Novel proteins mediate an interaction between clathrin‐coated vesicles and polymerizing actin filaments

Abstract

A monoclonal antibody, A-7C11, was generated which reacts with two polypeptides of 40 kDa and 80 kDa associated with the coat proteins of purified brain clathirn-coated vesicles. The 40-kDa antigen was purified and found to display actin-binding properties. Negative-staining electron microscopy showed that one of the antigens reactive with A-7C11 appears to mediate the association of isolated clathrin-coated vesicles with assembling actin filaments in vitro. Immunofluorescence microscopy of cultured fibroblasts with A-7C11 revealed the antigens aligned with both actin filaments and as punctate structures near the plasma membrane. The data suggest that the interaction between clathrin-coated vesicles and the actin cytoskeleton is mediated by antigens identified by monoclonal antibody A-7Cll.Clathrin-coated vesicles (CCV) associated with the plasma membrane participate in the clustering and internalization of receptor-ligand complexes [l, 21. Those associated with the Golgi cisternae mediate the processing [3] and secretion [4] of certain polypeptides, while those associated with synaptic terminals [2, 51 and the secretory regions of cells [6] may be involved in membrane recovery. Clathrin-coated vesicles have been isolated from a variety of tissues, and some of the major protein components of the vesicles' protein coat have been described (reviewed in [7]). On the other hand, a coherent understanding of how CCV participate in selecting their cargo, navigate the subcellular milieu, identify and interact with their target organelles has not emerged.In previous reports we showed that clathrin coat proteins displayed binding affinity for structural, anchoring and regulatory molecules of the cytoskeleton, namely actin, a-actinin and calmodulin [8, 91. More recently, the binding sites on clathrin for a-actinin and calmodulin were also defined [lo]. Immunofluorescence and ultrastructural studies by us and others suggested that a correlation exists in the distribution of clathrin-coated vesicles with actin microfilaments [11 -141. Moreover, a neuronal protein of 185 kDa (NP185), found associated with bovine brain clathrin-coated vesicles, displayed binding affinity for tubulin which was regulated by the casein kinase I1 phosphorylation of tubulin [15, 161. Thus, the identification of novel molecules in CCV which could mediate and regulate clathrin-coated vesicle functions related to the cytoskeleton could yield valuable insights into the biochemical basis of selective molecules and membrane transport.The generation of monoclonal antibodies (mAb) provided sensitive probes to identify and characterize individual components present in different types of vesicles [17] ual components of the mixture were then separated by the generation and cloning of hybridoma cells. The mAb A-7C11 was generated in this manner, using the dissociated coat proteins of bovine brain CCV as an immunogen. Among them, two previously unobserved components of 40 kDa and 80 kDa present in bovine brain CCV were found to be the cross-reactive antignes of...

Actin-, myosin- and ubiquitin-dependent endocytosis

“…A result that supports this view is the suppression of the pinocytic defect in D. discoideum double myosin null mutants when the amoebas are allowed to attach to substrate [60]. It is important to note that morphological and biochemical data suggest interactions between clathrin coats and the actin cytoskeleton [71,72]. The mechanism by which actin and myosin drive the formation of pinocytic vesicles is still completely unknown.…”

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…Morphological and biochemical data also suggest interactions between clathrin-coated vesicles and the actin cytoskeleton (Kohtz et al 1990;Salisbury et al 1980). Involvement of the actin cortex in the mobility of GFP-Iabelled clathrin-coated pits was recently shown in living mammalian (Gaidarov et al 1999) and D. discoideum cells (Damer et al 1998; Mol Bioi Cell 9: 130a).…”

A Myosin I Is Involved in Membrane Recycling from Early Endosomes

“…The ADP-ribosylation factor (ARF)-6 GTPase, which was shown to be important in modeling the plasma membrane and cortical actin cytoskeleton (Song et al, 1998), was recently implicated in the targeted delivery of recycling en-1014 dosomal vesicles to the plasma membrane (D' Souza-Schorey et al, 1998). Despite accumulating evidence for interactions between actin and coat proteins (Kohtz et al, 1990;Gaidarov et al, 1999), much less is known about the involvement of actin-dependent motor proteins. The discovery of a great diversity of myosins associated with many different organelles suggests that, in addition to bona fide transport, the actomyosin cytoskeleton may play a role in sorting processes and the modulation of membrane structures (Coluccio, 1997).…”