Use of transduction proteins to target trabecular meshwork cells: outflow modulation by profilin I.
OBJECTIVE
Fusion proteins containing a protein transduction domain (PTD4) are able to cross biological membranes. We tested the applicability of the protein transduction method for study of the aqueous humor trabecular outflow pathway by targeting the actin cytoskeleton, which is known to be involved in outflow facility regulation.


METHODS
Expression vectors useful for generating fusion proteins with the PTD4 domain and the actin-binding protein Profilin I were constructed. The transductional and functional properties of these proteins were tested in bovine trabecular meshwork cells in culture. The effects of PTD4-Profilin I on outflow facility were evaluated in perfused bovine anterior segments. PTD4-beta-galactosidase was used to visually check correct delivery of fusion proteins to trabecular meshwork cells.


RESULTS
The fusion proteins generated were characterized by western blot. Immunocytochemistry experiments showed intracellular staining for PTD4-Profilin I in trabecular meshwork cells in culture. The fusion protein was found in the cytoplasm associated with actin filaments and in the leading edge of the cellular membrane. In contrast, control Profilin I, without the PTD4 domain, was unable to cross the cell membrane. In perfused anterior segments, 2 microM PTD4-Profilin I increased trabecular outflow facility in a reversible manner, while Profilin I had no significant effect. Anterior segments perfused with PTD4-beta-galactosidase showed positive staining in the trabecular meshwork tissue.


CONCLUSIONS
Protein transduction technology is a valuable tool for targeting trabecular meshwork tissue, not only for performing physiological studies, but also as a potential drug-delivery method. Profilin I action on the actin cytoskeleton further reinforces the importance of this structure in outflow facility regulation.
Gomez-Cabrero
Azucena
A
Department of Physiological Sciences, I-August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Faculty of Medicine, University of Barcelona, Barcelona, Spain.
Comes
Nuria
N
Gonzalez-Linares
Javier
J
de Lapuente
Joaquin
J
Borras
Miquel
M
Pales
Jordi
J
Gual
Arcadi
A
Gasull
Xavier
X
Morales
Miguel
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D000869:Anterior Eye Segment / Q000378:metabolism*; D001082:Aqueous Humor / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015696:Gene Products, tat / Q000378:metabolism*; D005822:Genetic Vectors; D026901:Membrane Transport Proteins / Q000502:physiology; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000378:metabolism; D051304:Profilins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*
D000199:Actins; D015696:Gene Products, tat; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D051304:Profilins; D011993:Recombinant Fusion Proteins