosensors revert back to their inactive form only in the dark, while others can be switched back using a second wavelength or if kept in the dark, we wrote ‘dark/λ₂’. Underscored are strategies that allow controlling endogenous proteins. The asterisk indicates an irreversible strategy. Symbols and abbreviations: λ₁, activating wavelength; λ₂, deactivating wavelength; Cyt, cytoplasm; Nuc, nucleus; AsLOV2, LOV2 domain of Avena sativa phototropin 1; POI, protein of interest; NLS, nuclear localization signal; NES, nuclear export signal; dark green/light green ovals, heterodimerizing protein pair; Nb, nanobody; PM, plasma membrane; Int^C, C-terminal fragment of a split intein; Int^N, N-terminal fragment of a split intein; D1 and D3, interacting domains involved in autoinhibition; D2, functional domain; P, phosphorylation; DBD, DNA binding domain; TAD, transactivation domain; PAL, photoreceptor from Nakamurella multipartite; DEGRON, degradation signal; IDR, intrinsically disordered region; PD, photosensing domain.

Fig. 2.

Optogenetic control of nuclear protein import and export and gene expression in living cells. (A and B) Representative fluorescence images of U2OS cells transiently transfected with NES-mCherry-LINuS (A) and NLS-mCherry-LEXY (B) before (left image) and after (right image) 8 min of blue light illumination. Blue light was administered in short pulses of 300 ms every 30 s for 8 min. Scale bar, 30 µm. (C) Schematic illustration of the bacterial photography workflow. For simplicity, the reporter construct, consisting of the gfp gene under a promoter activated by the light-inducible TF, was omitted. (D) Photomask used to produce the bacteriograph in (E). The photomask representing the logo of the University of Freiburg was printed out and attached to the lid of a common agar plate. (E) Bacteriograph created following the procedure described in (C) and using the photomask described in (D). Images of the resulting bacterial lawn are taken with a fluorescent microscope and subsequently assembled in a single image. (F and G) Zoom-in on two parts of the bacteriograph in (E). Scale bar, 1 cm.