Sparc localizes to the blebs of hobit cells and human primary osteoblasts.
Secreted protein acidic and rich in cystein (SPARC) is a secreted glycoprotein involved in several biological processes such as tissue remodeling, embryonic development, cell/extracellular matrix interactions, and cell migration. In particular, SPARC affects bone remodeling through the regulation of both differentiation/survival of osteoblasts and bone extracellular matrix synthesis/turnover. Here, we investigated SPARC subcellular localization in the human osteoblastic HOBIT cell line by immunocytochemistry and western blot analysis. We show that, under normal exponential cell growth conditions, SPARC localized both to cell nucleus and to cytoplasm, with no co-localization on actin stress fibers. However, in colchicine-treated HOBIT cells and human primary osteoblasts undergoing blebs formation, SPARC showed a different cellular distribution, with an additional marked compartmentalization inside the blebs, where it co-localized with globular actin and actin-binding proteins such as alpha-actinin, cortactin, and vinculin. Moreover, we demonstrate by an in vitro assay that the addition of SPARC to actin and alpha-actinin inhibited the formation of cross-linked actin filaments and disrupted newly formed filaments, most likely due to a direct interaction between SPARC and alpha-actinin, as indicated by immunoprecipitation assay. The specific silencing of SPARC RNA expression markedly decreased the ability of colchicine-treated HOBIT cells to undergo blebbing, suggesting a direct role for SPARC in cell morphology dynamics during cytoskeletal reorganization.
Baldini
Giovanna
G
Department of Biomedicine, Section of Morphology, University of Trieste, via Manzoni 16, 34138 Trieste, Italy.
Ponti
Cristina
C
Bortul
Roberta
R
Narducci
Paola
P
Grill
Vittorio
V
Martelli
Alberto M
AM
D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D022081:Cell Surface Extensions / Q000378:metabolism* / Q000648:ultrastructure; D051356:Cortactin / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing; D006801:Humans; D010006:Osteoblasts / Q000378:metabolism*; D015676:Osteonectin / Q000378:metabolism*; D021381:Protein Transport; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D051356:Cortactin; D005353:Fibronectins; D015676:Osteonectin; D000185:Actinin; D016596:Vinculin