Mietske Wijers

The Dendritic Cell-Specific Adhesion Receptor DC-SIGN Internalizes Antigen for Presentation to T Cells

Dendritic cells (DCs) capture Ags or viruses in peripheral tissue to transport them to lymphoid organs to induce cellular T cell responses. Recently, a DC-specific C-type lectin was identified, DC-specific ICAM-grabbing non-integrin (DC-SIGN), that functions as cell adhesion receptor mediating both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R that captures HIVgp120 and facilitates DC-induced HIV transmission of T cells. Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function of DC-SIGN as endocytic receptor. In this study we demonstrate that on DCs DC-SIGN is rapidly internalized upon binding of soluble ligand. Mutating a putative internalization motif in the cytoplasmic tail reduces ligand-induced internalization. Detailed analysis using ratio fluorescence imaging and electron microscopy showed that DC-SIGN-ligand complexes are targeted to late endosomes/lysosomes. Moreover, ligands internalized by DC-SIGN are efficiently processed and presented to CD4+ T cells. The distinct pattern of expression of C-type lectins on DCs in situ and their nonoverlapping Ag recognition profile hint to selective functions of these receptors to allow a DC to recognize a wide variety of Ags and to process these to induce T cell activation. These data point to a novel function of the adhesion receptor DC-SIGN as an efficient DC-specific Ag receptor that can be used as a target to induce viral and antitumor immunity.

Multimodal imaging of nanovaccine carriers targeted to human dendritic cells

Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy against cancer and infectious diseases. The targeted delivery of nanovaccine particles (NPs) to DCs in vivo is a promising strategy to enhance immune responses. Here, targeted nanovaccine carriers were generated that allow multimodal imaging of nanocarrier-DC interactions from the subcellular to the organism level. These carriers were made of biodegradable poly(D,L-lactide-co-glycolide) harboring superparamagnetic iron oxide particles (SPIO) and fluorescently labeled antigen in a single particle. Targeted delivery was facilitated by coating the NPs with antibodies recognizing the DC-specific receptor DC-SIGN. The fluorescent label allowed for rapid analysis and quantification of specific versus nonspecific uptake of targeted NPs by DCs compared to other blood cells. In addition, it showed that part of the encapsulated antigen reached the lysosomal compartment of DCs within 24 h. Moreover, the presence of fluorescent label did not prevent the antigen from being presented to antigen-specific T cells. The incorporated SPIO was applied to track the NPs at subcellular cell organel level using transmission electron microscopy (TEM). NPs were found within endolysosomal compartments, where part of the SPIO was already released within 24 h. Furthermore, part of the NPs seemed to localize within the cytoplasm. Ex vivo loading of DCs with NPs resulted in efficient labeling and detection by MRI and did not abolish cell migration within collagen scaffolds. In conclusion, incorporation of two imaging agents within a single carrier allows tracking of targeted nanovaccines on a subcellular, cellular and possibly organism level, thereby facilitating rational design of in vivo targeted vaccination strategies.

The dendritic cell-derived protein DC-STAMP is highly conserved and localizes to the endoplasmic reticulum.

Recently, we described the molecular identification of dendritic cell‐specific TrAnsMembrane protein (DC‐STAMP), a multimembrane‐spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC‐STAMP (mDC‐STAMP) as well as the characterization of the DC‐STAMP protein. The results demonstrate that mDC‐STAMP is over 90% homologous to hDC‐STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC‐STAMP expression is enhanced by interleukin‐4 and down‐regulated upon DC maturation. Analysis of differently tagged DC‐STAMP proteins further demonstrates that hDC‐STAMP and mDC‐STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC‐STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC‐STAMP‐green fluorescent protein fusion protein. These data imply that DC‐STAMP may exert its effect in the ER.

Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria

Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study, we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility, and matrix protein diffusion. To this end, we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity, and the rate of matrix-targeted enhanced yellow fluorescent protein (mitoEYFP) diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria were two to three times higher in patient cells than in control cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI-deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI-deficient patients become more glycolytic, allowing a higher mitochondrial velocity.

Divergent Mitochondrial and Endoplasmic Reticulum Association of DMPK Splice Isoforms Depends on Unique Sequence Arrangements in Tail Anchors

ABSTRACT Myotonic dystrophy protein kinase (DMPK) is a Ser/Thr-type protein kinase with unknown function, originally identified as the product of the gene that is mutated by triplet repeat expansion in patients with myotonic dystrophy type 1 (DM1). Alternative splicing of DMPK transcripts results in multiple protein isoforms carrying distinct C termini. Here, we demonstrate by expressing individual DMPKs in various cell types, including C2C12 and DMPK −/− myoblast cells, that unique sequence arrangements in these tails control the specificity of anchoring into intracellular membranes. Mouse DMPK A and C were found to associate specifically with either the endoplasmic reticulum (ER) or the mitochondrial outer membrane, whereas the corresponding human DMPK A and C proteins both localized to mitochondria. Expression of mouse and human DMPK A—but not C—isoforms in mammalian cells caused clustering of ER or mitochondria. Membrane association of DMPK isoforms was resistant to alkaline conditions, and mutagenesis analysis showed that proper anchoring was differentially dependent on basic residues flanking putative transmembrane domains, demonstrating that DMPK tails form unique tail anchors. This work identifies DMPK as the first kinase in the class of tail-anchored proteins, with a possible role in organelle distribution and dynamics.

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

A tail-anchored myotonic dystrophy protein kinase isoform induces perinuclear clustering of mitochondria, autophagy, and apoptosis.

Background Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. Methodology/Principal Findings We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the proteins tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. Conclusion/Significance Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients.

Subcellular localization and differentiation-induced redistribution of the protein tyrosine phosphatase PTP-BL in Neuroblastoma cells.

Summary1.In cells of epithelial origin the protein tyrosine phosphatase PTP-BL is predominantly localized at the apical membrane of polarized cells. This large submembranous multidomain PTP is also expressed in cells of neuronal origin. We studied the localization of PTP-BL in mouse neuroblastoma cells utilizing EGFP-tagged versions of the protein.2.In proliferating Neuro-2a cells, immunofluorescence and immuno-electron microscopy revealed a submembranous FERM domain-dependent localization at cell–cell boundaries for EGFP-PTP-BL. Additionally, significant amounts of EGFP-PTP-BL are located in the cytoplasm as well as in nuclei. Upon serum depletion-induced differentiation of Neuro-2a cells, a partial shift of EGFP-PTP-BL from a cortical localization to cytoskeleton-like F-actin-positive structures is observed. Parallel biochemical studies corroborate this finding and reveal a serum depletion-induced shift of EFGP-PTP-BL from a membrane(-associated) fraction to an NP40-soluble cytoskeletal fraction.3.Different pools of PTP-BL-containing protein complexes can be discerned in neuronal cells, reflecting distinct molecular microenvironments in which PTP-BL may exert its function.

Glycolytic Metabolism is Differentially Coupled to Proliferative Potential and Morphodynamic Capacity in RAW 264.7 and Mafb/C-Maf Deficient Macrophage Lineages

Background: Macrophages are highly specialized immune cells of different developmental origin, which occur in a continuum of diverse functional states in almost all tissues. In order to fulfil their complex role in tissue homeostasis and defence against pathogens they must be able to live with heterogeneous extrinsic nutrient conditions in tissue niches and handle variation in intrinsic metabolic demand that is determined by differentiation state, functional specialization and immune challenge. The purpose of the present study was to gain more insight in how metabolic specialization and versatility in fate and immune effector function of macrophages are coupled. Methods: In vitro phenotypic characteristics of two macrophage cell lineages of profoundly different developmental origin and polarization capacity, RAW 264.7 and Maf-DKO cells, were analysed. By use of biochemical and cell biological approaches and scanning electron microscopy, we studied the metabolic profiles of these two types of macrophages in relation to proliferative capacity, morphological appearance of cell surface and cell shape, and phagocytic activity as index of morphodynamic potential. Results: Comparison of gross features of carbohydrate metabolism, including levels of glycolytic enzymes Hexokinase (HK), Pyruvate Kinase (PK-M2), lactate dehydrogenase and nicotinamide phosphoribosyltransferase (Nampt), glucose and oxygen consumption and lactate production rates, and intracellular concentrations and redox ratios of NAD(P)(H) demonstrated that RAW 264.7 and Maf-DKO cells are conspicuously similar in that they both rely heavily on the use of glycolysis. In this respect they share many characteristics with primary macrophages. Strikingly, this uniform metabolic signature does not translate in behavioral-functional similarities as RAW 264.7 cells have a significantly higher proliferation rate, whereas Maf-DKO cells appear to be morphodynamically more active, form significantly more surface membrane protrusions and phagocytose complement opsonized particles much more efficiently. Conclusion: We conclude that the global rate of glycolysis in intermediary carbohydrate metabolism is similar for the two cell lineages, but that they can make differential use of this important pathway, either to fuel high morphodynamic activity in Maf-DKO cells, or for the sustenance of cell growth in fast proliferating RAW 264.7 cells. Our findings are in keeping with the idea that macrophages may uniformly prefer use of the rapid response time of glycolysis because this pathway provides them with the ability to meet any possible short-timescale energy demand required for immune function.