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Tunable light and drug induced depletion of target proteins.

ABSTRACT: Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.

SUBMITTER: Deng W

PROVIDER: S-EPMC6965615 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Tunable light and drug induced depletion of target proteins.

Deng Wen W Bates Jack A JA Wei Hai H Bartoschek Michael D MD Conradt Barbara B Leonhardt Heinrich H

Nature communications 20200116 1

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to in ...[more]

PMID: 31949141

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Project description:Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, ?-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Dataset Information

Tunable light and drug induced depletion of target proteins.

Publications

Tunable light and drug induced depletion of target proteins.

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