Michele A. West

A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits.

BACKGROUND Clathrin-coated pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol. Little is known about the regulation and mechanism of this assembly process. RESULTS We have used an in vitro assay for clathrin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new coated pits at the cell surface in permeabilised cells, the Rab5-GDI complex is required for ligand sequestration into clathrin-coated pits. CONCLUSIONS We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.

Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation

BACKGROUND Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells. RESULTS We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis. CONCLUSIONS Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.

TLR signalling regulated antigen presentation in dendritic cells.

Recent evidence suggests that TLR signalling in dendritic cells (DCs) transiently enhances antigen endocytosis and autophagy, augments the assembly of key antigen transport and processing systems, qualitatively modulates protein translation and induces a temporary cessation of DC motility. These rapid changes require activation of the MAP kinases, PI3-kinase and downstream signalling pathways and are observed in both myeloid DC and, with variations on the theme, in plasmacytoid DC.

TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure.

Membrane ruffling, macropinocytosis and antigen presentation in the absence of gelsolin in murine dendritic cells

Previous studies have shown that mice lacking the actin‐severing and capping protein gelsolin have defects in leukocyte and platelet function. Moreover, dermal fibroblasts from gelsolin knockout (Gsn–) mice showed substantially reduced motility, membrane ruffling and pinocytosis. We have generated dendritic cells (DC) from spleens of Gsn– mice to investigate the importance of gelsolin in antigen endocytosis and processing. We show here that Gsn– DC produce apparently normal membrane ruffles which can resolve to form large macropinosomes. Moreover, presentation of exogenous antigens on both MHC class II and class I molecules was equivalent in Gsn– and wild‐type DC. Thus the major rearrangements of the actin cytoskeleton needed for DC antigen uptake and presentation can proceed in the absence of a major actin filament regulatory protein.

A role for ARF6 in dendritic cell podosome formation and migration

ADP‐ribosylation factor 6 (ARF6) is a widely expressed GTPase that influences both membrane traffic and actin cytoskeleton function. Its role in dendritic cells (DC) has not previously been investigated. We analysed the effect of retroviral expression of ARF6 GDP/GTP binding and other functional mutants in primary murine DC. Maturation in response to lipopolysaccharide (LPS) proceeded normally in DC expressing ARF6 mutants and production of inflammatory cytokines was similarly unaffected. Although LPS‐stimulated macropinocytosis was suppressed by expression of the GTP‐binding Q67L ARF6 mutant we detected no overall activation of ARF6 by LPS. The ability of immature DC to migrate towards CCL3 and to a lesser extent, of mature DC to migrate towards CCL19, was compromised by expression of either the Q67L or the GDP‐binding T44N mutant. Examination of the actin cytoskeleton in these cells revealed that both mutants strongly inhibited the formation of F‐actin‐rich podosomes, providing a possible explanation for the effects of ARF6 mutants on DC migration. Thus, these studies identify responses in DC that require normal ARF6 function, though not necessarily further ARF6 activation. They reveal for the first time a role for ARF6 in podosome formation and demonstrate functional effects of the T44N ARF6 mutant.

A crucial role for β2 integrins in podosome formation, dynamics and Toll-like-receptor-signaled disassembly in dendritic cells.

ABSTRACT The dynamic properties of podosomes, their ability to degrade the underlying matrix and their modulation by Toll-like receptor (TLR) signaling in dendritic cells (DCs) suggests they have an important role in migration. Integrins are thought to participate in formation and dynamics of podosomes but the multiplicity of integrins in podosomes has made this difficult to assess. We report that murine DCs that lack &bgr;2 integrins fail to form podosomes. Re-expression of &bgr;2 integrins restored podosomes but not when the membrane proximal or distal NPxF motifs, or when an intervening triplet of threonine residues were mutated. We show that &bgr;2 integrins are remarkably long-lived in podosome clusters and form a persistent framework that hosts multiple actin-core-formation events at the same or adjacent sites. When &bgr;2 integrin amino acid residues 745 or 756 were mutated from Ser to Ala, podosomes became resistant to dissolution mediated through TLR signaling. TLR signaling did not detectably modulate phosphorylation at these sites but mutation of either residue to phospho-mimetic Asp increased &bgr;2 integrin turnover in podosomes, indicating that phosphorylation at one or both sites establishes permissive conditions for TLR-signaled podosome disassembly.

Antigen Processing in B Lymphocytes

There is considerable current interest in how the events occurring during antigen processing and presentation via class II MHC glycoproteins are organised with respect to the endocytic pathway. This follows the discovery that most antigens require an intracellular processing step in order to produce a suitable ligand for binding to MHC Class II glycoproteins and, as a complex, to the T cell receptor. The available evidence strongly suggests that following endocytosis, processing and unfolding take place in intracellular compartments and that suitable MHC/peptide complexes are then returned to the cell surface. Using T cells to assay for the appearance of processed determinants it has been established that these antigen processing events take approximately one hour, are sensitive to chloroquine and fixation of the antigen presenting cells (APC) and to inhibitors of endosomal/lysosomal proteases (Ziegler and Unanue 1982; Lanzavecchia 1985; Buus and Werdelin 1986).