Possible Role of Direct Rac1-Rab7 Interaction in Ruffled Border Formation of Osteoclasts

Abstract

Rab7 has been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts, it is involved in formation of the ruffled border, which is a late endosomal-like compartment in the plasma membrane. Here we report a new Rab7-interacting protein, Rac1, another small GTPase protein that binds to the GTPform of Rab7 as found with a two-hybrid system. We demonstrate further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, partial colocalization is perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments enabling endosomal vesicles to switch tracks and may thus also regulate ruffled border formation in osteoclasts.Skeletal modeling during growth and bone remodeling in the adult skeleton are dependent on bone resorption. Osteoclasts are the cells that are responsible for the degradation of the organic and inorganic bone matrix. These multinucleated cells mature from a monocyte/ macrophage lineage of precursor cells and are mainly located in the vicinity of the bone surface in bone marrow (1). When bone resorption is induced, osteoclasts migrate to the site of the resorption, become highly polarized (2), and form four distinct membrane domains: the ruffled border, sealing zone, functional secretory domain, and basolateral domain (2-4). During the process of osteoclast activation, the cells attach to the bone matrix through a ring-like structure called a sealing zone. The membrane-facing bone matrix inside the sealing zone is the ruffled border formed via rapid fusion of intracellular acidic vesicles to the plasma membrane (5, 6). This results in acidification and release of proteinases into the space between the bone matrix and the ruffled border known as the resorption lacuna. As a result, bone matrix is digested (7). The mineral is dissolved by acid and collagen matrix degraded by proteinases, the degradation products are internalized locally, and transcytosed to the functional secretary domain at the top of the polarized osteoclasts for secretion (8, 9). Thus, the ruffled border, which is assembled by fusion with endosomal membranes, shows not only characteristics of the plasma membrane but also those of the late endosomal/lysosomal membranes (5). Indeed, many proteins present at the ruffled border are also found in endosomal and/or lysosomal membranes, including Rab7, vacuolar H ϩ -ATPase, cathepsin K, and others.We have previously shown that the ruffled border is divided functionally into two subdomains, the fusion zone for secretion and the uptake zone for endocytosis of degraded products (10).Recent studies have shown that Ras-related small GTPases of the Rab family control the endocytic, secretory, and recycling traffic of intracellular vesicles in mammalian cells (11)(12)(13)(14). Approximately 60 Rab proteins have been identified in the human gen...

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