Identification of the molecular mechanisms contributing to polarized trafficking in osteoblasts.
The directionality of matrix deposition in vivo is governed by the ability of a cell to direct vesicularflow to a specific target site. Osteoblastic cells direct newly synthesized bone matrix proteins toward the bone surface. In this study, we dissect the molecular mechanisms underlying the polarized trafficking of matrix protein in osteoblasts. We demonstrate using TEM, immunocytochemistry, and cDNA analysis, the ability of osteoblastic cells in culture to form tight junction-like structures and report the expression of the tight junction associated proteins occludin and claudins 1-3 in these cells. We identify intercellular contact sites and the leading edge of migratory osteoblasts as major target sites of vesicular trafficking in osteoblasts. Proteins required for this process, rsec6, NSF, VAMP1, and syntaxin 4, as well as the bone matrix protein, osteopontin, localize to these sites. We demonstrate that osteoblasts in vivo possess VAMP1 and, furthermore, report the expression of two VAMP1 splice variants in these cells. In addition, osteoblasts express the NSF attachment protein alpha-SNAP and the t-SNARE SNAP23. Thus, cell-to-cell contact sites and the leading edge of migratory osteoblasts contain a unique complement of proteins required for SNARE mediated membrane fusion.
Prêle
Cecilia M
CM
Bone and Mineral Centre, Royal Free and University College Medical School, The Rayne Institute, London, WC1E 6JJ, United Kingdom.
Horton
Michael A
MA
Caterina
Paul
P
Stenbeck
Gudrun
G
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism / Q000648:ultrastructure; D002450:Cell Communication / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D062445:Claudin-1; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D005333:Fetus; D008565:Membrane Proteins / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D062793:Occludin; D010006:Osteoblasts / Q000378:metabolism* / Q000648:ultrastructure; D010012:Osteogenesis / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology*; D050683:R-SNARE Proteins; D051381:Rats; D017208:Rats, Wistar; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D019108:Tight Junctions / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D062445:Claudin-1; C568114:Cldn1 protein, rat; C096102:Exoc3 protein, rat; C076326:Exoc4 protein, rat; D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins