Erik Bos

Laminin-α4 and Integrin-Linked Kinase Mutations Cause Human Cardiomyopathy Via Simultaneous Defects in Cardiomyocytes and Endothelial Cells

Background— Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. Methods and Results— We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-&agr;4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (Kd=5±3 &mgr;mol/L) and Arg1073X LAMA4 (Kd=1±0.2 &mgr;mol/L) mutants compared with the wild-type LAMA4 protein (Kd=440±20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. Conclusions— This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

The Transferrin Receptor: A Potential Molecular Imaging Marker for Human Cancer

Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR) has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI) probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date.

An ACAP1-containing clathrin coat complex for endocytic recycling.

Whether coat proteins play a widespread role in endocytic recycling remains unclear. We find that ACAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF) 6, is part of a novel clathrin coat complex that is regulated by ARF6 for endocytic recycling in two key physiological settings, stimulation-dependent recycling of integrin that is critical for cell migration and insulin-stimulated recycling of glucose transporter type 4 (Glut4), which is required for glucose homeostasis. These findings not only advance a basic understanding of an early mechanistic step in endocytic recycling but also shed key mechanistic insights into major physiological events for which this transport plays a critical role.

Human CD8+ T Cells Store RANTES in a Unique Secretory Compartment and Release It Rapidly after TcR Stimulation

The chemokine RANTES is secreted rapidly after activation of human CD8+ T cells, with a cycloheximide-resistant burst during the first hour. This pattern was observed in purified memory and effector phenotype CD8+ cells from blood as well as in blasts. In contrast, secretion of other chemokines and interferon-gamma by these cells was sensitive to cycloheximide and detectable only after a lag. Immunofluorescence microscopy of CD8+ memory and effector cells and blasts showed RANTES present in intracellular vesicles that do not significantly colocalize with cytotoxic granule markers or other markers of defined cytoplasmic compartments. Immunoelectron microscopy confirmed that RANTES is stored in small vesicles distinct from the lysosomal secretory granules. RANTES+ vesicles polarize rapidly in response to TcR engagement and are more rapidly depleted from the cytoplasm. These results show that CD8+ T cells have two distinct TcR-regulated secretory compartments characterized by different mobilization kinetics, effector molecules, and biological function.

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at −150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.

Three-Dimensional Electron Microscopic Imaging of Membrane Invaginations in Escherichia coli Overproducing the Chemotaxis Receptor Tsr

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images. Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50nm, vitreous cryo-sections of Saccharomyces cerevisiae.

The Translocon Protein Sec61 Mediates Antigen Transport from Endosomes in the Cytosol for Cross-Presentation to CD8+ T Cells

The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER.

Cryo‐Immunogold Electron Microscopy

This unit describes subcellular localization of proteins/antigens using high‐resolution cryo‐immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar‐coated grids. This unit is accompanied by eleven videos that illustrate many of the procedures used in this unit.