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.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Cytological Techniques , Light , Ubiquitin-Protein Ligases/drug effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/radiation effects , Animals , Apoptosis , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/drug effects , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/radiation effects , Caspases/drug effects , Caspases/metabolism , Caspases/radiation effects , Cell Engineering/methods , DNA Damage , DNA Ligase ATP , DNA Repair , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins , HeLa Cells , Humans , Lamin Type A/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods.