Peptide-based targeting of fluorophores to organelles in living cells.
Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A peptide carrying the nuclear leader sequence of the simian virus SV40 large tumor antigen, KKKRK, was used to direct the fluorophore to the nucleus. The fluorescent peptides for peroxisomes, ER, and the TGN spontaneously incorporate into living fibroblasts at 37 degrees C and accumulate in their target organelles within minutes. The uptake is still significant at 4 degrees C, indicating that endocytosis is not required for internalization. The highly charged nuclear peptide (net charge +4) does not spontaneously internalize. However, by transient permeabilization of the plasma membrane, this fluorescent peptide was found to rapidly accumulate in the nucleus. These fluorescent peptides open new opportunities to follow various aspects of specific organelles such as their morphology, biogenesis, dynamics, degradation, and their internal parameters (pH, redox).
Pap
E H
EH
Institute of Biomembranes, Department of Biochemistry of Lipids, Utrecht University, 3508 TB Utrecht, The Netherlands.
Dansen
T B
TB
van Summeren
R
R
Wirtz
K W
KW
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D001896:Boron Compounds / Q000378:metabolism*; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D003208:Concanavalin A / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D018613:Microscopy, Confocal; D015388:Organelles / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D051381:Rats
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D015415:Biomarkers; D001896:Boron Compounds; D002518:Ceramides; D005456:Fluorescent Dyes; D010455:Peptides; D021382:Protein Sorting Signals; D003208:Concanavalin A; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan