Tracking exogenous intracellular casp‐3 using split GFP

Abstract

Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH-sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein-modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self-proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase-3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild-type caspase-3. Only in the presence of cytosolic GFP strands 1-10 will the tagged caspase-3 generate fluorescence to signal a non-endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomallyentrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp-3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge-property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.

Evaluating Endosomal Escape of Caspase-3-Containing Nanomaterials Using Split GFP

“…C3-11 maintains properties of native casp-3 and can report on cytosolic localization upon reassembly with GFP1–10, which is constitutively expressed cytosolically (Scheme ). We often used a catalytically dead variant generated by knocking out (KO) the active site cysteine residue generating C3 KO -11 . Using this catalytically dead variant allowed us to track cytosolic levels of casp-3 without inducing apoptosis.…”

“…Accordingly, imaging of dual-labeled NG (Cy3-polymer, Cy5-protein) demonstrates punctate colocalization of the cargo and delivery vehicle (Figure C), implying great complex internalization. Despite this endosomal entrapment, it is clear that the amount of casp-3 that does escape the endosome is sufficient to induce cell death, , but enhancing endosomal escape is a warranted investigation to optimize efficacy.…”

“…Generation of C3 KO -11 and C3-11 variants have been described previously . pET23b plasmid encoding human WT casp-3, or variant, was transformed into BL21­(DE3) E.…”

“…Moreover, the process of cell fixation allows redistribution of endocytotic vesicles, often compromising membrane integrity. − On the other hand, flow cytometry or total cell lysate analysis solely reveal total internalization, regardless of cytosolic or endosomal localization. Therefore, alternative methods, such as the use of split GFP, have recently been investigated to reveal true cytosolic translocation of protein cargos and antibodies. − …”

“…This approach facilitates optimization of nanogel (NG) polymer composition and surface functionalization for endosomal escape (Scheme ). While the approach holds promise for many protein cargos, we focused this project on caspases because of their potential for targeted cell killing, in the context of proliferative diseases, including cancer, rheumatoid arthritis, and scleroderma. Furthermore, caspase-3 has been delivered by many materials, − in addition to our redox-responsive systems, , underlying merit for this development.…”