Emtb 3xgfp

We synthesized mRNAs for microinjections with Ambion’s SP6 mMESSAGEmMACHINE kit. The capped mRNAs produced were diluted in nuclease-free water and used for microinjections in order to detect fluorescence signal in early M. crozieri embryos. Nuclei were marked and followed using histone H2A-mCherry (H2A-mCh) and GFP-Histone (H2B-GFP). The plasmids carrying the nuclear marker pCS2-H2B-GFP (GFP-Histone) and pDestTol2pA2-H2A-mCherry [37 (link)] were transformed, purified and concentrated as described before and then linearized with the restriction enzymes NotI and BglII, respectively. To follow live microtubules, we used a GFP fusion of the microtubule binding domain of ensconsin (EMTB-3XGFP). These clones were the gift of the Bement Lab (University of Wisconsin) [8 (link), 56 (link)] and were commercially ordered from http://addgene.org (EMTB-3XGFP: https://www.addgene.org/26741/).

Cells were grown overnight in 24-well plates at 37°C in a 5% CO₂ atmosphere. After reaching over 80% confluence, the plasmids mRuby-Clathrin (Addgene #55852), pEGFP-Sec23A (Addgene #66609), ZsGreen-Rab5 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.), EGFP-Rab7A (Addgene #28047), GFP-LAMP1 (Addgene #16290), EMTB-3XGFP (Addgene #26741), pSNAPf-Cox8A (Addgene #101129), or pSNAPf-TOMM20 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.) was transfected into the cells using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 24 h, the transfected cells were digested with trypsin-EDTA and seeded into Nunc Glass Bottom Dishes (Φ 12 mm, Thermo Fisher Scientific, Inc.) at a density of 1.5 ~ 2.0 × 10⁴ per well in growth medium (150 μL). The cells were grown for an additional 12 ~ 24 h before incubation with the indicated probes.

Plasmid amplification was performed via transformation in E. coli (XL1-Blue) and DNA isolation via MIDI-prep (NucleoBond® Xtra Midi, Macherey & Nagel, #740410). The respective amber stop mutants were generated by introducing a TAG codon through PCR-based site-directed mutagenesis. The plasmid for expression of the TNF-receptor pTNFR1-tdEOS was a gift from Mike Heilemann (Addgene plasmid #98273). The plasmid for the expression of the Kainate receptor (pcDNA3-GluK2) was a gift from Dr. Peter Seeburg (Max-Planck Institute for Medical Research, Heidelberg, Germany). An AgeI restriction site was introduced at the C-terminus of the GluK2 coding sequence in front of the STOP-codon. Subsequently, GluK2 was cloned into the MCS of the pEGFP-N1 plasmid (Clontech #U55762) using the restriction enzymes EcoRI and AgeI. The plasmid for expression of the microtubule-associated protein Ensconsin (EMTB-3xGFP) was a gift from William Bement (Addgene plasmid #26741). The plasmid for the expression of the tRNA/tRNA-synthetase pair (pCMV tRNA^Pyl/NESPylRS^AF) was kindly provided by Edward Lemke^{53 (link)}.