Curated Optogenetic Publication Database

Abstract: Several light-inducible hetero-dimerization tools have been developed to spatiotemporally control subcellular localization and activity of target proteins or their downstream signaling. In contrast to other genetic technologies, such as CRISPR-mediated genome editing, these optogenetic tools can locally control protein localization on the second timescale. In addition, these tools can be used to understand the sufficiency of target proteins' function and manipulate downstream events. In this chapter, I will present methods for locally activating cytoplasmic dynein at the mitotic cell cortex in human cells, with a focus on how to generate knock-in cell lines and set up a microscope system.

Abstract: The transcription factor p53 is a stress sensor that turns specific sets of genes on to allow the cell to respond to the stress depending on its severity and type. p53 is classified as tumor suppressor because its function is to maintain genome integrity promoting cell cycle arrest, apoptosis, or senescence to avoid proliferation of cells with damaged DNA. While in many human cancers the p53 gene is itself mutated, there are some in which the dysfunction of the p53 pathway is caused by the overexpression of negative regulators of p53, such as Mdm2, that keep it at low levels at all times. Here we develop an optogenetic approach to control endogenous p53 levels with blue light. Specifically, we control the nuclear localization of the Mmd2-binding PMI peptide using the light-inducible export system LEXY. In the dark, the PMI-LEXY fusion is nuclear and binds to Mdm2, consenting to p53 to accumulate and transcribe the target gene p21. Blue light exposure leads to the export of the PMI-LEXY fusion into the cytosol, thereby Mdm2 is able to degrade p53 as in the absence of the peptide. This approach may be useful to study the effect of localized p53 activation within a tissue or organ.

Abstract: To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.