Transduction of TAT fusion proteins into the human and bovine trabecular meshwork.
OBJECTIVE
To examine the applicability of TAT (the protein transduction domain of transactivating transcription polypeptide)-mediated protein-transduction technology, in introducing proteins of interest into trabecular meshwork (TM) cells in various culture systems.


METHODS
Normal human TM cell cultures, human tissues in organ cultures, and bovine eyes in perfusion organ cultures were incubated or perfused for various lengths of time with TAT- and hemagglutinin (HA)-tagged fusion proteins, TAT-HA-beta-galactosidase (TAT-HA-beta-gal), TAT-HA-myocilin, and TAT-HA-myocilin-EGFP. Transduction of TAT-HA-beta-gal was detected by X-gal staining. Transduction of myocilin or myocilin-EGFP was evaluated by immunostaining or fluorescence. beta-Gal and EGFP proteins were used as the negative control.


RESULTS
Blue X-gal staining, signifying beta-gal activity resulting from transduction, was observed in cultured TM cells in a concentration- and time-dependent manner. TAT-HA-beta-gal was also transduced into cells in all regions of TM tissues in organ cultures. TM cell cultures, after TAT-HA-myocilin incubation, showed an enhanced myocilin staining compared with the control cultures. Stronger myocilin or HA staining was also noted in TM tissues of TAT-HA-myocilin-incubated or -perfused eyes. Myocilin transduction resulted in a loss of actin stress fibers and focal adhesions in TM cells in culture. The level of phosphorylated myosin light chain was reduced. Human and bovine TM tissues after TAT-myocilin transduction also exhibited a diminished actin and paxillin-vinculin staining.


CONCLUSIONS
TAT fusion proteins can be efficiently transduced into TM cells and tissues. The TAT-mediated protein transduction technology may be valuable in studies of proteins such as myocilin in the TM.
Sakai
Hiroshi
H
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Park
Bum-Chan
BC
Shen
Xiang
X
Yue
Beatrice Y J T
BY
D000199:Actins / Q000378:metabolism; D000328:Adult; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015696:Gene Products, tat / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D009924:Organ Culture Techniques; D051419:Paxillin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*; D014161:Transduction, Genetic; D016596:Vinculin / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism; D000099179:Myocilin
D000199:Actins; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D015696:Gene Products, tat; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D006388:Hemagglutinins; D051419:Paxillin; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D001616:beta-Galactosidase; D000099179:Myocilin; C485184:enhanced green fluorescent protein