Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.
The association of actin filaments with the plasma membrane maintains cell shape and adhesion. Here, we show that the plasma membrane ion exchanger NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton. This occurs through a previously unrecognized structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM). NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, have impaired organization of focal adhesions and actin stress fibers, and an irregular cell shape. We propose a structural role for NHE1 in regulating the cortical cytoskeleton that is independent of its function as an ion exchanger.
Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NCI were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously described 7-S collagen domain. The pepsin-solubilized form lacked domain NCI and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NCI domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NCI) was estimated to be about 800 nm comprising the length of two molecules.Type IV collagen is a unique member among the collagenous proteins and is considered to be the major structural component of basement membranes [I 1. Biosynthetic studies have shown that the constituent chains of type IV collagen ( M , about 180000) are larger than those of interstitial collagens and procollagens, and that they are not substantially processed when deposited in the matrix [2-51. A further unique feature are frequent interruptions of the triple helix as indicated by sequence analysis [6]. This explains the protease sensitivity of native type IV collagen [7,8] and possibly causes a greater flexibility of the large triple-helical segments. These molecules also possess another short triple-helical segment (7-S domain) which appears to be part of a rather compact fragment named 7-S collagen [9,10].Electron microscopical studies have demonstrated that basement membranes have a rather amorphous appearance [I I], quite different from the cross-striated fibrillar structures of collagenous proteins observed in interstitial connective tissue. X-ray diffraction studies of stretched lens capsules have indeed indicated a poorly ordered fibrillar array [12]. On the basis of these observation Kefalides [I] suggested a model for the basement membrane matrix which envisions sheets of type IV collagen to be cross-linked to alternating layers of non-collagenous proteins. Ot...
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