Dryland endolithic Chroococcidiopsis and temperate fresh water Synechocystis have distinct membrane lipid and photosynthesis acclimation strategies upon desiccation and temperature increase.

An effect of climate change is the expansion of drylands in temperate regions, predicted to affect microbial biodiversity. Photosynthetic organisms being at the base of ecosystem's trophic networks, we compared an endolithic desiccation-tolerant Chroococcidiopsis cyanobacteria isolated from gypsum rocks in the Atacama Desert, with a freshwater desiccation-sensitive Synechocystis. We sought whether some acclimation traits in response to desiccation and temperature variations were shared, to evaluate the potential of temperate species to possibly become resilient to future arid conditions. When temperature varies, Synechocystis tunes the acyl composition of its lipids, via a homeoviscuous acclimation mechanism known to adjust membrane fluidity, whereas no such change occurs in Chroococcidiopsis. Vice versa, a combined study of photosynthesis and pigment content shows that Chroococcidiopsis remodels its photosynthesis components and keeps an optimal photosynthetic capacity at all temperatures, whereas Synechocystis is unable to such adjustment. Upon desiccation on a gypsum surface, Synechocystis is rapidly unable to revive, whereas Chroococcidiopsis is capable to recover after three weeks. Using X-ray diffraction, we found no evidence that Chroococcidiopsis could use water extracted from gypsum crystal in such conditions, as a surrogate of missing water. The sulfolipid sulfoquinovosyldiacylglycerol becomes the prominent membrane lipid in both dehydrated cyanobacteria, highlighting an overlooked function for this lipid. Chroococcidiopsis keeps a minimal level of monogalactosyldiacylglycerol, which may be essential for the recovery process. Results support that two independent adaptation strategies have evolved in these species to cope with temperature and desiccation increase, and suggest some possible scenarios for microbial biodiversity change triggered by climate change.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

PepS: An Innovative Microfluidic Device for Bedside Whole Blood Processing before Plasma Proteomics Analyses.

Immunoassays have been used for decades in clinical laboratories to quantify proteins in serum and plasma samples. However, their limitations make them inappropriate in some cases. Recently, mass spectrometry (MS) based proteomics analysis has emerged as a promising alternative method when seeking to assess panels of protein biomarkers with a view to providing protein profiles to monitor health status. Up to now, however, translation of MS-based proteomics to the clinic has been hampered by its complexity and the substantial time and human resources necessary for sample preparation. Plasma matrix is particularly tricky to process as it contains more than 3000 proteins with concentrations spanning an extreme dynamic range (1010). To address this preanalytical challenge, we designed a microfluidic device (PepS) automating and accelerating blood sample preparation for bottom-up MS-based proteomics analysis. The microfluidic cartridge is operated through a dedicated compact instrument providing fully automated fluid processing and thermal control. In less than 2 h, the PepS device allows bedside plasma separation from whole blood, volume metering, depletion of albumin, protein digestion with trypsin, and stabilization of tryptic peptides on solid-phase extraction sorbent. For this first presentation, the performance of the PepS device was assessed using discovery proteomics and targeted proteomics, detecting a panel of three protein biomarkers routinely assayed in clinical laboratories (alanine aminotransferase 1, C-reactive protein, and myoglobin). This innovative microfluidic device and its associated instrumentation should help to streamline and simplify clinical proteomics studies.

Mass Spectrometry-Based Proteomics Reveal Alcohol Dehydrogenase 1B as a Blood Biomarker Candidate to Monitor Acetaminophen-Induced Liver Injury.

Acute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death, and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to screen omics databases and identify potential biomarkers for hepatocyte cell death. Then, discovery proteomics was harnessed to select from among these candidates those that were specifically detected in the blood of acetaminophen-induced ALI patients. Among these candidates, the isoenzyme alcohol dehydrogenase 1B (ADH1B) was massively leaked into the blood. To evaluate ADH1B, we developed a targeted proteomics assay and quantified ADH1B in serum samples collected at different times from 17 patients admitted for acetaminophen-induced ALI. Serum ADH1B concentrations increased markedly during the acute phase of the disease, and dropped to undetectable levels during recovery. In contrast to alanine aminotransferase activity, the rapid drop in circulating ADH1B concentrations was followed by an improvement in the international normalized ratio (INR) within 10-48 h, and was associated with favorable outcomes. In conclusion, the combination of omics data exploration and proteomics revealed ADH1B as a new blood biomarker candidate that could be useful for the monitoring of acetaminophen-induced ALI.

A proteomics assay to detect eight CBRN-relevant toxins in food.

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS.

DIGESTIF: a universal quality standard for the control of bottom-up proteomics experiments.

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.

Adaptive traits of cysts of the snow alga Sanguina nivaloides unveiled by 3D subcellular imaging.

Sanguina nivaloides is the main alga forming red snowfields in high mountains and Polar Regions. It is non-cultivable. Analysis of environmental samples by X-ray tomography, focused-ion-beam scanning-electron-microscopy, physicochemical and physiological characterization reveal adaptive traits accounting for algal capacity to reside in snow. Cysts populate liquid water at the periphery of ice, are photosynthetically active, can survive for months, and are sensitive to freezing. They harbor a wrinkled plasma membrane expanding the interface with environment. Ionomic analysis supports a cell efflux of K+, and assimilation of phosphorus. Glycerolipidomic analysis confirms a phosphate limitation. The chloroplast contains thylakoids oriented in all directions, fixes carbon in a central pyrenoid and produces starch in peripheral protuberances. Analysis of cells kept in the dark shows that starch is a short-term carbon storage. The biogenesis of cytosolic droplets shows that they are loaded with triacylglycerol and carotenoids for long-term carbon storage and protection against oxidative stress.

Mass spectrometry-based absolute protein quantification: PSAQ™ strategy makes use of "noncanonical" proteotypic peptides.

Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.

Introducing AAA-MS, a rapid and sensitive method for amino acid analysis using isotope dilution and high-resolution mass spectrometry.

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.

Targeted Proteomics Analysis of Staphylococcal Superantigenic Toxins in Menstrual Fluid from Women with Menstrual Toxic Shock Syndrome (mTSS).

Menstrual toxic shock syndrome (mTSS) is a rare life-threatening febrile illness that occurs in women using intravaginal menstrual protection. It is caused by toxic shock syndrome toxin 1 (TSST-1) produced by Staphylococcus aureus, triggering a sudden onset of rash and hypotension, subsequently leading to multiple organ failure. Detecting TSST-1 and S. aureus virulence factors in menstrual fluid could accelerate the diagnosis and improve therapeutic management of mTSS. However, menstrual fluid is a highly complex matrix, making detection of bacterial toxins challenging. Here, we present a mass-spectrometry-based proteomics workflow for the targeted, quantitative analysis of four S. aureus superantigenic toxins in menstrual fluids (TSST-1, SEA, SEC, and SED). This method was applied to characterize toxin levels in menstrual fluids collected from patients with mTSS and healthy women. Toxins were detectable in samples from patients with mTSS and one healthy donor at concentrations ranging from 0 to 0.46 µg/mL for TSST-1, and 0 to 1.07 µg/mL for SEC. SEA and SED were never detected in clinical specimens, even though many S. aureus strains were positive for the corresponding genes. The method presented here could be used to explore toxin production in vivo in users of intravaginal devices to improve the diagnosis, understanding, and prevention of mTSS.

Sorting of the v-SNARE VAMP7 in Dictyostelium discoideum: a role for more than one Adaptor Protein (AP) complex.

Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism.

High-density rafts preferentially host the complement activator measles virus F glycoprotein but not the regulators of complement activation.

The fusion (F) protein of measles virus (MeV) activates the alternative pathway of human complement in the presence of both CD46 and CD55 which regulate the complement activation [Devaux, P., Christiansen, D., Plumet, S., Gerlier, D., 2004. Cell surface activation of the alternative complement pathway by the fusion protein of measles virus. J. Gen. Virol. 85, 1665-1673]. The original observation of cold detergent-resistant membranes sedimenting at a higher density than the membrane rafts lead us to analyse the respective distribution of F, CD46 and C55 molecules in what we call heavy rafts (HRs) and in the classical low-density membrane rafts (Rs). Membrane rafts were isolated after cold TX100 solubilization and flotation on a sucrose gradient. The denser fractions collected from the lower part of the gradient could be further separated into a translucent pellet (HR) and a soluble supernatant (S). HR and R were both sensitive to TX100 solubilization after cholesterol depletion and solubilized by octyl-d-glucoside but differed in their lipid and protein composition. A proteomic analysis revealed that the HR fraction was derived from heterogeneous cellular membranes including plasma membrane, early endosomes and rough endoplasmic reticulum. Interestingly, CD55 and CD46 almost exclusively associated with R and S fractions, respectively, while after MeV infection or transient expression, MeV-F distributed almost equally between R, HR and S fractions. However more immature MeV-F(0) than mature MeV-F(1) proteins was associated with the HR fraction whereas this ratio was reverse in R and S fractions. After activation of the alternative pathway of human complement by F expressing cells, both C3b and F protein associated with R, HR and S fractions. When four or five of the five cysteines located in the transmembrane and cytoplasmic tail of F protein were substituted with serine residues, the mutated F distributed almost exclusively in HR fractions and was still efficient in activating the complement. We propose that the partitioning of F, CD46 and CD55 molecules in different membrane microdomains could account for the ability of F to escape complement regulation by the CD55 and CD46 regulators.

Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

Protein Biomarker Discovery in Non-depleted Serum by Spectral Library-Based Data-Independent Acquisition Mass Spectrometry.

In discovery proteomics experiments, tandem mass spectrometry and data-dependent acquisition (DDA) are classically used to identify and quantify peptides and proteins through database searching. This strategy suffers from known limitations such as under-sampling and lack of reproducibility of precursor ion selection in complex proteomics samples, leading to somewhat inconsistent analytical results across large datasets. Data-independent acquisition (DIA) based on fragmentation of all the precursors detected in predetermined isolation windows can potentially overcome this limitation. DIA promises reproducible peptide and protein quantification with deeper proteome coverage and fewer missing values than DDA strategies. This approach is particularly attractive in the field of clinical biomarker discovery, where large numbers of samples must be analyzed. Here, we describe a DIA workflow for non-depleted serum analysis including a straightforward approach through which to construct a dedicated spectral library, and indications on how to optimize chromatographic and mass spectrometry analytical methods to produce high-quality DIA data and results.

Interactions of an essential Bacillus subtilis GTPase, YsxC, with ribosomes.

YsxC is a small GTPase of Bacillus subtilis with essential but still unknown function, although recent works have suggested that it might be involved in ribosome biogenesis. Here, purified YsxC overexpressed in Escherichia coli was found to be partly associated with high-molecular-weight material, most likely rRNA, and thus eluted from gel filtration as a large complex. In addition, purification of ribosomes from an E. coli strain overexpressing YsxC allowed the copurification of the YsxC protein. Purified YsxC was shown to bind preferentially to the 50S subunit of B. subtilis ribosomes; this interaction was modulated by nucleotides and was stronger in the presence of a nonhydrolyzable GTP analogue than with GTP. Far-Western blotting analysis performed with His(6)-YsxC and ribosomal proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that YsxC interacted with at least four ribosomal proteins from the 50S subunit. Two of these putative protein partners were identified by mass spectrometry as L1 and L3, while the third reactive band in the one-dimensional gel contained L6 and L10. The fourth band that reacted with YsxC contained a mixture of three proteins, L7/L12, L23, and L27, suggesting that at least one of them binds to YsxC. Coimmobilization assays confirmed that L1, L6, and L7/L12 interact with YsxC. Together, these results suggest that YsxC plays a role in ribosome assembly.

Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization.

Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and alpha-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of alpha-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1(-) mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron-sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1-KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family.

Introducing plasma/serum glycodepletion for the targeted proteomics analysis of cytolysis biomarkers.

A major class of clinical biomarkers is constituted of intracellular proteins which are leaking into the blood following ischemia, exposure to toxic xenobiotics or mechanical aggression. Their ectopic presence in plasma/serum is an indicator of tissue damage and raises a warning signal. These proteins, referred to as cytolysis biomarkers, are generally of cytoplasmic origin and as such, are devoid of glycosylation. In contrast, most plasma/serum proteins originate from the hepatic secretory pathway and are heavily glycosylated (at the exception of albumin). Recent advances in targeted proteomics have supported the parallelized evaluation of new blood biomarkers. However, these analytical methods must be combined with prefractionation strategies that reduce the complexity of plasma/serum matrix. In this article, we present the glycodepletion method, which reverses the hydrazide-based glycocapture concept to remove plasma/serum glycoproteins from plasma/serum matrix and facilitates the detection of cytolysis biomarkers. Glycodepletion was integrated to a targeted proteomics pipeline to evaluate 4 liver cytolysis biomarker candidates in the context of acetaminophen-induced acute hepatitis.

Multiplex and accurate quantification of acute kidney injury biomarker candidates in urine using Protein Standard Absolute Quantification (PSAQ) and targeted proteomics.

There is a need for multiplex, specific and quantitative methods to speed-up the development of acute kidney injury biomarkers and allow a more specific diagnosis. Targeted proteomic analysis combined with stable isotope dilution has recently emerged as a powerful option for the parallelized evaluation of candidate biomarkers. This article presents the development of a targeted proteomic assay to quantify 4 acute kidney injury biomarker candidates in urine samples. The proteins included in the assessed panel consisted of myo-inositol oxygenase (MIOX), phosphoenolpyruvate carboxykinase 1 (PCK1), neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP). The proteomic assay combined an antibody-free sample preparation and a liquid chromatography-selected reaction monitoring (LC-SRM) analysis pipeline. For accurate quantification of the selected candidates, we used PSAQ (Protein Standard Absolute Quantification) standards which are isotopically labeled versions of the target proteins. When added directly to the biological samples, these standards improve detection specificity and quantification accuracy. The multiplexed assay developed for the 4 biomarker candidates showed excellent analytical performance, in line with the recommendations of health authorities. Tests on urine from two small patient cohorts and a group of healthy donors confirmed the relevance of NGAL and L-FABP as biomarkers for AKI diagnosis. The assay is readily adaptable to other biomarker candidates and should be very useful for the simultaneous and accurate quantification of multiple biomarkers.

Pycnosomes: Condensed Endosomal Structures Secreted by Dictyostelium Amoebae.

Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.