THE HUMAN CELL


"Vesicles" is a collective term for cytoplasmic organelles that are often too small to have distinct features when imaged by light microscopy. The majority of the vesicles are membrane-bound organelles, however, also large protein complexes can fall under this category, as they are difficult to distinguish from cytosolic bodies. Examples of organelles with a vesicle annotation are the members of the endolysosomal pathway, transport vesicles including secretory granules, peroxisomes, or lipid droplets.

The biological function of an organelle is defined by its proteome (see Figure 1 for examples of proteins with a vesicular staining). In the Cell Atlas, 1838 (9%) of all human proteins have been experimentally shown to localize to vesicles (Figure 2). Analysis of the vesicle proteome shows highly enriched terms for biological processes related to lipid metabolic reactions and protein transport. About 59% (n=1088) of the vesicle proteins localize to one or more additional locations, with nucleoplasm, cytosol and the Golgi apparatus as the most common ones.

SNX1 - HeLa
CLTA - U-251 MG
AP2B1 - U-2 OS

Figure 1. Examples of proteins localized to the vesicles. SNX1 is part of the retromer complex that mediates the retrograde transport of cargo proteins from endosomes to the trans-Golgi network (TGN) and is involved in endosome-to-plasma membrane transport for cargo protein recycling (detected in HeLa cells). CLTA is a structural element in clathrin-coated vesicles, which are required for the receptor-mediated endocytosis at the plasma membrane (detected in U-251 MG cells). AP2B1 is a component of the adaptor protein complex 2, which is involved in clathrin-dependent endocytosis (detected in U-2 OS cells).

  • 9% (1838 proteins) of all human proteins have been experimentally detected in the vesicles by the Human Protein Atlas.
  • 455 proteins in the vesicles are supported by experimental evidence and out of these 116 proteins are validated by the Human Protein Atlas.
  • 1088 proteins in the vesicles have multiple locations.
  • 166 proteins in the vesicles show a cell to cell variation. Of these 148 show a variation in intensity and 19 a spatial variation.
  • Proteins are mainly involved in lipid metabolism and protein transport.

Figure 2. 9% of all human protein-coding genes encode proteins localized to vesicles. Each bar is clickable and gives a search result of proteins that belong to the selected category.

The structure of the vesicles


Substructures

  • Vesicles: 1771
  • Peroxisomes: 21
  • Endosomes: 17
  • Lysosomes: 17
  • Lipid droplets: 35

    The general structure of organelles annotated as vesicles is a round membrane-enclosed lumen that is less than 1 μm in diameter. Hence, there are only few differences in the structure of the vesicles which can be seen by light microscopy that allow a classification of the organelle, e.g. size, number or the position related to other organelles (Figure 3). Therefore, the true identity of the organelle is often only revealed by the detection of specific marker proteins in immunofluorescence images (see Table 1). Structural information about the organelles can be elucidated by electron microscopy and biochemical analyses.

    Table 1. Selection of proteins suitable as markers for different vesicular organelles.

    Gene

    Description

    Substructure

    ANKFY1 Ankyrin repeat and FYVE domain containing 1 Endosomes
    RAB5C RAB5C, member RAS oncogene family Endosomes
    AGPS Alkylglycerone phosphate synthase Peroxisomes
    ACBD5 Acyl-CoA binding domain containing 5 Peroxisomes
    RAB7A RAB7A, member RAS oncogene family Lysosomes
    PLIN3 Perilipin 3 Cytosol
    Lipid droplets

    Endosomes
    Endosomes can be further sub-classified into early, recycling, and late endosomes, and there is a continuous transition between these classes. Each of them can be defined by their function, by a distinct set of proteins (e.g. members of the Rab-family of proteins (Stenmark H. 2009)), or by morphological differences. The early endosome (EE) has a pleomorphic structure, which consists of cisternae. From these cisternae, two distinct subdomains emerge: vesicular structures with internal invaginations (300-400 nm diameter) and tubular extensions (60 nm diameter) (Gruenberg J. 2001). These two subdomains give rise to either recycling endosomes in the case of the tubular extension or form multivesicular bodies, which transport cargo to the late endosomes. Late endosomes (LE) have cisternal, tubular, and vesicular regions with numerous membrane invaginations, similar to those found in the EE (Griffiths G et al, 1988).

    Lysosomes
    The Belgian Nobel laureate de Duve discovered the lysosome in 1955 and named it after the richness in hydrolytic enzymes (De Duve C et al, 1955). Lysosomes have a tubular morphology of about 0.1-1.2 μm in size and a characteristic acidic pH-value of 4.5-5, which is ideal for the enzymes in the lysosomal lumen. The membrane of lysosomes is rich in glycoproteins and consists of an unusual lipid composition as a protection from the digestive enzymes (Schwake M et al, 2013).

    Peroxisomes
    The peroxisomes is another organelle discovered by de Duve in 1966 (De Duve C et al, 1966), and he named them because of their involvement in peroxidase reactions. Peroxisomes originate from the ER, but they are able to replicate themselves by division. They differ in size from 0.1-1 μm and have a dynamic structure, which is usually spherical. But the shape can change and become more elongated prior to division or in adaption to different conditions (Smith JJ et al, 2013). Elongated peroxisomes can help to distinguish peroxisomes from other vesicles in IF.

    Lipid droplets
    Lipid droplets (LDs) were known for a long time, but were believed to be a rather inert storage for lipids. The discovery of the first LD-associated protein in 1991 by Londos and coworkers (Greenberg AS et al, 1991) changed this view, and today LDs are considered as organelles. LDs are formed at the ER and have a simple, yet conserved structure: a hydrophobic core containing the lipids is surrounded by a membrane monolayer (instead of a bilayer found in all other organelles) to which proteins are attached (Walther TC et al, 2012). The size of LDs ranges from hundreds of nanometers to the single 100 micrometer large LD that fills adipocytes. Under normal conditions, cells have none or few small LDs, but if those few LDs are large enough, LD-associated proteins appear in perfectly round rings and the protein location can be annotated more precisely.

    RAB5C - U-2 OS
    LAMTOR4 - U-2 OS
    ABCD3 - A-431


    PLIN3 - A-431
    EPS15L1 - MCF7
    C19orf60 - U-2 OS

    Figure 3. Examples of the different types of vesicles found in the Cell Atlas. Endosomal protein RAB5C in U-2 OS cells. Lysosomal protein LAMTOR4 in U-2 OS cells. Peroxisomal protein ABCD3 in A-431 cells. LD-associated protein PLIN3 in A-431 cells. Clathrin-coated vesicle protein EPS15L1 in MCF7 cells. Vesicle-front forming protein C19orf60 in U-2 OS cells.

    The function of the vesicles


    Endosomes and lysosomes
    Endocytic vesicles containing material such as receptors and their bound ligands are transported to the early endosome. An efficient sorting takes place in the endosomes. Some of the receptors are sent back to the plasma membranes for reuse, while other receptors are sorted into multivesicular bodies and transported to LEs, which later fuse with lysosomes for the degradation of the material. Many of the highest expressed proteins with a vesicular staining are involved in catabolic processes in the lysosome, e.g. PSAP (Table 2). Since endosomes and lysosomes take part in the regulation of receptors and transporters in the plasma membrane as well as in further distribution of endocytic material, a dysfunction of these organelles can cause human diseases. One example is the Niemann-Pick Type C Disease, where the intracellular cholesterol transport is disturbed leading to a neurologically progressive disease (Peake KB et al, 2010).

    Peroxisomes
    Peroxisomes are multifunctional organelles that harbor a variety of enzymes and are involved in several anabolic and catabolic cellular pathways. The main function of peroxisomes is β-oxidation of long- and very long-chain fatty acids. They also contribute to the utilization and production of reactive oxygen species in the cell. In addition, peroxisomes carry out important reactions such as phospholipid biosynthesis, chemical detoxification or oxidation of purines, polyamines, and some amino acids (Antonenkov VD et al, 2010).

    Lipid droplets
    Nearly all cells are able to form LDs and use them as the main storage site for cellular neutral lipids. These lipids, mainly triacylglycerol and cholesterol, are utilized for the generation of energy or serve as building blocks for the synthesis of other lipids. LDs are linked to a growing number of diseases, but most prominent is their role in obesity and diabetes (Walther TC et al, 2012).

    Table 2. Highly expressed single localizing proteins with a vesicular staining across different cell lines.

    Gene

    Description

    Average TPM

    PSAP Prosaposin 713
    CD63 CD63 molecule 684
    PPA1 Pyrophosphatase (inorganic) 1 253
    RAB7A RAB7A, member RAS oncogene family 203
    TMED2 Transmembrane p24 trafficking protein 2 194
    RAB5C RAB5C, member RAS oncogene family 157
    CTSC Cathepsin C 154
    LAMTOR4 Late endosomal/lysosomal adaptor, MAPK and MTOR activator 4 146
    CTSA Cathepsin A 144
    PICALM Phosphatidylinositol binding clathrin assembly protein 133

    Gene Ontology (GO)-based enrichment analysis of genes encoding proteins that localize mainly to vesicles reveals several functions associated with the group of organelles comprised of vesicles. In addition, it also indicates the organelles that are represented by the term vesicles. The highly enriched terms for the GO domain Biological Process are related mainly to lipid metabolism, which is connected to processes in peroxisomes, and processes related to the function of endosomes (Figure 4a). For the GO domain Molecular Function, vesicle proteins are enriched for sterol transport and receptor related actions (Figure 4b).

    Figure 4.a Gene Ontology-based enrichment analysis for the vesicles proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.

    Figure 4.b Gene Ontology-based enrichment analysis for the vesicles proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.

    Vesicles proteins with multiple locations


    Approximately 59% (n=1088) of the vesicle proteins detected in the Cell Atlas also localize to other compartments in the cell. The network plot (Figure 5) shows that the most overrepresented locations with vesicles are nucleoplasm, ER, and the Golgi apparatus. Given the function of vesicles, these multiple locations could highlight a higher activity in the secretory pathway. Examples of multilocalizing proteins within the proteome of vesicles can be seen in Figure 6.

    Figure 5. Interactive network plot of vesicle-associated proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to vesicles and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the vesicle proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p≤0.05). Note that this calculation is only done for proteins with dual localizations. Each node is clickable and results in a list of all proteins that are found in the connected organelles.

    VTI1B - U-2 OS
    GPRC5A - U-2 OS
    EPN3 - HaCaT

    Figure 6. Examples of multilocalizing proteins in the vesicle proteome. The examples show common or overrepresented combinations for multilocalizing proteins in the proteome. VTI1B promotes the fusion of vesicles with the target membrane at the Golgi apparatus (detected in U-2 OS cells). GPRC5A, detected at the plasma membrane and vesicles, is an orphan receptor, which might be involved in the interaction between retinoic acid and G-protein signaling pathways (detected in U-2 OS cells). EPN3 co-localizes with clathrin-coated vesicles and shuttles into the nucleus (detected in HaCaT cells).

    Expression levels of vesicles proteins in tissue


    The transcriptome analysis (figure 7) shows that vesicle proteins are less likely to be expressed in all tissues, but more likely to be enhanced or enriched in certain tissues compared to a background of all genes with protein data in the Cell Atlas. These results potentially reflect the variety of functions involving vesicle proteins, particularly in the transport of secretory proteins and other biomolecules to the outside of the cell.

    Figure 7. Bar plot showing the distribution of expression categories, based on the gene expression in tissues, for vesicles-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks statistically significant deviation(s) (p≤0.05) from all other organelles based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.

    Relevant links and publications


    Antonenkov VD et al, 2010. Peroxisomes are oxidative organelles. Antioxid Redox Signal.
    PubMed: 19958170 DOI: 10.1089/ars.2009.2996

    De Duve C et al, 1966. Peroxisomes (microbodies and related particles). Physiol Rev.
    PubMed: 5325972 

    DE DUVE C et al, 1955. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J.
    PubMed: 13249955 

    Greenberg AS et al, 1991. Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J Biol Chem.
    PubMed: 2040638 

    Griffiths G et al, 1988. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell.
    PubMed: 2964276 

    Gruenberg J. 2001. The endocytic pathway: a mosaic of domains. Nat Rev Mol Cell Biol.
    PubMed: 11584299 DOI: 10.1038/35096054

    Peake KB et al, 2010. Defective cholesterol trafficking in Niemann-Pick C-deficient cells. FEBS Lett.
    PubMed: 20416299 DOI: 10.1016/j.febslet.2010.04.047

    Schwake M et al, 2013. Lysosomal membrane proteins and their central role in physiology. Traffic.
    PubMed: 23387372 DOI: 10.1111/tra.12056

    Smith JJ et al, 2013. Peroxisomes take shape. Nat Rev Mol Cell Biol.
    PubMed: 24263361 DOI: 10.1038/nrm3700

    Stenmark H. 2009. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol.
    PubMed: 19603039 DOI: 10.1038/nrm2728

    Walther TC et al, 2012. Lipid droplets and cellular lipid metabolism. Annu Rev Biochem.
    PubMed: 22524315 DOI: 10.1146/annurev-biochem-061009-102430