Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter.
BACKGROUND
The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag, to the extracellular domain of a truncated integrin.


RESULTS
Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture.


CONCLUSIONS
The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.
Svendsen
Soshana
S
Promega Corporation 2800 Woods Hollow Road, Madison, WI 53711, USA. soshana.svendsen@promega.com
Zimprich
Chad
C
McDougall
Mark G
MG
Klaubert
Dieter H
DH
Los
Georgyi V
GV
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D016023:Integrins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins