Wang Yongqiang, Brooks Patricia Joyce, Jang Janet Jinyoung, Silver Alexandra Shade, Arora Pamma D, McCulloch Christopher A, Glogauer Michael
Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
Biochim Biophys Acta. 2015 Jul;1853(7):1715-24. doi: 10.1016/j.bbamcr.2015.04.001. Epub 2015 Apr 12.
Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.
细胞融合过程是破骨细胞生成过程中的一个关键限速步骤，但调节丝状伪足形成的机制尚未明确。我们对源自稳定转染了一种质粒的细胞培养的前破骨细胞中的丝状伪足生成进行了表征，该质粒表达一种与mEGFP融合的短肌动蛋白丝结合肽（Lifeact），可使肌动蛋白丝在活细胞中定位。融合始于丝状伪足，丝状伪足是宽度大于2μm的细胞延伸部分，其延伸末端对肌球蛋白-X进行免疫染色。丝状伪足在较大迁移细胞的前沿以及相邻较小细胞的尾部形成，这两种细胞都沿相同方向迁移。DC-STAMP的染色是环绕性的，并不定位于细胞-细胞融合位点。与野生型细胞相比，Rac1基因缺失的单核细胞丝状伪足数量减少6倍，形成的多核破骨细胞数量减少4倍。从延时图像中我们发现，融合在时间上与肌动蛋白丝的连贯且空间隔离的条带形成相关，这些条带起源于细胞体并延伸到丝状伪足中。在接近的细胞形成初始接触后，这些肌动蛋白丝条带参与细胞融合。我们得出结论，丝状伪足的形成受Rac1调节，以在破骨细胞生成过程中启动细胞间接触。这一步骤之后是丝状伪足中肌动蛋白丝条带的严格调控组装，这导致细胞间间隙的闭合，最终实现细胞融合。这些新的、依赖肌动蛋白的过程对破骨细胞生成中的融合过程很重要。
