Cytoskeletal reorganization by soluble Wnt-3a protein signalling.
BACKGROUND
Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles.


RESULTS
We prepared mouse L cells transfected with Wnt-3a cDNA, and found that their beta-catenin protein level was up-regulated. When conditioned medium (CM) was collected from cultures of the transfectants and added to nontransfected L cells, the beta-catenin level of the latter was also increased. Approximately 50% of the Wnt-3a proteins synthesized by the transfectants were secreted into the CM in a soluble form. These secreted Wnt-3a proteins formed an activity gradient in the environment surrounding the transfectants. Then, we studied whether Wnt-3a had any effect on cellular behaviour in vitro. When the CM containing Wnt-3a (W3a-CM) was added to cultures of C57MG mammary epithelial cells, their morphology was altered to exhibit closer intercellular contacts. Immunostaining for various adhesion and cytoskeletal proteins showed that the actin-microfilamental system was re-organized by the W3a-CM treatment. It induced a directional alignment of actin stress fibres and other actin-associated proteins. Moreover, villin, localized only at the perinuclear regions in untreated C57MG cells, was re-distributed to the leading edges of the cells, co-localizing with F-actin, in the presence of Wnt-3a.


CONCLUSIONS
Our findings suggest that Wnt-3a protein, in the soluble form, can act to re-organize cytoskeletal structures.
Shibamoto
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan.
Higano
K
K
Takada
R
R
Ito
F
F
Takeichi
M
M
Takada
S
S
D000199:Actins / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007739:L Cells; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D009024:Morphogenesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D015534:Trans-Activators; D014162:Transfection; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin
D000199:Actins; C495267:CTNNB1 protein, mouse; D015820:Cadherins; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D011506:Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; C026742:plastin; C026615:villin