DC sentinel function depends on the ability of these cells to take up a wide variety of antigenic materials. This is achieved through a combination of phagocytosis, macropinocytosis, and receptor-mediated endocytosis (11
). We have found that HS1 plays an important role in this process, by promoting receptor-mediated endocytosis of protein antigens. DCs from HS1−/−
mice show diminished receptor-mediated endocytosis of multiple proteins and impaired presentation of exogenous protein-derived antigens on both MHC Class I and MHC class II.
The requirement for HS1 in antigen trafficking and presentation appears to be restricted to receptor-mediated endocytic pathway. HS1−/−
DCs presented peptide antigens efficiently, indicating that HS1 is not required for actin-dependent events at the DC side of the immunological synapse (77
). Moreover, while there is strong evidence that actin polymerization is important for macropinocytosis and phagocytosis (12
), HS1 expression was not required for these processes. By extension, since we find no cortactin expression in murine BMDCs (28
), cortactin family members generally are dispensible. Both macropinocytosis and phagocytosis are downregulated upon DC maturation (65
), while receptor-mediated endocytosis proceeds unabated in mature DCs (13
). Phagocytosis and macropinocytosis depend on the actin nucleating proteins WASp and WAVE, and the downregulation of these processes is linked to diminished activation of the Rho family GTPase CDC42 upstream of these events (13
). Interestingly, HS1 differs from WASp and WAVE in that it does not depend on activation by Rho GTPases, and so is not subject to downregulation at this level. Indeed, we find that HS1 expression is modestly upregulated upon DC maturation. Moreover, HS1 is enriched in podosomes, which are largely dissolved on DC maturation (28
), presumably increasing the pool of HS1 available to support endocytic functions in mature DCs.
Our analysis of HS1−/− DCs shows a 2-fold decrease in the maximal uptake of endocytosed proteins, yet these cells require 30–100 fold more protein antigen to achieve the same level of T cell activation as wild type DCs. The assay we used for receptor-mediated endocytosis measures only a single, synchronized round of protein uptake. When multiplied by the many rounds of endocytosis that take place within the 4-hour period of the antigen pulse, the defects we observe could readily account for at least 30–50 fold reduction in overall antigen uptake. Thus, the requirement for HS1 to facilitate endocytosis is likely to be the primary cause of the diminished antigen presentation that we observe in HS1-deficient DCs. The finding that antigens taken up by phagocytosis and macropinocytosis are presented efficiently further supports this conclusion. Nonetheless, it is possible that HS1 is also required for additional intracellular steps of antigen trafficking. Testing this possibility will require quantitative analysis of the fate of the endocytosed pool of protein.
While the DC literature tends to focus on phagocytosis and macropinocytosis, receptor-mediated endocytosis is also an important means of antigen uptake. Typically, it is argued that receptor-mediated endocytosis is particularly important at low ligand concentrations. While this is no doubt true, our side-by-side comparison of presentation of ovalbumin-derived peptides following uptake by receptor-mediated endocytosis and macropinocytosis shows that receptor-mediated uptake is dominant even at very high antigen doses. DCs express numerous cell surface receptors that preferentially bind non-self molecules and take them up in a highly efficient manner. Ovalbumin is taken up by the mannose receptor (65
and ), which is important for uptake of pathogen-derived glycoproteins (82
). Uptake and cross-presentation of GRP94-peptide complexes also require receptor-mediated endocytosis, though the relevant receptor(s) remain controversial (67
). Other receptors also play an important physiological role. Among these are the C-type lectin DC-SIGN, which promotes the endocytosis of HIV and other viruses and their presentation on MHC Class I (85
). The multi-lectin receptor DEC205 plays a similar role (89
). Even Fcγ receptors, which are best known for their role in phagocytosis, carry out receptor-mediated endocytosis of small immune complexes (91
). CD1b recycling occurs via receptor-mediated endocytosis, and continues in mature DCs (81
), a phenomenon that has been proposed to be the basis of effective presentation of lipid antigens from mycobacteria, under conditions where presentation of antigens on MHC Class II fails (95
). We show here that HS1 is required for receptor-mediated uptake via three distinct receptors, and it seems likely that this is a general requirement. Going forward, it will be interesting to ask if HS1 expression has an important impact on DC function in vivo
, particularly in the context of viruses, lipid antigens, or DC-vaccines that rely on receptor-mediated uptake. Moreover, since HS1 is also expressed in B cells, it will be interesting to ask if BCR-dependent uptake of antigens is also HS1-dependent.
HS1 functions both as an actin regulatory protein and an adapter molecule, and it seems likely that both of these functions are called into play to promote receptor-mediated endocytosis. We show that HS1 interacts with dynamin 2 through binding of its C-terminal SH3 domain to the proline-rich domain of dynamin 2, the same mechanism used to mediate cortactin-dynamin 2 interaction (41
). Interestingly, however, the endocytic phenotype of HS1−/−
DCs does not mirror that of cells treated with the dynamin GTPase inhibitor dynasore. In keeping with studies in other cell types (76
), we found that treatment of DCs with dynasore led to the accumulation of deep endocytic invaginations. The accumulation of wide-necked (U-shaped) structures was predominant, though narrow-necked (O-shaped) endocytic invaginations were also observed. The latter structures are also seen in cells expressing dominant negative dynamin mutants, and are thought to represent a requirement for dynamin in the fission of fully formed coated pits (9
). The U-shaped invaginations represent an earlier endocytic intermediate, and are proposed to represent an additional requirement for dynamin in driving membrane deformation (76
). In contrast to dynamin-inhibited cells, HS1-deficient DCs exhibited abnormally low numbers of both U-shaped and O-shaped endocytic invaginations. The simplest interpretation of this finding is that HS1 is required for an early step in membrane invagination, preceding the deep invaginations formed by dynamin activity. These early intermediates are thought to be metastable (97
), perhaps explaining why no clear accumulation of shallow pits was observed in HS1-deficient DCs. This interpretation is consistent with a two-step model for dynamin function, in which dynamin first serves as regulatory GTPase to ensure vectorial coat assembly and enhance membrane curvature, and subsequently, in an assembled state, uses GTPase-driven mechano-chemical activity to drive vesicle scission (96
). According to this model, SH3 domain containing proteins such as HS1 sense the status of cargo loading, membrane curvature and/or coat assembly, and regulate this functional switch.
In addition to binding dynamin 2, HS1 activates the Arp2/3 complex, binds actin filaments, and interacts with the actin regulatory proteins WASp and WIP (30
). Thus, it is also likely that HS1 promotes actin-dependent aspects of endocytosis. The literature regarding the requirement for actin polymerization in endocytosis is conflicting. In S. cerevisae, actin recruitment occurs after clathrin coat formation and provides an essential force for membrane deformation (99
). In mammalian cells, however, results are variable and depend on the cell type and the nature of the endocytic structure (100
). Recent work from the Kirchhausen group has shown that actin regulatory proteins are recruited to long-lived endocytic structures and are particularly important for internalization of large structures, such as clathrin plaques and some virus particles (104
). On the basis of this work, it seems that actin polymerization provides the force needed to generate oversized clathrin-coated structures, and to overcome other obstacles, such as high membrane tension (107
). Since HS1 promotes the formation of branched-actin filaments, it may play a key role in this process. Importantly, HS1’s interactions with the actin cytoskeleton and dynamin are likely to be coupled. Indeed, we find that interactions between HS1 and dynamin are dramatically enhanced by deletion of the N-terminal actin regulatory region of HS1. This, and similar observations regarding HS1 binding to WASp and WIP (28
), are compatible with a model in which the N-terminus of HS1 partially restricts availability of the SH3 domain, such that interaction with the actin cytoskeleton releases the SH3 domain and promotes binding to dynamin 2 and other molecules. This model explains our inability to co-immunoprecipitate endogenous dynamin 2 and HS1, since the pool of active HS1 would be selectively associated with the insoluble actin cytoskeleton. A positive feedback loop may also be at play, since engagement of the dynamin proline-rich domain by SH3 domains has been shown to promote dynamin GTPase activity (108
). Such a mechanism would promote the coupled assembly of actin and dynamin at endocytic invaginations.
An important unresolved question is to what extent HS1 function is distinct from that of its more widely expressed homologue cortactin. In non-hematopoietic cells, the weight of the evidence indicates that cortactin plays a key role in endocytic uptake (51
), though some studies challenge this view (112
). Cortactin is recruited to long-lived clathrin-rich regions of the membrane, and kinetic analysis shows that cortactin recruitment peaks at a late time point, close to the time of dynamin recruitment (72
). Furthermore, phosphorylation of cortactin promotes its association with dynamin (74
) and the tyrosine phosphorylation sites that have been linked to regulated cortactin function are conserved in HS1. Given all the biochemical and cell biological similarities between cortactin and HS1, why did hematopoietic lineages evolve a distinct family member? There are, in fact, some important functional differences between HS1 and cortactin. The structural features necessary for F-actin binding are distinct (31
). Moreover, HS1 is less efficient than cortactin at driving Arp2/3 complex-nucleated actin polymerization (30
). Actin binding promotes cortactin interaction with dynamin (74
), and our evidence suggests that the same is true for HS1. Thus, the difference in mode of actin binding between HS1 and cortactin may translate into important differences in the mechanisms by which these proteins link the actin cytoskeleton to dynamin function. Since BMDCs from HS1−/−
mice lack both HS1 and cortactin, they provide an ideal experimental system in which to carry out comparative reconstitution studies to address this possibility.