Distinct Intracellular Compartments Research Articles

Monosaccharide coating modulate the intracellular trafficking of gold nanoparticles in dendritic cells

Dendritic cells (DCs) have emerged as a promising target for drug delivery and immune modulation due to their pivotal role in initiating the adaptive immune response. Gold nanoparticles (AuNPs) have garnered interest as a platform for targeted drug delivery due to their biocompatibility, low toxicity and precise control over size, morphology and surface functionalization. Our investigation aimed to elucidate the intracellular uptake and trafficking of AuNPs coated with different combinations of monosaccharides (mannose, galactose, and fucose) in DCs. We used 30 unique polymer-tethered monosaccharide combinations to coat 16 nm diameter spherical gold nanoparticles and investigated their effect on DCs phenotype, uptake, and intracellular trafficking.DCs internalised AuNPs coated with 100% fucose, 100% mannose, 90% mannose + 10% galactose, and 80% mannose + 20% galactose with highest efficiency. Flow cytometry analysis indicated that 100% fucose-coated AuNPs showed increased lysosomal and endosomal contents compared to other conditions and uncoated AuNPs. Imaging flow cytometry further demonstrated that 100% fucose-coated AuNPs had enhanced co-localisation with lysosomes, while 100% mannose-coated AuNPs exhibited higher co-localisation with endosomes.Furthermore, our data showed that the uptake of carbohydrate-coated AuNPs predominantly occurred through receptor-mediated endocytosis, as evidenced by a marked reduction of uptake upon treatment of DCs with methyl-β-cyclodextrins, known to disrupt receptor-mediated endocytosis. These findings highlight the utility of carbohydrate coatings to enable more targeted delivery of nanoparticles and their payload to distinct intracellular compartments in immune cells with potential applications in drug delivery and immunotherapy.

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

Spatiotemporal Dynamic Regulation of Organelles During Meiotic Development, Insights From Fungi.

Eukaryotic cell development involves precise regulation of organelle activity and dynamics, which adapt the cell architecture and metabolism to the changing developmental requirements. Research in various fungal model organisms has disclosed that meiotic development involves precise spatiotemporal regulation of the formation and dynamics of distinct intracellular membrane compartments, including peroxisomes, mitochondria and distinct domains of the endoplasmic reticulum, comprising its peripheral domains and the nuclear envelope. This developmental regulation implicates changes in the constitution and dynamics of these organelles, which modulate their structure, abundance and distribution. Furthermore, selective degradation systems allow timely organelle removal at defined meiotic stages, and regulated interactions between membrane compartments support meiotic-regulated organelle dynamics. This dynamic organelle remodeling is implicated in conducting organelle segregation during meiotic differentiation, and defines quality control regulatory systems safeguarding the inheritance of functional membrane compartments, promoting meiotic cell rejuvenation. Moreover, organelle remodeling is important for proper activity of the cytoskeletal system conducting meiotic nucleus segregation, as well as for meiotic differentiation. The orchestrated regulation of organelle dynamics has a determinant contribution in the formation of the renewed genetically-diverse offspring of meiosis.

SGPL1 stimulates VPS39 recruitment to the mitochondria in MICU1 deficient cells

ObjectiveMitochondrial “retrograde” signaling may stimulate organelle biogenesis as a compensatory adaptation to aberrant activity of the oxidative phosphorylation (OXPHOS) system. To maintain energy-consuming processes in OXPHOS deficient cells, alternative metabolic pathways are functionally coupled to the degradation, recycling and redistribution of biomolecules across distinct intracellular compartments. While transcriptional regulation of mitochondrial network expansion has been the focus of many studies, the molecular mechanisms promoting mitochondrial maintenance in energy-deprived cells remain poorly investigated. MethodsWe performed transcriptomics, quantitative proteomics and lifespan assays to identify pathways that are mechanistically linked to mitochondrial network expansion and homeostasis in Caenorhabditis elegans lacking the mitochondrial calcium uptake protein 1 (MICU-1/MICU1). To support our findings, we carried out biochemical and image analyses in mammalian cells and mouse-derived tissues. ResultsWe report that micu-1(null) mutations impair the OXPHOS system and promote C. elegans longevity through a transcriptional program that is independent of the mitochondrial calcium uniporter MCU-1/MCU and the essential MCU regulator EMRE-1/EMRE. We identify sphingosine phosphate lyase SPL-1/SGPL1 and the ATFS-1-target HOPS complex subunit VPS-39/VPS39 as critical lifespan modulators of micu-1(null) mutant animals. Cross-species investigation indicates that SGPL1 upregulation stimulates VPS39 recruitment to the mitochondria, thereby enhancing mitochondria-lysosome contacts. Consistently, VPS39 downregulation compromises mitochondrial network maintenance and basal autophagic flux in MICU1 deficient cells. In mouse-derived muscles, we show that VPS39 recruitment to the mitochondria may represent a common signature associated with altered OXPHOS system. ConclusionsOur findings reveal a previously unrecognized SGPL1/VPS39 axis that stimulates intracellular organelle interactions and sustains autophagy and mitochondrial homeostasis in OXPHOS deficient cells.

Rational Design of Surface-State Controlled Multicolor Cross-Linked Carbon Dots with Distinct Photoluminescence and Cellular Uptake Properties.

We disclose for the first time a facile synthetic methodology for the preparation of multicolor carbon dots (CDs) from a single source barring any chromatographic separations. This was achieved via sequential intraparticle cross-linking of surface abundant carboxylic acid groups on the CDs synthesized from a precursor to control their photoluminescence (PL) spectra as well as affect their degree of cellular internalization in cancer cells. The change in PL spectra with sequential cross-linking was projected by theoretical density functional theory (DFT) studies and validated by multiple characterization tools such as X-ray photoelectron spectroscopy (XPS), PL spectroscopy, ninhydrin assay, etc. The variation in cellular internalization of these cross-linked CDs was demonstrated using inhibitor assays, confocal microscopy, and flow cytometry. We supplemented our findings with high-resolution dark-field imaging to visualize and confirm the colocalization of these CDs into distinct intracellular compartments. Finally, to prove the surface-state controlled PL mechanisms of these cross-linked CDs, we fabricated a triple-channel sensor array for the identification of different analytes including metal ions and biologically relevant proteins.

Protein Mimicry and the Design of Bioactive Cell-Penetrating Peptides: The Genesis of STOPSPERM Bioportides.

The mature spermatozoon, a highly differentiated cell equipped for the sole purpose of fertilization, lacks the protein machinery required for conventional endocytotic mechanisms. Perhaps contrary to expectation, cell-penetrating peptides (CPPs) rapidly translocate across the unique sperm plasma membrane to accrete within distinct intracellular compartments. Confocal microscopy, employing red-fluorescent CPPs and bioportides, is a convenient platform to study this membrane translocation process. In the virtual absence of genetic expression, rapid physiological responses of human sperm are dependent upon protein-protein interactions that may be regulated by posttranslational modifications including phosphorylation. This chapter provides an outline of the design of bioactive CPPs, or bioportides, which include protein-mimetic sequences from the interaction domains of sperm proteins. Protocols are included which enable the biological assessment of the impact of bioportides upon the viability and motility of spermatozoa.

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Neuronal Trafficking of the Amyloid Precursor Protein-What Do We Really Know?

Alzheimer’s disease (AD) is the most common type of dementia, contributing to 60–80% of cases. It is a neurodegenerative disease that usually starts symptomless in the first two to three decades and then propagates into a long-term, irreversible disease, resulting in the progressive loss of memory, reasoning, abstraction and language capabilities. It is a complex disease, involving a large number of entangled players, and there is no effective treatment to cure it or alter its progressive course. Therefore, a thorough understanding of the disease pathology and an early diagnosis are both necessary. AD has two significant pathological hallmarks: extracellular senile plaques composed of amyloid β-peptide (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein, and the aggregation of Aβ, which starts in earlier stages, is usually claimed to be the primary cause of AD. Secretases that cleave Aβ precursor protein (APP) and produce neurotoxic Aβ reside in distinct organelles of the cell, and current concepts suggest that APP moves between distinct intracellular compartments. Obviously, APP transport and processing are intimately related processes that cannot be dissociated from each other, and, thus, how and where APP is transported determines its processing fate. In this review, we summarize critical mechanisms underlying neuronal APP transport, which we divide into separate parts: (1) secretory pathways and (2) endocytic and autophagic pathways. We also include two lipoprotein receptors that play essential roles in APP transport: sorting-related receptor with A-type repeats and sortilin. Moreover, we consider here some major disruptions in the neuronal transport of APP that contribute to AD physiology and pathology. Lastly, we discuss current methods and technical difficulties in the studies of APP transport.

Protein Kinase C-Dependent Von Willebrand Factor Secretion Is a Dual-Phased Process That Originates from 2 Distinct Intracellular Compartments

Introductionvon Willebrand factor (VWF) and VWF-propeptide (VWFpp) are stored for secretion within specialized endothelial cell secretory organelles called Weibel-Palade bodies (WPBs). Protein kinase C (PKC) is a key signaling intermediary for a number of endothelial agonists that trigger the release of VWF, such as Vascular Endothelial Growth Factor (VEGF) and Shiga toxin Stx1B. While both agonists elevate intracellular free Ca2+ ([Ca2+]i) and simultaneously either signal through the novel, Ca2+-independent PKCδ (VEGF) or utilize the classical, Ca2+-dependent PKCα (Stx1B), there has been speculation whether an additional effector pathway may be operating during PKC-mediated VWF secretion. Phorbol 12-myristate 13-acetate (PMA), an activator of both classical and novel protein kinase C (PKC) isoforms, is also a potent VWF secretagogue, and it is generally assumed that PMA-evoked VWF release arises from exocytosis of WPBs.ResultsHere we present evidence that PMA elicits a dual-phased secretory response with an early (0-15 minutes) and a late (>15 minutes) stage. A large fraction of the early phase of PMA-evoked VWF and VWFpp secretion arises from a cycloheximide (CHX)-sensitive (i.e., nascent or non-storage) compartment, and can be blocked by pre-treatment with the broad spectrum PKC inhibitor bisindolylmaleimide I (BIS I). Histamine-induced VWFpp secretion during the early phase is insensitive to CHX or BIS I. VEGF induced a sharp rise of VWFpp secretion in the early phase and a much slower increase in the later phase. The early phase was also substantially reduced by CHX and BIS I, suggesting release from the same non-storage pool that is mobilized by PMA.High speed imaging of WPB exocytosis and [Ca2+]i in cells expressing VWFpp-EGFP targeted to WPBs revealed that the majority of PMA- and VEGF-induced VWFpp secretion during the early phase did not originate from WPBs. Histamine stimulation was associated with a sharp rise in [Ca2+]i, followed by rapid exocytosis within seconds of 41.4±4.0% (mean±sem, n=12 cells, 322 fusion events) of fluorescent WPBs. PMA did not elevate [Ca2+]i, led to a slow gradual increase in fusion events after a long and highly variable delay (357±63 seconds) and resulted in release of 12.4±3.3% (n=13 cells, 133 fusion events) of VWFpp-EGFP labeled WPBs. VEGF induced a complex [Ca2+]i increase, and after considerable delay (37.8 ±5.9 seconds) this was followed by WPB release of 18.2±3.5% (n=14 cells, 181 fusion events) of fluorescent WPBs. Because all three stimuli release quantitatively similar amounts of VWFpp during the early phase, the discrepancy in the fractions of WPBs undergoing degranulation suggests that PKC signaling mobilizes VWFpp from a compartment other than WPBs.Analysis of PMA-evoked VWFpp secretion showed that pre-treatment with the potent phospholipase D1/2 inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) significantly reduced the early, CHX-sensitive component of PMA-evoked VWFpp secretion, which suggests that this pool originates from the trans -Golgi network. Silencing of syntaxin-6, which we identify as a novel immature WPB-SNARE, and the recycling endosome regulator Rab11 revealed that PMA-evoked VWFpp secretion does not traverse through a non-classical post-Golgi compartment such as the recycling endosome on its way out.During the late phase PMA leads to almost complete degranulation of WPBs, but the mechanism has so far remained elusive. Time-lapse imaging of PMA-, histamine- and VEGF-stimulated WPB degranulation showed that this was insensitive to CHX treatment. In support of a role in sensitizing WPB release towards [Ca2+]i we found that a short pre-treatment with PMA enhanced the ability of ionomycin to induce Ca2+-driven VWFpp secretion at sub-maximal but not maximal doses. PMA-potentiated, ionomycin-induced VWFpp secretion was insensitive to CHX, underscoring that this originated from WPBs.ConclusionsOur data show that two pathways operate for rapid regulated secretion of VWF, one involving WPB exocytosis and the other, regulated by PKC activation, that accesses VWF and VWFpp in a late biosynthetic compartment, most likely the trans -Golgi network. Our findings also have significant implications for the mechanistic interpretation of data obtained from biochemical studies of WPB exocytosis induced by PMA, where secreted VWF has been assumed to arise solely from WPBs. DisclosuresNo relevant conflicts of interest to declare.

Endocytic trafficking and signaling of proton‐sensing receptor GPR65

Proton-sensing G-protein coupled receptors (GPCRs) allow cells to sense and respond to changes in extracellular pH. Although pH disturbances have profound effects on cell function and disease progression, little is known about how proton-sensing GPCR activation, trafficking and signaling from distinct intracellular compartments contribute to cellular responses. Here we use GPR65, a proton-sensing GPCR that contributes to pathological conditions characterized by acidic tissue microenvironments such as cancer and chronic inflammatory conditions, as a prototype to study these questions. We use live imaging of HEK293 cells expressing tagged versions of our protein of interest, GPR65, to visualize receptor trafficking, sorting to intracellular compartments, and receptor activation using signaling and conformational biosensors in real time. Our preliminary results indicate that GPR65 localizes to intracellular compartments at steady state and recruits conformational biosensors to both the plasma membrane and intracellular compartments when exposed to neutral and acidic pH. Characterizing GPR65 trafficking and activation within the endosomal pathway will help us understand how proton-sensing receptors contribute to normal physiology and disease and provide new strategies to treat pathological conditions associated with acidic microenvironments.

Compartment-specific opioid receptor signaling is selectively modulated by different dynorphin peptides.

Many signal transduction systems have an apparent redundancy built into them, where multiple physiological agonists activate the same receptors. Whether this is true redundancy, or whether this provides an as-yet unrecognized specificity in downstream signaling, is not well understood. We address this question using the kappa opioid receptor (KOR), a physiologically relevant G protein-coupled receptor (GPCR) that is activated by multiple members of the Dynorphin family of opioid peptides. We show that two related peptides, Dynorphin A and Dynorphin B, bind and activate KOR to similar extents in mammalian neuroendocrine cells and rat striatal neurons, but localize KOR to distinct intracellular compartments and drive different post-endocytic fates of the receptor. Strikingly, localization of KOR to the degradative pathway by Dynorphin A induces sustained KOR signaling from these compartments. Our results suggest that seemingly redundant endogenous peptides can fine-tune signaling by regulating the spatiotemporal profile of KOR signaling.

CTP synthase forms cytoophidia in archaea

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.

Dynamics of intracellular mannan and cell wall folding in the drought responses of succulent Aloe species.

Plants have evolved a multitude of adaptations to survive extreme conditions. Succulent plants have the capacity to tolerate periodically dry environments, due to their ability to retain water in a specialized tissue, termed hydrenchyma. Cell wall polysaccharides are important components of water storage in hydrenchyma cells. However, the role of the cell wall and its polysaccharide composition in relation to drought resistance of succulent plants are unknown. We investigate the drought response of leaf‐succulent Aloe (Asphodelaceae) species using a combination of histological microscopy, quantification of water content, and comprehensive microarray polymer profiling. We observed a previously unreported mode of polysaccharide and cell wall structural dynamics triggered by water shortage. Microscopical analysis of the hydrenchyma cell walls revealed highly regular folding patterns indicative of predetermined cell wall mechanics in the remobilization of stored water and the possible role of homogalacturonan in this process. The in situ distribution of mannans in distinct intracellular compartments during drought, for storage, and apparent upregulation of pectins, imparting flexibility to the cell wall, facilitate elaborate cell wall folding during drought stress. We conclude that cell wall polysaccharide composition plays an important role in water storage and drought response in Aloe.

Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells.

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101’s action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

Intracellular Follicle-Stimulating Hormone Receptor Trafficking and Signaling.

Models of G protein-coupled receptor (GPCR) signaling have dramatically altered over the past two decades. Indeed, GPCRs such as the follicle-stimulating hormone receptor (FSHR) have contributed to these new emerging models. We now understand that receptor signaling is highly organized at a spatial level, whereby signaling not only occurs from the plasma membrane but distinct intracellular compartments. Recent studies in the role of membrane trafficking and spatial organization of GPCR signaling in regulating gonadotropin hormone receptor activity has identified novel intracellular compartments, which are tightly linked with receptor signaling and reciprocally regulated by the cellular trafficking machinery. Understanding the impact of these cell biological mechanisms to physiology and pathophysiology is emerging for certain GPCRs. However, for FSHR, the potential impact in both health and disease and the therapeutic possibilities of these newly identified systems is currently unknown, but offers the potential to reassess prior strategies, or unveil novel opportunities, in targeting this receptor.

Anaplasmataceae Subversion of the Lysosomal Activity

Pathogenic bacteria belonging to the family Anaplasmataceae include species of the genera Ehrlichia Anaplasma and Neorickettsia These bacterial obligate intracellular parasites have evolved diverse mechanisms for evasion of host cellular defenses One of these mechanisms involves adaptations for survival in distinct intracellular compartments that allow their replication in seclusion from lysosomal killing Here I review the intracellular niches inhabited by these obligate intracellular parasites such as arrested early endosomes lysosomes and vesicles that do not fuse with the endosomal compartment but intersect with an exocytic pathway

ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments for Uncoating.

Of the more than 150 human rhinovirus (RV) serotypes, 89 utilize intercellular adhesion molecule-1 (ICAM-1) for cell entry. These belong either to species A or B. We recently demonstrated that RV-B14 and RV-A89, despite binding this same receptor, are routed into distinct endosomal compartments for release of their RNA into the cytosol. To gain insight into the underlying mechanism we now comparatively investigate the port of entry, temperature-dependence of uncoating, and intracellular routing of RV-B3, RV-B14, RV-A16, and RV-A89 in HeLa cells. The effect of various drugs blocking distinct stages on the individual pathways was determined via comparing the number of infected cells in a TissueFaxs instrument. We found that RV-B14 and RV-A89 enter via clathrin-, dynamin-, and cholesterol-dependent pathways, as well as by macropinocytosis. Drugs interfering with actin function similarly blocked entry of all four viruses, indicating their dependence on a dynamic actin network. However, uniquely, RV-A89 was able to produce progeny when internalized at 20 °C followed by neutralizing the endosomal pH and further incubation at 37 °C. Blocking dynein-dependent endosomal transport prevented uncoating of RV-A16 and RV-A89, but not of RV-B3 and RV-B14, indicative for routing of RV-A16 and RV-A89 into the endocytic recycling compartment for uncoating. Our results call for caution when developing drugs aimed at targeting entry or intracellular trafficking of all rhinovirus serotypes.

Multiparametric detection of autoantibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous disease with respect to disease manifestations, disease progression and treatment response. Therefore, strategies to identify biomarkers that help distinguishing SLE subgroups are a major focus of biomarker research. We reasoned that a multiparametric autoantibody profiling approach combined with data mining tools could be applied to identify SLE patient clusters. We used a bead-based array containing 86 antigens including diverse nuclear and immune defense pathway proteins. Sixty-four autoantibodies were significantly (p < 0.05) increased in SLE (n = 69) compared to healthy controls (HC, n = 59). Using binary cut-off thresholds (95% quantile of HC), hierarchical clustering of SLE patients yields five clusters, which differ qualitatively and in their total number of autoantibodies. In two patient clusters the overall accumulated autoantibody reactivity of all antigens tested was 31% and 48%, respectively. We observed a positive association between the autoantibody signature present in these two patient clusters and the clinical manifestation of glomerulonephritis (GLMN). In addition, groups of autoantibodies directed against distinct intracellular compartments and/or biological motifs characterize the different SLE subgroups. Our findings highlight the relevant potential of multiparametric autoantibody detection and may contribute to a deeper understanding of the clinical and serological diversity of SLE.

Identification of a Major Lipid Droplet Protein in a Marine Diatom Phaeodactylum tricornutum.

Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets.