The Emerging World of Membrane Vesicles: Functional Relevance, Theranostic Avenues and Tools for Investigating Membrane Function

Abstract

Lipids are essential components of cell membranes and govern various membrane functions. Lipid organization within membrane plane dictates recruitment of specific proteins and lipids into distinct nanoclusters that initiate cellular signaling while modulating protein and lipid functions. In addition, one of the most versatile function of lipids is the formation of diverse lipid membrane vesicles for regulating various cellular processes including intracellular trafficking of molecular cargo. In this review, we focus on the various kinds of membrane vesicles in eukaryotes and bacteria, their biogenesis, and their multifaceted functional roles in cellular communication, host-pathogen interactions and biotechnological applications. We elaborate on how their distinct lipid composition of membrane vesicles compared to parent cells enables early and non-invasive diagnosis of cancer and tuberculosis, while inspiring vaccine development and drug delivery platforms. Finally, we discuss the use of membrane vesicles as excellent tools for investigating membrane lateral organization and protein sorting, which is otherwise challenging but extremely crucial for normal cellular functioning. We present current limitations in this field and how the same could be addressed to propel a fundamental and technology-oriented future for extracellular membrane vesicles.

Keywords: diagnosis; drug delivery; exosomes; host-pathogen interactions; lipid biomarkers; lipids; membrane organization; membrane vesicles.

FIGURE 1

Membrane vesicles of variable sizes and origin. Schematic representation of various kinds of natural membrane vesicles from eukaryotic and bacterial cells. of the kinds of vesicles obtained from different cell types. Artificial membrane vesicles, giant plasma membrane vesicle (GPMV), synthesized from eukaryotic cells by chemical blebbing from plasma membranes of wild type or genetically modified cells. The exogenously expressed fluorescent tagged proteins in genetically modified cells are incorporated into the cell plasma membrane before GPMVs formation, forming the so-called engineered GPMVs. Giant Endoplasmic Reticulum Vesicles (GERs) represent membrane vesicles derived from endoplasmic reticulum (ER). OMV, outer membrane vesicle; CMV, cytoplasmic membrane vesicle; O-IMV, outer inner membrane vesicle; GPMV, giant plasma membrane vesicle and GERV, Giant endoplasmic reticulum vesicle.

FIGURE 2

Compositional heterogeneity in membrane vesicles guide their membrane biophysical properties. (A) Fold change in the abundance of specific lipid species in exosomes compared to the donor cells across various cell lines. Data reprinted from Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Llorente, A et al. Molecular lipidomics of exosomes released by PC-3 prostate cancer cells, 2013, 1831, 1,302–1,309. Copyright (2013), with permission from Elsevier (B) Relative changes in the abundance of indicated lipid constituents in the exosomes derived from PC3 cells. The data has been adapted with from Skotland et al. (2019) (C). Schematic representation of biophysical attributes such as rigidity, membrane thickness and fluidity of cell-derived membrane vesicles dependent on the lipid composition. Enrichment of cholesterol and saturated lipid confer high rigidity and higher membrane thickness, while enrichment of unsaturated lipids leads to increased fluidity and higher membrane deformability.

FIGURE 3

Multifaceted functions of membrane vesicles. (A) Pathogen derived membrane vesicles (MV) aid in communication, in the delivery of virulence factors to host cells during infection, as well as in altering interaction with the host and the use of the bacterial OMVs for the development of immunization against the pathogen. (B) Specific applications of eukaryotic membrane vesicles for 1) intracellular signalling and transport of chemicals, 2) to serve as minimally invasive diagnostic markers against various diseases by using the eukaryotic pathogen derived MV-enclosed molecular cargos such as DNA, proteins, lipids, and mi-RNA and 3) in recombinant cancer therapy via the combination of exosomes and nanotherapy for targeted cancer therapy. Exosomes derived from target cells are tagged with bioactive molecules (red) or functionalized protein (green) and injected into cancer cells or animal models for anti tumor therapy or help in immunization and vaccination against a particular cancer type.

FIGURE 4

Giant plasma membrane vesicles for investigating membrane organization and proteins localization. (A) Fluorescent images showing phase segregation into liquid ordered (l_o) and liquid disordered (l_d) regions in cell-derived GPMVs labeled with NDB-DPPE (green, preferentially partitions into l_o) and Rh-DOPE (red, partitions into l_d), scale bar: 5 μm. Image reprinted from Biochimica et Biophysica Acta (BBA), Biomembranes, Sengupta, P et al. Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles, 2008, 1778, 20–32. Copyright (2008), with permission from Elsevier (B) Representative Laurdan two photon microscopy images of GPMVs derived from A431 cells. Ch1 denotes the emission from Laurdan at 425/50 nm and ch2 at 525/70 nm, with GP image calculated using (Ch1- G * Ch2)/(Ch1+ G * Ch2) in the region. G denotes the calibration factor, scale bar 10 μm. GP scale ranges from −1.0 to +1.0. Image reproduced with permission from Kaiser, HJ et al. Proceedings of the National Academy of Sciences, 2009, 106 (39) 16,645–16,650. The GP values at each pixel are extracted and corresponding GP histogram is generated that shows bimodal GP distribution indicative of at least two lipid phases with distinct order. (C) Representative fluorescence images showing partitioning of GPI-anchored protein (green, YFP-GL-GPI) and transmembrane protein (green, mLAT-EGFP) in the l_o and l_d region, respectively, within phase segregated GMPVs. The GMPVs were also labeled with l_d marker (red, Rh-DOPE). Image reprinted from Biochimica et Biophysica Acta (BBA) - Biomembranes, Sengupta, P. et al. Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles, 2008, 1778, 20–32. Copyright (2008), with permission from Elsevier Representative fluorescence images showing partitioning of PMP22 (green) into the l_o region of phase segregated GPMVs derived from Hela cells. The GPMVs were also labeled with l_d marker (red, DilC12). Image reproduced with permission from Marinko, JT et al. Proceedings of the National Academy of Sciences, 2020, 117 (25) 14,168–14,177. Copyright (2020) Marinko, Kenworthy, Sanders (D) Schematic representation of atomic force microscopy and spectroscopy workflow for studying mechanical membrane properties. Left, solid supported bilayer (SSB) formed from GPMVs fusion on solid mica support. Topographical AFM image of such a SSB and the corresponding force maps, depicting breakthrough force distribution across bilayer surface. AFM Image reprinted from Biopyhiscal Journal, Adhyapak, P. et al. Dynamical Organization of Compositionally Distinct Inner and Outer Membrane Lipids of Mycobacteria, 2020, 118, 1,279–1,291. Copyright (2020), with permission from Elsevier. Breakthrough force corresponds to force required to pierce through the bilayer surface. Histogram depiction of the breakthrough force distribution showing bimodal force distribution indicative of at least two mechanically distinct lipid regions.