Unique kinetics of Oct3/4 microlocalization following dissociation of human embryonic stem cell colonies.
To investigate the effects of the Rho-dependent protein kinase (ROCK) inhibitor Y-27632 on the kinetics of E-cadherin, F-actin, and Oct3/4 distributions in dissociated human embryonic stem (hES) cells and to analyze their interactions morphologically, Y-27632-treated [R(i) (+)] and untreated [R(i) (-)] cells were immunohistochemically stained for E-cadherin and Oct3/4 within 24h of dissociation and also for F-actin. Furthermore, the gene expression of E-cadherin, Oct3/4, and RhoA was confirmed by quantitative real-time RT-PCR. E-cadherin expression intensified linearly along the membranes of R(i) (+) cells or intercellular junctions in cell clusters. F-actin accumulated along the periphery of cells and expanded in a web-like manner along junctions in cell clusters, and Oct3/4 was restricted to the nucleus within few hours of dissociation. However, R(i) (-) cells exhibited deformation and blebbing and appeared to die over time. E-cadherin exhibited a punctate pattern along the periphery, after which it accumulated on one or both sides of the cytoplasm. Actin filaments were concentrated at the bleb bases. Oct3/4 was detected in the cytoplasm, not in the nucleus the recovery of integrated E-cadherin distribution. Quantitative real-time RT-PCR revealed RhoA upregulation and E-cadherin downregulation at 12h after dissociation. Oct3/4 gene expression was unaffected by ROCK inhibition. These results revealed that the cooperative nature of hES cells is maintained by the E-cadherin-actin cytoskeleton system along with the restricted distribution of Oct3/4 in the nucleus. RhoA activation followed by dissociation disorders this system and accelerates cell death, which is partially suppressed by ROCK inhibition.
Department of Histology and Embryology, Shinshu University School of Medicine, Matsumoto, Nagano, Japan.
Hinako
H
Ichikawa
Yoshiya
Y
Kanoh
Sakiko
S
Shirasawa
Tadayuki
T
Yokoyama
Fengming
F
Yue
Daihachiro
D
Tomotsune
Katsunori
K
Sasaki
D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000648:ultrastructure*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D027701:Organic Cation Transport Proteins / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors; D000099254:Organic Cation Transporter 3
D000199:Actins; D000577:Amides; D015820:Cadherins; D004791:Enzyme Inhibitors; D027701:Organic Cation Transport Proteins; D011725:Pyridines; D054460:rho-Associated Kinases; D000099254:Organic Cation Transporter 3; C108830:Y 27632