B cell receptors and complement receptors target the antigen to distinct intracellular compartments

The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation.     

Distribution of insulin receptors between plasmalemma and intracellular compartments: effect of amino acids

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.     

Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.     

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.     

Dendritic molecular transporters provide control of delivery to intracellular compartments

Novel biocompatible macromolecular vectors were developed that not only enable transport of bioactive cargo across the cell membrane but also control the delivery into defined intracellular compartments. This work describes the synthesis and design of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. The internalization of the fluorescein-labeled molecular transporter into mammalian cells showed strong subcellular localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. We observed that the subcellular distribution of these vectors varied significantly, as one of the vectors concentrates in the nucleus (FD-1) while the other (FD-2) concentrates in the cytosol. All experiments performed with NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC) showed similar results. The differential localization patterns of the two molecular transporters can be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations, utilizing this practical approach, might overcome limitations in drug delivery and pioneer future technologies in drug transport.     

Chaperone proteins as single component reagents to assess antibody nonspecificity

Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few “false negatives” (assuming a complex cell extract as the “true” value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.     

Regulation of anterograde transport of alpha2-adrenergic receptors by the N termini at multiple intracellular compartments

The studies on the intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly focused on the C termini of the receptors. In this report we determined the role of the extracellular N termini of alpha(2)-adrenergic receptors (alpha(2)-ARs) in the anterograde transport from the endoplasmic reticulum (ER) through the Golgi to the cell surface. The N-terminal-truncated alpha(2B)-AR mutant is completely unable to target to the cell surface. A single Met-6 residue is essential for the export of alpha(2B)-AR from the ER, likely through modulating correct alpha(2B)-AR folding in the ER. The Tyr-Ser motif, highly conserved in the membrane-proximal N termini of all alpha(2)-AR subtypes, is required for the exit of alpha(2A)-AR and alpha(2B)-AR from the Golgi apparatus, thus representing a novel Tyr-based motif modulating GPCR transport at the Golgi level. These data provide the first evidence indicating an essential role of the N termini of GPCRs in the export from distinct intracellular compartments along the secretory pathway.     

Steroid hormone receptors: intracellular distribution

Recent reports, both biochemical and morphological, have challenged the widely accepted two-step model of steroid hormone action. This model proposed that steroid hormone receptors existed under two different forms: the unliganded receptor in the cytoplasm and the hormone-bound receptor complex in the nucleus. A nuclear translocation mechanism was hypothesized as a necessary link between the two forms. In contradiction with this model, new studies have concluded to the absence of receptor in the cytoplasm and its presence in the nucleus under all hormonal conditions, thus rendering the hypothetical nuclear translocation unnecessary. In this review, we discuss how our concept of the mechanism of action of steroid hormone ought to be revised in the light of the new data.     

Binding of lectin-fluorescein conjugates to intracellular compartments of growth-plate chondrocytes in situ

In this study, lectin-binding techniques are applied to growth-plate cartilage to analyze the intracellular localization of lectin-binding glycoconjugates of chondrocytes in situ. The binding of ten fluorescein-conjugated lectins is analyzed on 1-micron-thick Epon-embedded, nondecalcified sections of growth plates from Yucatan swine. Comparisons are made to intracellular binding in chondrocytes of tracheal, articular, and auricular cartilage. Ear epithelium, tracheal epithelium, and kidney are used as positive control tissues for the specificity of lectin binding. Only the mannose-binding lectins had affinity for the RER and nuclear envelope. Eight lectins reacted within the Golgi complex with characteristic patterns which ranged from localized fine linear strands to extensive vesicular accumulations. When cartilage slabs were exposed before embedment to the ionophore monensin to disrupt intracellular transport through the Golgi, it was possible to define differential subcompartments of the Golgi complex, based upon sites of sugar addition. Also, it was possible to characterize the cytoplasmic deposits of reserve-zone chondrocytes which were positive with concanavalin A as glycogen, based upon their sensitivity to amylase. This method allows resolution at the light-microscopic level of lectin-binding glycoconjugates with localization to specific organelles. Patterns of intracellular binding were consistent with biochemical data relating to the subcellular localization of processing steps of complex carbohydrates prior to secretion.     

Peripherin-2: an intracellular analogy to viral fusion proteins

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.     

Chaperone function and mechanism of small heat-shock proteins

Small heat-shock proteins （sHSPs） are ubiquitous ATP-independent molecular chaperones that play crucial roles in protein quality control in cells. They are able to prevent the aggregation and/or inactivation of various non-native sub- strate proteins and assist the refolding of these substrates independently or under the help of other ATP-dependent chaperones. Substrate recognition and binding by sHSPs are essential for their chaperone functions. This review focuses on what natural substrate proteins an sHSP pro- tects and how it binds the substrates in cells under fluctuat- ing conditions. It appears that sHSPs of prokaryotes, although being able to bind a wide range of cellular pro- teins, preferentially protect certain classes of functional proteins, such as translation-related proteins and metabolic enzymes, which may well explain why they could increase the resistance of host cells against various stresses. Mechanistically, the sHSPs of prokaryotes appear to possess numerous multi-type substrate-binding residues and are able to hierarchically activate these residues in a temperature-dependent manner, and thus act as tempera- ture-regulated chaperones. The mechanism of hierarchical activation of substrate-binding residues is also discussed regarding its implication for eukaryotic sHSPs.     

The transmembrane domain directs TLR9 to intracellular compartments that contain TLR3

Toll-like receptors (TLRs) are type I transmembrane (TM) proteins indispensable for sensing microbial and viral infection. Despite their conserved primary structures, some TLRs that detect pathogen-derived nucleic acids (TLR3, TLR7, TLR8, and TLR9) are retained in the cytoplasm. The intracellular localization of TLR9 is important for its ability to discriminate self- and non-self DNA, but the mechanism by which it is retained in the cytoplasm is unclear. In the present study, we found that the TM domain of TLR9 directs its intracellular localization. The TM domain of TLR9 also targets CD25, a heterologous type I TM protein, to intracellular compartments that contain TLR9. We also found that TLR9 generally co-localizes with TLR3, although its linker region, not its TM domain, directs intracellular localization of TLR3. These data demonstrate that the TM domain of TLR9 is a critical regulatory element that targets TLR9 to its intracellular location.     

Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.     

Pattern recognition receptors: from the cell surface to intracellular dynamics

Detection of potentially infectious microorganisms is essential for plant immunity. Microbial communities growing on plant surfaces are constantly monitored according to their conserved microbe-associated molecular patterns (MAMPs). In recent years, several pattern-recognition receptors, including receptor-like kinases and receptor-like proteins, and their contribution to disease resistance have been described. MAMP signaling must be carefully controlled and seems to involve receptor endocytosis. As a further surveillance layer, plants are able to specifically recognize microbial effector molecules via nucleotide-binding site leucine-rich repeat receptors (NB-LRR). A number of recent studies show that NB-LRR translocate to the nucleus in order to exert their activity. In this review, current knowledge regarding the recognition of MAMPs by surface receptors, receptor activation, signaling, and subcellular redistribution are discussed.     

Shapes of discoid intracellular compartments with small relative volumes

A prominent feature of many intracellular compartments is a large membrane surface area relative to their luminal volume, i.e., the small relative volume. In this study we present a theoretical analysis of discoid membrane compartments with a small relative volume and then compare the theoretical results to quantitative morphological assessment of fusiform vesicles in urinary bladder umbrella cells. Specifically, we employ three established extensions of the standard approach to lipid membrane shape calculation and determine the shapes that could be expected according to three scenarios of membrane shaping: membrane adhesion in the central discoid part, curvature driven lateral segregation of membrane constituents, and existence of stiffer membrane regions, e.g., support by protein scaffolds. The main characteristics of each scenario are analyzed. The results indicate that even though all three scenarios can lead to similar shapes, there are values of model parameters that yield qualitatively distinctive shapes. Consequently, a distinctive shape of an intracellular compartment may reveal its membrane shaping mechanism and the membrane structure. The observed shapes of fusiform vesicles fall into two qualitatively different classes, yet they are all consistent with the theoretical results and the current understanding of their structure and function.     

Targeting of aequorin for calcium monitoring in intracellular compartments

We have recently developed a new method for monitoring Ca²⁺ concentrations in defined cell compartments. The cDNA encoding the Ca²⁺-sensitive photoprotein aequorin has been modified in order to include specific targeting sequences and expressed in eukaryotic cells; the recombinant protein, specifically located inside the cells, has allowed the direct study of mitochondrial and nuclear Ca²⁺ concentrations in living cells. The principles, and the application, of this new methodology are discussed in this article.