Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

The balance of myogenic and adipogenic differentiation is crucial for skeletal muscle homeostasis. Given the vital role of membrane proteins (MBPs) in cell signal perception, membrane proteomics was conducted to delineate mechanisms regulating differentiation of adipogenic and myogenic precursors in skeletal muscle. Adipogenic and myogenic precursors with divergent differentiation potential were isolated from the longissimus dorsi muscle of neonatal pigs by the preplate method. A total of 85 differentially expressed MBPs ( P < 0.05 and fold change ≥1.2 or ≤0.83) between 2 precursors were detected via isobaric tags for relative and absolute quantitation (iTRAQ) assay, including 67 up-regulated and 18 down-regulated in myogenic precursors. Functional enrichment analysis uncovered that myogenic and adipogenic precursors showed significant differences in cytoskeleton organization, syncytium formation, environmental information processing, and organismal systems. Furthermore, key MBPs in regulating cell differentiation were also characterized, including ITGB3, ITGAV, ITPR3, and EPHA2. Noteworthily, EPHA2 was required for myogenic differentiation, and it may promote myogenic differentiation through ERK signaling. Collectively, our study provided an insight into the distinct MBP profile between myogenic and adipogenic precursors in skeletal muscle and served as a solid basis for supporting the role of MBPs in regulating differentiation.-Zhang, X., Wang, L., Qiu, K., Xu, D., Yin, J. Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

Cell surface profiling of cultured cells by direct hydrazide capture of oxidized glycoproteins.

Glycoproteins are a particularly interesting subset of the cellular proteome as a high proportion of proteins present on the extracellular cell surface are glycosylated. These cell surface proteins are ideal targets for biologic drug therapies or for diagnostics tests. Here, we describe a modification of the well-described Cell Surface Capture (CSC) method for the selective isolation and identification of cell surface glycoproteins that contain N-linked carbohydrates. This modification, which we refer to as Direct Cell Surface Capture (D-CSC), is based on oxidation of cell surface glycans on intact cells, followed by direct conjugation of the oxidized oligosaccharides to a solid support using hydrazide chemistry, with no biotinylation step. As a proof-of-principle, we applied D-CSC to the analysis of cell surface membrane proteins of three adherent cancer cell lines (A549, OVCAR3, and U87MG) and compared our results to those published using the well-established Cell Surface Capture (CSC) method, demonstrating comparable selectivity for cell surface proteins. •A method enabling the identification of cell surface proteins from cells in culture is described.•Application of this method to profile the cell surface on three different cancer cell lines is included.

Evaluating Cell Membrane Localization and Intracellular Transport of Proteins by Biotinylation.

Protein translocation to the cell membrane and transport through intracellular compartments are dynamic processes frequently altered in cancer cells. Abnormal protein localization can affect key cell functions, including transduction of extracellular signals and organization of the cytoskeleton, significantly affecting oncogenicity and therapeutic responses. In this chapter, we describe a surface protein biotinylation method that allows the study of membrane localization and endosomal transport of membrane-associated proteins. Surface biotinylation can be used to evaluate baseline protein levels at the membrane, and other processes such as internalization, recycling, and degradation of proteins in response to different treatments or as a consequence of oncogenic mutations. Further, the combination of this technique with other strategies, such as treatments with transport inhibitors, allows investigation of specific steps of protein trafficking through the cell.

Reticulocyte Maturation.

Changes to the membrane proteins and rearrangement of the cytoskeleton must occur for a reticulocyte to mature into a red blood cell (RBC). Different mechanisms of reticulocyte maturation have been proposed to reduce the size and volume of the reticulocyte plasma membrane and to eliminate residual organelles. Lysosomal protein degradation, exosome release, autophagy and the extrusion of large autophagic-endocytic hybrid vesicles have been shown to contribute to reticulocyte maturation. These processes may occur simultaneously or perhaps sequentially. Reticulocyte maturation is incompletely understood and requires further investigation. RBCs with membrane defects or cation leak disorders caused by genetic variants offer an insight into reticulocyte maturation as they present characteristics of incomplete maturation. In this review, we compare the structure of the mature RBC membrane with that of the reticulocyte. We discuss the mechanisms of reticulocyte maturation with a focus on incomplete reticulocyte maturation in red cell variants.

An Efficient GUI-Based Clustering Software for Simulation and Bayesian Cluster Analysis of Single-Molecule Localization Microscopy Data.

Ligand binding of membrane proteins triggers many important cellular signaling events by the lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma membrane. This local clustering can lead to the co-enrichment of molecules that create an intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium towards the execution of a signaling event. In this way, clustering can serve as a cellular switch. The underlying uneven distribution and local enrichment of the signaling cluster's constituting membrane proteins can be used as a functional readout. This information is obtained by combining single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly distinguish clusters from fluctuations in the background noise to generate quantitative data on this complex process. Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative field, and several algorithms and software solutions have been put forward. However, in most cases, such cluster algorithms require multiple analysis parameters to be defined by the user, which may lead to biased results. Furthermore, most cluster algorithms neglect the individual localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it has entailed high computational cost, increasing runtime drastically. Finally, most software is challenging to use as they require advanced technical knowledge to operate. Here we combined three advanced cluster algorithms with the Bayesian approach and parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing than for previous approaches. Our work will simplify access to a well-controlled analysis of clustering data generated by SMLM and significantly accelerate data processing. The inclusion of a simulation mode aids in the design of well-controlled experimental assays.

The emerging role of cell surface receptor and protein binding radiopharmaceuticals in cancer diagnostics and therapy.

Targeting specific cell membrane markers for both diagnostic imaging and radionuclide therapy is a rapidly evolving field in cancer research. Some of these applications have now found a role in routine clinical practice and have been shown to have a significant impact on patient management. Several molecular targets are being investigated in ongoing clinical trials and show promise for future implementation. Advancements in molecular biology have facilitated the identification of new cancer-specific targets for radiopharmaceutical development.

Red cell membrane protein abnormalities as defined by sds-page among patients with anemia in a West African region hospital practice.

BACKGROUND: Erythrocytes require an ability to deform and withstand shear stress while negotiating microcirculation. These properties are largely due to their excess surface area per volume and the characteristics of the membrane's protein. Deficiencies of these proteins are associated with chronic hemolysis. METHODS: This was a cross-sectional study aimed at determining the prevalence of red cell membrane protein abnormalities as determined by sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE) among patients with anemia attending the outpatient clinics of the hospital. RESULTS: A total of 823 participants were recruited into the study with a mean age of 34±14 years. There were 410 (49.8%) participants with hematocrit ≥ 36% and 413 with hematocrit ≤ 35.9% of which 192 participants (23.3%) had abnormal red cell indices. Following SDS-PAGE, 21 (10.9%) of the 192 participants had deficient PAGE tracing. Abnormal spectrin band was observed in 17 (81%) of the 21 participants. The hematocrit was significantly lower while the reticulocyte count and red cell distribution width were higher in participants with red cell membrane abnormalities. CONCLUSION: One in ten patients with mild anemia and abnormal red cell indices in clinical practice may be having hereditary red cell membrane protein defect. Presence of raised reticulocyte count, family history of mild anemia, increased red cell distribution width and red cell morphology may be used to screen for membrane deficiency.

Internalization Characterization of Si Nanorod with Camouflaged Cell Membrane Proteins Reveals ATXN2 as a Negative Regulator.

The fabrication of shape-controlled nanocarriers is critical for efficient delivery of biomolecules across the cell membrane. Surface coating of the nanocarrier can improve internalization efficiency. Here, we developed a facile method of silicon nanorod fabrication leading to a controlled size and shape. We then systematically evaluated five surface modifications with membrane proteins from different cancer cell lines including MCF7, MD231, Hela, Panc-PDX, and Panc-1. We demonstrated that silicon nanorods coated with either a homolytic or heterolytic membrane protein coating have significantly improved internalization efficiency as compared with uncoated Si nanorods. To elucidate the molecular mechanism of the improved efficiency associated with a modified coating, we analyzed the coating membrane proteins derived from five cell lines with proteomics and identified 601 proteins shared by different cell sources. These proteins may function as cell-substrate adhesion molecules that contribute to the enhanced internalization. We also tested the internalization efficiency of nanorods with different coatings in each of the five cell lines to determine the influencing factors from target cells. We found that the internalization efficiency varied among different target cells, and the ranking of the average efficiency was as follows: Hela > Panc-PDX > MD231 > MCF7 > Panc-1. The bioinformatics analysis suggested that the low internalization efficiency in Panc-1 cells might be associated with the upregulation of ATXN2, which is a negative regulator of endocytosis. We further demonstrated that ATXN2 knockdown with specific siRNA significantly improved nanorod internalization efficiency in Panc-1 cells suggesting that ATXN2 can be a reference for efficiency prediction of nanoparticle delivery to tumor cells. Thus, we studied the effect of different cancer cell membrane proteins on nanorod uptake efficiencies. These results can improve nanorod internalization to cancer cells, including a fundamental understanding of the internalization efficiency of cancer cells.

Characterization of Cell Glycocalyx with Mass Spectrometry Methods.

The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques.

Biomimetic nanoparticles for inflammation targeting.

There have been many recent exciting developments in biomimetic nanoparticles for biomedical applications. Inflammation, a protective response involving immune cells, blood vessels, and molecular mediators directed against harmful stimuli, is closely associated with many human diseases. As a result, biomimetic nanoparticles mimicking immune cells can help achieve molecular imaging and precise drug delivery to these inflammatory sites. This review is focused on inflammation-targeting biomimetic nanoparticles and will provide an in-depth look at the design of these nanoparticles to maximize their benefits for disease diagnosis and treatment.

Alterations of membrane protein expression in red blood cells of Alzheimer's disease patients.

Preventive measures, prognosis, or selected therapy in multifactorial maladies, including Alzheimer's disease (AD), require the application of a wide range of diagnostic assays. There is a large unmet need for relatively simple, blood-based biomarkers in this regard. We have recently developed a rapid and reliable flow cytometry and antibody-based method for the quantitative measurement of various red blood cell (RBC) membrane proteins from a drop of blood. Here, we document that the RBC expression of certain membrane proteins, especially that of the GLUT1 transporter and the insulin receptor (INSR), is significantly higher in AD patients than in age-matched healthy subjects. The observed differences may reflect long-term metabolic alterations relevant in the development of AD. These findings may pave the way for a diagnostic application of RBC membrane proteins as relatively stable and easily accessible personalized biomarkers in AD.