Journal: bioRxiv

Article Title: Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

doi: 10.1101/2021.10.10.463806

Figure Lengend Snippet: a A conceptual schema describing rapidly inducible manipulation of intracellular organelles based on the integration of chemically- or optically-triggered hetero-dimerization and phospholipid remodeling by PLAAT enzymes. This strategy anticipates rapid switching of the PLAAT activity right at the target membrane-bound organelles (e.g., mitochondria) to induce their deformation as well as defunctionalization. b Fluorescence images of COS-7 cells expressing CFP-FRB-MoA (mitochondrial anchor, cyan) along with either mCherry-FKBP-PLAAT3-FL (red) or mCherry-FKBP-PLAAT3-FL-LD (lipase-dead PLAAT3, red) before and 35 mins after addition of 100 nM rapamycin. Enlarged images to highlight mitochondria morphology are shown on the right. Experimental timeline and schematic drawing of constructs used in this experiment are shown on top. c A summary of PLAAT3 domain structures (top), as well as C-terminal sequences of full-length PLAAT3 along with its lipase-dead mutant (LD) and a series of truncation mutants (bottom). Properties such as efficiency of inducible deformation, subcellular localization and cell viability were scored based on experiments described below, and indicated for each mutant tested (right). Pro: proline-rich domain, LRAT: Lecithin-retinol acyltransferase (also responsible for PLA 1 /A 2 activity), TM: putative transmembrane domain, CT: C-terminus domain, FL: full length of human PLAAT3, LD: lipase dead harboring C113S point mutation, 20TM, 19TM, 18TM, and 15TM: PLAAT3 mutant with truncations in TM and a full defect of CT, dTM: PLAAT3 mutant totally defective of TM and CT. d A fraction of COS-7 cells indicating fully-deformed mitochondria was calculated before (grey) and after (light brown) rapamycin treatment, and presented for cells expressing mCherry-FKBP-FL-LD, mCherry-FKBP-FL, mCherry-FKBP-2CT, mCherry-FKBP-dCT, mCherry-FKBP-18TM, or mCherry-FKBP-dTM and CFP-FRB-MoA. n = 104, 110, 121, 96, 138, 115, 103, 102, 97, 93, 125, and 111 cells from left to right; analyzed from three individual experiments. e Fluorescence images of cells expressing YFP, YFP-FL, YFP-FL-LD, or YFP-18TM, obtained at 48-hr post-transfection. These cells were stained with Hoechst. Insets are zoom-in images. Scale bar = 100 μm. f A number of YFP positive cells shown in e was calculated and used as a measure of cell viability. n = 591, 556, 499 and 601 cells from left to right; analyzed from three individual experiments. Error bars indicate means ± s.d..

Article Snippet: Human Plaat cDNAs were purchased from ORIGENE [RC208444 for PLAAT1 (NM_020386), RC212578 for PLAAT2 (NM_017878), RC200242 for PLAAT3 (NM_007069), RC201923 for PLAAT4 (NM_004585), and RC228184 for PLAAT5 (NM_054108)].

Techniques: Activity Assay, Membrane, Fluorescence, Expressing, Construct, Mutagenesis, Transfection, Staining