In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

https://www.cell.com/developmental-cell/pdf/S1534-5807(11)00207-3.pdf

The ESCRT Pathway

http://gene-quantification.net/cocucci-meldolesi-exosomes-ectosomes-2015.pdf

Ectosomes and exosomes: shedding the confusion between extracellular vesicles

Exosomes and microvesicles are extracellular nanovesicles released by most but not all cells. They are specifically equipped to mediate intercellular communication via the transfer of genetic information, including the transfer of both coding and non-coding RNAs, to recipient cells. As a result, both exosomes and microvesicles play a fundamental biological role in the regulation of normal physiological as well as aberrant pathological processes, via altered gene regulatory networks and/or via epigenetic programming. For example, microvesicle-mediated genetic transfer can regulate the maintenance of stem cell plasticity and induce beneficial cell phenotype modulation. Alternatively, such vesicles play a role in tumor pathogenesis and the spread of neurodegenerative diseases via the transfer of specific microRNAs and pathogenic proteins. Given this natural property for genetic information transfer, the possibility of exploiting these vesicles for therapeutic purposes is now being investigated. Stem cell-derived microvesicles appear to be naturally equipped to mediate tissue regeneration under certain conditions, while recent evidence suggests that exosomes might be harnessed for the targeted delivery of human genetic therapies via the introduction of exogenous genetic cargoes such as siRNA. Thus, extracellular vesicles are emerging as potent genetic information transfer agents underpinning a range of biological processes and with therapeutic potential.

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Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy

When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the endosomal sorting complex required for transport (ESCRT) complexes 0, I, II and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inwards by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, -I, -II and -III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. Here we show that ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and ESCRT-II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and ESCRT-II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction.

/pdf/molecular-mechanism-of-multivesicular-body-biogenesis-by-2vad3ckdrr.pdf

Molecular mechanism of multivesicular body biogenesis by ESCRT complexes

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins – hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/CHMP6, and hSnf7-1/CHMP4a – to each other, studying the effects of deleting predicted α-helical domains on their behavior in transfected cells. Surprisingly, removing ∼40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA+ adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default ‘closed’ state and an activated ‘open’ state under control of sequences at their C-terminus and associated factors.

Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain.

Cataracts are a clinically diverse and genetically heterogeneous disorder of the crystalline lens and a leading cause of visual impairment. Here we report linkage of autosomal dominant “progressive childhood posterior subcapsular” cataracts segregating in a white family to short tandem repeat (STR) markers D20S847 (LOD score [Z] 5.50 at recombination fraction [θ] 0.0) and D20S195 (Z=3.65 at θ=0.0) on 20q, and identify a refined disease interval (rs2057262–(3.8 Mb)–rs1291139) by use of single-nucleotide polymorphism (SNP) markers. Mutation profiling of positional-candidate genes detected a heterozygous transversion (c.386A→T) in exon 3 of the gene for chromatin modifying protein-4B (CHMP4B) that was predicted to result in the nonconservative substitution of a valine residue for a phylogenetically conserved aspartic acid residue at codon 129 (p.D129V). In addition, we have detected a heterozygous transition (c.481G→A) in exon 3 of CHMP4B cosegregating with autosomal dominant posterior polar cataracts in a Japanese family that was predicted to result in the missense substitution of lysine for a conserved glutamic acid residue at codon 161 (p.E161K). Transfection studies of cultured cells revealed that a truncated form of recombinant D129V-CHMP4B had a different subcellular distribution than wild type and an increased capacity to inhibit release of virus-like particles from the cell surface, consistent with deleterious gain-of-function effects. These data provide the first evidence that CHMP4B, which encodes a key component of the endosome sorting complex required for the transport-III (ESCRT-III) system of mammalian cells, plays a vital role in the maintenance of lens transparency.

CHMP4B, a Novel Gene for Autosomal Dominant Cataracts Linked to Chromosome 20q

Cells contain compartments called organelles that are enclosed within membranes similar to the plasma membrane that surrounds the cell itself. Cells police the proteins on their membranes and move old or damaged proteins into a type of organelle called an endosome. This involves the membrane folding in on itself to form a multivesicular body. The multivesicular bodies deliver their contents to organelles called lysosomes where the old proteins are destroyed. Although it is known that over 30 proteins are involved in the formation of multivesicular bodies, many aspects of how they operate are not well understood. Moreover, disruptions to this process contribute to a large number of diseases including forms of cancer and neurodegeneration. Importantly, the same proteins are hijacked by viruses such as HIV to help them escape from the cells they have infected. Most of the proteins involved in forming multivesicular bodies are part of the ESCRT (Endosomal Sorting Complex Required for Transport) system of proteins. A special set of these proteins—ESCRT-III—is thought to cut the membrane to release vesicles and viruses, as well as helping the membrane to deform. Previously, researchers have been unsure how the ESCRT-III complex works because it has a short lifespan and is too small to see on cellular membranes using standard techniques. Now Cashikar, Shim et al. have used a technique called deep-etch electron microscopy in combination with gene knockdown strategies to reveal the structure of the ESCRT-III complex inside cells. A protein called Vps4 is known to recycle ESCRT-III complexes, so Cashikar, Shim et al. studied cells in which the levels of Vps4 had been depleted in order to increase the lifespan of ESCRT-III complexes. In these cells filaments made of ESCRT-III complexes tended to form conical spirals that matched the size and shape of the vesicles and viruses ESCRT-III is thought to produce. ESCRT-III filaments also accumulated as rings around the molecules destined for incorporation into a vesicle or virus. This indicated a new role for Vps4: it separates ESCRT-III from the contents of the vesicle, leaving it free to form a spiral that drives release of the vesicle or virus from the cell. The next challenge will be to test the predictions of this model using techniques that can capture individual vesicle formation events in real time. Understanding the function of ESCRT-III in greater detail may suggest ways to manipulate this pathway to limit the replication of viruses or the degradation of membrane proteins.

https://cdn.elifesciences.org/articles/02184/elife-02184-v2.pdf

Structure of cellular ESCRT-III spirals and their relationship to HIV budding

Cell biology of the ESCRT machinery.

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2–1, and CHMP3/hVps24 but not CHMP4A/hSnf7–1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with “MIT interacting motifs” (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

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Soomin Shim

Papers

Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain.

CHMP4B, a Novel Gene for Autosomal Dominant Cataracts Linked to Chromosome 20q

Structure of cellular ESCRT-III spirals and their relationship to HIV budding

Cell biology of the ESCRT machinery.

Novel interactions of ESCRT-III with LIP5 and VPS4 and their implications for ESCRT-III disassembly.