Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology

Abstract

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Assembly principles and stoichiometry of a complete human kinetochore module

“…The apparent colinearity of binding motifs in CENP-C with the outer-inner kinetochore axis prompted us to define this protein as a blueprint for kinetochore assembly ( 12 , 14 , 15 , 19 ). To gain deeper insights into how CENP-C shapes the kinetochore-centromere interface, we harnessed a protein ligation technique ( 37 ) to obtain a full-length version of human CENP-C. Electroporated in cells acutely depleted of endogenous CENP-C, the fusion protein was functional, showcasing the power of electroporation for targeted protein delivery and functional studies with recombinant proteins in cells ( 64 ).…”

BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis

“…Depletion of endogenous CENP-OQU was achieved with two rounds of Lipofectamine RNAiMAX transfection, for a total of 72 hours, with the following siRNA oligos at a total concentration of 100 nM: CENP-Q (GAGUUAAUGACUGGGAAUA; AUGGAAAGGGCACGAAGACA; ACAAAGCACACUAACCUAA; UGUCAGAGAAUAAGGUUAG; GGUCUGGCAUUACUACAGGAAGAAA), CENP-U: (GAAAGCCAUCUGCGAAAUA; GAAAAUAAGUACACAACGU; GGGAAGAUAUCUCAUGACA; GCGCAAGACGUUCAAAGAA) and CENP-O (GUACGAAGCCCUUGCAUCA; AAGCCAUUCUGCAGGCAUA; GCUCACACAAUCCACGAACA; CUAGAUUGCUGUAUAAGGA). For rescue experiments, 48 hours into the siRNA depletion of endogenous OQU, we performed electroporation of Alexa488 labeled recombinant CENP-OPQUR at a concentration of 4 μM as previously described ( Alex et al., 2019 ; Pesenti et al., 2018 )(Neon Transfection System, Thermo Fisher). As control we used Alexa488.…”

Altered conformation of α-synuclein drives dysfunction of synaptic vesicles in a synaptosomal model of Parkinson’s disease

“…Because we were interested in the behavior of αSyn inside synaptosomes, we designed a modified version of in-cell NMR ( Theillet et al, 2016 ) in which protein electroporation ( Ramanathan et al, 2000 ) is used to deliver either 15 N or 13 C isotope-enriched human αSyn into purified synaptosomes. To establish whether the observed NMR signals came from the αSyn inside the synaptosomes, immediately after the electroporation pulses, we performed a trypsin treatment to remove non-internalized, extra-synaptosomal protein ( Alex et al, 2019 ), followed by a sodium carbonate (pH 11) wash to remove membrane-associated proteins, and a final wash with NMR buffer (see STAR Methods ). After these steps, the electroporated exogenous αSyn remained inside the intact synaptosomes and was shielded from trypsin cleavage ( Figure 3A ).…”