Robust transient anchorage was observed with the maximal cross-linking protocol. The preassembled complexes clustering 135 or fewer Thy-1 GPIAPs are shown to be sufficient to trigger transient anchorage comparable with the clusters formed by maximal cross-linking. Cholesterol is essential for gold particle–bound clusters to be transiently anchored on the cell surface, suggesting that a cholesterol-dependent nanodomain is formed under maximally cross-linked gold particles. The cross-linking–triggered signaling events involve SFK cascades, PI3 kinase, and caveolin-1. What molecular mechanisms might account for this phenomenon? The broad outlines of how different modes of transient anchorage might occur can be gleaned from recent reviews. Simons and Toomre (2000)
suggested that the cross-linking of receptors might be a key to effecting signal transduction by either altering partitioning into existing raft domains or bringing smaller rafts together. Kusumi et al. (2004)
suggested that one mode of coupling clustered GPIAPs may involve an unspecified transmembrane protein and recruitment of small inner leaflet rafts containing lipid-linked signaling molecules to the site of the cluster raft on the outer leaflet.
To focus future experiments, we propose a somewhat more specific working hypothesis () using these ideas as a starting point. In this model, cross-linking induces cholesterol-dependent nanodomains (cluster rafts) to form on both the inner and outer leaflet. Such clusters would include key transmembrane proteins bridging the inner and outer leaflets so that signals could be transmitted across the membrane in discrete locations. These proteins would be included from the outset and/or incorporated after collision of the cluster with the transmembrane protein. Whether initial anchoring is assisted by oligomerization-induced trapping (Kusumi et al., 2005
) is an open issue. When an activated SFK randomly partitions into such a nanodomain on the inner leaflet, phosphorylation by SFK induces a resident transmembrane molecule to attach to the actin cytoskeleton through adaptor proteins. The attachment results in a transient anchorage event that continues until the SFK is deactivated and a recruited phosphatase dephosphorylates the resident linking molecule.
Figure 6. Transient anchorage hypothesis. Cross-linking causes nanodomains to form on both the inner and outer leaflet, with a transmembrane protein bridging the two leaflets to transduce the signal. When an activated SFK randomly partitions into such a nanodomain, (more ...)
Specific molecular players can be accommodated by this generic hypothesis. A novel SFK substrate, C-terminal Src kinase–binding protein (Cbp; or PAG), was identified recently as a transmembrane protein-binding partner for C-terminal Src kinase, a kinase that regulates SFKs by phosphorylation at their C-terminal regulatory site (Matsuoka et al., 2004
). There is also evidence indicating that Thy-1 is associated with Cbp (Durrheim et al., 2001
). Furthermore, after phosphorylation by proximate SFKs, Cbp binds the ERM (ezrin/radixin/moesin)-binding protein (EBP50) via its PDZ domain and, thus, associates with the cytoskeleton through ERM proteins (Itoh et al., 2002
). Therefore, Cbp is a plausible candidate for the transmembrane protein component of the hypothesis. Indeed, we have obtained results that are at least consistent with the transient anchorage of GPIAPs proceeding via the EBP50-ERM-actin cytoskeleton linkage: an EBP50-binding transmembrane protein, CFTR, exhibits transient anchorage without using the maximal cross-linking protocol, and removal of the C-terminal PDZ-binding domain of CFTR, which binds EBP50, in the Δ4 mutant (Gentzsch et al., 2004
) abrogates transient anchorage (). Therefore, our hypothesis suggests that there is a common mode of cytoskeletal binding for both CFTR and GPIAPs (via EBP50-ezrin) but two different ways to couple to these adaptors, either directly (CFTR) or indirectly (GPIAP) via the formation of nanodomains and SFK regulation. Indeed, a regulatable ezrin linkage to Cbp was recently hypothesized to link rafts containing the B cell receptor to the lymphocyte cytoskeleton (Gupta et al., 2006
Previous studies have shown that caveolin-1 deficiency diminishes both the number of caveolae (Drab et al., 2001
) and caveolin-2 expression (Razani et al., 2001
). Therefore, the diminution of transient anchorage in caveolin-1−/−
cells is consistent with some of it occurring in caveolar structures. Alternatively or additionally, other cellular functions of caveolin-1 and -2 may be associated with transient anchorage. In their studies of the mobility of Simian virus 40 (SV40) on the cell surface before and just after its entry into the cell, Damm et al. (2005)
also found a partial dependence on caveolin-1. Their findings can be compared and contrasted with our study. After diffusing, SV40 often stops as opposed to being transiently anchored as a prelude to internalization via caveolin-1–dependent or –independent pathways. However, only the caveolin-1–independent internalization pathway of SV40 requires tyrosine kinase activity and cholesterol.
In our study, transient anchorage does not exclusively depend on caveolin-1, suggesting at least two pathways to anchorage, but both pathways depended on cholesterol and SFK. Damm et al. (2005)
also used SPT to study the diffusion behavior of mouse polyoma viruslike particles (which are 45 nm in diameter and similar to the size of gold particles) bound to live cell membranes and found cholesterol-dependent but SFK-independent confinement (Ewers et al., 2005
). Viruslike particles exhibited confined diffusion in small zones 30–60 nm in diameter, which differs from transient anchorage in the fact that neither SFKs nor caveolin is required for confinement. Confinement requires the cross-linking of viral receptor (ganglioside), which promotes linkage to the actin cytoskeleton in an as yet undefined way. In general, based on the extensive study of viral interactions with the cell membrane, it could be anticipated that multiple modes of transient anchorage would exist (Marsh and Helenius, 2006
Unexpectedly, inhibition of the conversion of PIP2
by PI3 kinase enhances transient anchorage and induces directional motion in a substantial fraction of trajectories. It is possible that PIP2
may preferentially partition into the inner leaflet microdomains induced by the cross-linked clusters of GPIAPs, and transient anchorage might then be formed via PIP2
to adaptor proteins that are associated with the actin cytoskeleton underneath the cell membrane, such as the ERM family proteins (Fievet et al., 2004
). The bidirectional movements observed in some of the trajectories suggests that PIP2
clusters that participate in transient anchorage might also bind to motor proteins through their FERM or pleckstrin homology domains, thus contributing to the directed transport of cross-linked clusters by walking along cytoskeletal filaments. Binding between clustered PIP2
and motor proteins also provides a possible explanation for the disappearance of longer stopping periods because the motor proteins are active most of the time and might be expected to move the cluster directionally with short pauses (Kural et al., 2005
Collectively, our data suggests that the transient anchorage phenotype may be regulated in different ways depending on the biological context. Moreover, the transient anchorage assay presented here should be valuable in defining precise linkages to the cytoskeleton and how they are regulated.