By using dual-view TIRF acquisition, we were able to simultaneously measure the steady-state diffusion of the two BCR isotypes expressed by naive B cells. We observed that diffusion of both IgM and IgD is restricted within the plasma membrane. Interestingly, the proportion of BCR that was very slow diffusing was greater for IgD compared to IgM. The reason for the increased immobility of IgD has yet to be identified but may be due to the presence of both transmembrane and GPI-anchored forms, which could alter microdomain association (Chaturvedi et al., 2002; Wienands and Reth, 1992
). Moreover, we previously observed that IgD appears to be preclustered (Depoil et al., 2008
) and such organization could influence diffusion dynamics. Indeed, it should be noted that although we are able to visualize single particles of BCR, we do not know whether these particles are monomers or oligomers (Schamel and Reth, 2000
) or even higher-order “protein islands” (Lillemeier et al., 2006
We have identified the intracellular domain of Igβ as a crucial element for the restriction in BCR diffusion. Simply substituting the intracellular domain of Igβ with that of MHC class I increased BCR diffusion nearly 3-fold. The intracellular domain of Igβ and MHC class I do not differ greatly in size, particularly in comparison to the whole protein, so it is unlikely that the difference in diffusion is merely size-dependent slowing. Instead, our data suggest that Igβ increases the efficiency of actin-mediated restriction in BCR diffusion. It remains to be determined whether this is due to a specific interaction between Igβ and actin, or whether a difference in the length or tertiary structure of the cytoplasmic domain of Igβ and MHC class I affects their trapping by the membrane skeleton (Edidin et al., 1994
). Also, it is possible that some of this difference in diffusion may be due to a difference in the net charge of the cytoplasmic domain, which could affect its affinity for proteins in the cytoplasm or electrostatic interactions with phospholipids in the plasma membrane (Xu et al., 2008
). The very small difference between the IgM-Mutβ chimeric receptor and the diffusion dynamics of the wild-type receptor suggests that the transmembrane domain may also influence BCR diffusion, possibly through interaction with other proteins, such as CD19 and/or CD21 (Carter et al., 1997
); the formation of BCR oligomers (Schamel and Reth, 2000
); or microdomain association, which could influence additional factors such as molecular crowding (Dix and Verkman, 2008; Zhou, 2009
A recent study examining the role of the extracellular Cμ4 domains in BCR clustering upon antigen stimulation noted that a proportion of BCR are immobile in unstimulated cells (Tolar et al., 2009
). However, the mechanism or functional relevance of this observation was not investigated. Here, simultaneous visualization of two parameters has allowed us to investigate the mechanism that restricts steady-state BCR diffusion. We find that BCR diffusion is highly reduced within actin-rich regions, which may consist of membrane compartments on the nanometer scale as suggested by the membrane skeleton fence model (Kusumi et al., 2005
). Moreover, we observe that this network defines micron-sized compartments, consistent with recent SPT observations of Fc
RI in a basophilic cell line (Andrews et al., 2008
). However, this work did not report highly restricted diffusion of Fc
RI within actin-rich regions, so it remains to be determined whether our observations reflect a general phenomenon for ITAM-containing immunoreceptors.
Here we directly visualize how the dynamic linkage of the plasma membrane to the actin cytoskeleton influences diffusion. We identify the ERM protein ezrin as an important component regulating this interaction and defining BCR diffusion. We find that ezrin defines compartments or “corrals” that restrict diffusion as proposed in early models (Saxton, 1995; Sheetz, 1983; Sheetz et al., 1980
). No doubt additional structural proteins of the membrane skeleton, such as gelsolin, villin, or spectrin, are likely to also participate in the regulation of diffusion. What's more, we observed rapid remodeling of the ezrin network and propose that ezrin provides a mechanism to very quickly modify membrane protein diffusion. Indeed, our visualization of ezrin while simultaneously tracking the BCR suggests that such proteins dynamically “gate” (Tsuji and Ohnishi, 1986
) the diffusion of membrane proteins and may permit the transitional “hop” between compartments observed in previous SPT studies (Fujiwara et al., 2002; Kusumi et al., 1993
). Moreover, receptor signaling could regulate such a linkage (Delon et al., 2001; Faure et al., 2004; Gupta et al., 2006
) and thus fine tune diffusion dynamics during activation. We posit that antigen-engaged BCRs probably trigger a localized dephosphorylation of ERM proteins and detachment of the membrane skeleton, thus altering diffusion of unengaged BCR in close proximity, which may then gain accessibility to ligand or BCR microclusters.
Importantly, we find that gross alteration of the actin cytoskeleton is sufficient to trigger B cell signaling to a similar extent as BCR crosslinking. Moreover, we demonstrate that this signaling is most probably mediated via the BCR and is correlated with increased BCR diffusion. It is therefore conceivable that the steady-state dynamism of the actin cytoskeleton, that is, small constitutive alterations in the organization of F-actin, provides a mechanism to generate low-intensity tonic BCR signaling. Such signals may then feedback into alterations of the actin cytoskeleton. Indeed, we know that ligand-induced BCR signaling rapidly alters the organization of F-actin (Hao and August, 2005
). Thus, we suggest that tonic BCR signaling may influence actin organization and dynamics. In line with this, B cells lacking key BCR signaling molecules have dramatically altered steady-state morphology (Weber et al., 2008
). Moreover, IgM diffusion is decreased in B cells deficient in PLCγ2 and Vav1 and 2. These results imply an interplay between BCR signaling and the actin cytoskeleton, which probably contributes to the regulation of tonic signaling in B cells.
At present, it is not clear how alteration of the actin cytoskeleton triggers BCR signaling. According to the oligomeric BCR complex model (Reth et al., 2000; Schamel and Reth, 2000
), association of antigen with pre-existing BCR oligomers induces a disruption of the oligomeric complex permitting accessibility and phosphorylation of Igα-β and the activation of signaling. Perhaps the actin cytoskeleton has a role in maintaining this complex. Interestingly, recent electron microscopy studies have shown that the TCR exists within higher-order protein islands that are connected to the actin cytoskeleton and depend on it for their formation and/or maintenance (Lillemeier et al., 2006
). It may be that the oligomeric BCR complex is linked to actin and alteration of F-actin causes a similar disruption to the oligomeric complex induced by antigen and thus triggers BCR signaling. Dynamic actin remodeling might then be important for subsequent internalization of active BCR from the cell surface resulting in signal termination, as previous studies have suggested (Stoddart et al., 2005
). It has also been suggested that the BCR undergoes clustering and a conformational opening of Igα-β upon antigen binding, which is dependent on association of the BCR with distinct lipid domains (Tolar et al., 2005
). It may be that disruption of the actin cytoskeleton alters the localization of BCR and lipid rafts, possibly bringing together these membrane domains.
However, our data suggest that signaling induced by disruption of actin may be related to a change in BCR diffusion. Two possible models can be envisaged to account for our results. One model is that the membrane skeleton restricts BCR mobility and may thus restrict the interaction between the BCR and coreceptors or activated signaling molecules. Disruption of the diffusion barrier increases the mobile fraction of the BCR and may thus increase the probability that the BCR will encounter an activated kinase or coreceptor such as CD19. Alternatively, it may be that the actin cytoskeleton affects the segregation of kinases or phosphatases from the BCR during the steady state. For example, the actin cytoskeleton may immobilize BCRs and phosphatases together and disruption of F-actin releases this inhibitory interaction as BCRs diffuse away. An important parameter to determine will be whether the number of cell surface BCRs alters the signaling induced by disruption of the actin cytoskeleton, as well as the location of tyrosine kinases, protein phosphatases, and coreceptors. Future research investigating the locations of these activating and inhibitory molecules in relation to actin may shed light on these potential models.
Our data provide convincing evidence that an ezrin- and actin-defined network influences steady-state BCR diffusion dynamics by creating barriers that restrict BCR diffusion. We identify the intracellular domain of Igβ as important for the efficiency of this restriction in BCR diffusion. Importantly, alteration of this network is sufficient to induce robust intracellular signaling, in the absence of antigen stimulation, which is concomitant with an increase in BCR mobility. Moreover, we show that this signaling is most probably initiated by the BCR. Thus, our results suggest that the membrane skeleton plays an important function in controlling BCR dynamics and thereby signaling in a way that could be important for understanding both tonic and antigen-induced signaling.