Over-expression of alpha-actinin with a GFP fusion protein is sufficient to increase whole-cell stiffness in human osteoblasts.
Osteoblasts respond to shear stress by simultaneously increasing their whole-cell stiffness and up-regulating the cytoskeletal crosslinking protein alpha-actinin. The stiffness of reconstituted cytoskeletal networks increases following the addition of alpha-actinin, but the effect of alpha-actinin on whole-cell mechanical behavior has not been investigated. The hypothesis of this study was that increasing alpha-actinin in the cytoskeleton would be sufficient to increase whole-cell stiffness. hFOB osteoblasts were transfected with a plasmid for GFP-tagged alpha-actinin, resulting in a 150% increase in the amount of alpha-actinin. The GFP-alpha-actinin fusion protein co-fractionated with the cytoskeleton and co-localized to the same regions of the cytoskeleton as endogenous alpha-actinin. Whole-cell mechanical behavior was measured by atomic force microscopy using a 25 mum diameter microsphere as an indenter. The whole-cell stiffness of cells over-expressing GFP-alpha-actinin was 60% higher than cells expressing only endogenous alpha-actinin (p < 0.002), which was within the range of mechanical behavior observed in osteoblastic cells exposed to 1 and 2 Pa of fluid shear. These results indicate that the up-regulation of alpha-actinin synthesis in osteoblasts is sufficient to alter the whole-cell mechanical behavior and highlights the potential role of alpha-actinin to reinforce cells against mechanical loads.
Jackson
Wesley M
WM
Department of Mechanical Engineering, Orthopaedic Biomechanics Laboratory, University of California, 6175 Etcheverry Hall, Mailstop 1740, Berkeley, CA 94720-1740, USA.
Jaasma
Michael J
MJ
Baik
Andrew D
AD
Keaveny
Tony M
TM
D000185:Actinin / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D004548:Elasticity; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D018625:Microscopy, Atomic Force; D010006:Osteoblasts / Q000502:physiology* / Q000648:ultrastructure; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D033081:Shear Strength; D013314:Stress, Mechanical; D014162:Transfection; D015854:Up-Regulation
D011993:Recombinant Fusion Proteins; D000185:Actinin; D049452:Green Fluorescent Proteins