Page 11 - 2014 Printable Abstract Book
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(2) secreted exosomes (resulting from exocytosis as the surface membrane of multivesicular bodies or
MVB fuses with the PM, spilling the exosomes into the extracellular space). Shed vesicles (SV)/ectosomes
and exosomes are distinguished by their size, density, protein composition, lipid profile and relative
amounts of exposed phosphatidyl serine. MV are distinct from membrane blebs (containing histones and
DNA) that are released during apoptosis and from ectodomains of PM-associated proteins that are shed
from the cell surface. Owing to the use of diverse protocols to isolate MV, mixtures of SV/ectosomes,
exosomes and apoptotic blebs may be unintentionally prepared and published in the literature as a single
class of MV, driving a need to standardize sample preparation and purification. The formation of
SV/ectosomes is initiated as a membrane bud. Membrane buds emerge in response to the high energetic
cost within microdomains from the association of dissimilar lipids having a height mismatch at a phase
boundary that forces a polar headgroup region to contact a hydrophobic region. The resulting line tension
is relieved by coalescence of microdomains into circular zones that grow until a critical size is achieved
(exceeding the Helfrich or curvature-dependent energy), resulting in deformation of the membrane out
of the plane, and reduction in the zone of contact. Most (but not all) PM microdomains involved in PM
budding are enriched in sterols and sphingolipids that form “rafts”. The buds elongate and ultimately,
pinch off at the base, resulting in SV/ectosome release. Vesicle shedding may be also facilitated by calcium
release from the endoplasmic reticulum into the cytoplasm, resulting in disassembly of actin filaments
beneath the PM and activation of enzymes that regulate phospholipid asymmetry across the PM. By
contrast, exosomes containing endocytic markers such as tetraspanins are secreted into the local
microenvironment and surrounding body fluids. A web-based database of the protein, lipid, mRNA and
miRNA content of purified exosomes has been developed (ExoCarta). In the case of synaptic signaling,
exosomes that have delivered their cargo are continually recycled via endocytosis at clathrin-coated pits
in the PM and its deep invaginations, after which endosomes, endocytic intermediates and MVB are
sequentially formed. Following their release, MV either break down within minutes, target neighboring
cells in the microenvironment (paracrine signaling) or diffuse into biological fluids, including the blood,
CSF and urine, where they move to distant sites (endocrine signaling). In addition to being phagocytosed,
MV may interact with a target cell through several mechanisms, including (1) fusion with its PM and
release of cargo (such as proteins, mRNA and miRNA) into the cytosol, (2) docking onto a specific surface
receptor with initiation of signal transduction and (3) endocytosis and subsequent fusion with endosome
membranes. The horizontal transfer of oncogenic and procoagulant activities to target cells via
SV/ectosomes is associated with flotillin-1, a protein marker for lipid rafts. Tetraspanins may serve as
target cell selection markers for exosomes. Intercellular signaling occurs via exocytosis, proteolytic
cleavage, juxtacrine signaling and gap junctions. It has been long recognized that MV released in vitro and
in vivo may traffic signals among cells. In 1982, we determined that SV mediate juxtacrine communication
among hematopoietic cells when we reported that an erythroid-directed growth factor (burst-promoting
activity; BPA) was present in the pellet after centrifugation at 40,000 x g, 30 min of serum-free medium
conditioned (CM) by resting lymphocytes (later identified as B-cells). Freeze-fracture EM showed the
pellet to contain numerous MV that were heterogeneous in size (0.1-0.4 u) and texture, many of which
contained pits representing integral membrane proteins. Subcellular fractionation showed BPA to localize
to the PM. Activity was enriched in MV, compared to that in the PM. MV contained 2 of the 4 major
glycoproteins present in PMs, consistent with a PM origin of MV with the exclusion of selected proteins.
Thus, MV are constituitively and preferentially exfoliated/shed (SV) from the cell surface. SV that were
centrifuged to equilibrium on continuous dextran gradients prepared in medium of different tonicities
behaved as osmometers and layered at p=1.030, consistent with their being sealed and spherical with an
9 | P a g e
MVB fuses with the PM, spilling the exosomes into the extracellular space). Shed vesicles (SV)/ectosomes
and exosomes are distinguished by their size, density, protein composition, lipid profile and relative
amounts of exposed phosphatidyl serine. MV are distinct from membrane blebs (containing histones and
DNA) that are released during apoptosis and from ectodomains of PM-associated proteins that are shed
from the cell surface. Owing to the use of diverse protocols to isolate MV, mixtures of SV/ectosomes,
exosomes and apoptotic blebs may be unintentionally prepared and published in the literature as a single
class of MV, driving a need to standardize sample preparation and purification. The formation of
SV/ectosomes is initiated as a membrane bud. Membrane buds emerge in response to the high energetic
cost within microdomains from the association of dissimilar lipids having a height mismatch at a phase
boundary that forces a polar headgroup region to contact a hydrophobic region. The resulting line tension
is relieved by coalescence of microdomains into circular zones that grow until a critical size is achieved
(exceeding the Helfrich or curvature-dependent energy), resulting in deformation of the membrane out
of the plane, and reduction in the zone of contact. Most (but not all) PM microdomains involved in PM
budding are enriched in sterols and sphingolipids that form “rafts”. The buds elongate and ultimately,
pinch off at the base, resulting in SV/ectosome release. Vesicle shedding may be also facilitated by calcium
release from the endoplasmic reticulum into the cytoplasm, resulting in disassembly of actin filaments
beneath the PM and activation of enzymes that regulate phospholipid asymmetry across the PM. By
contrast, exosomes containing endocytic markers such as tetraspanins are secreted into the local
microenvironment and surrounding body fluids. A web-based database of the protein, lipid, mRNA and
miRNA content of purified exosomes has been developed (ExoCarta). In the case of synaptic signaling,
exosomes that have delivered their cargo are continually recycled via endocytosis at clathrin-coated pits
in the PM and its deep invaginations, after which endosomes, endocytic intermediates and MVB are
sequentially formed. Following their release, MV either break down within minutes, target neighboring
cells in the microenvironment (paracrine signaling) or diffuse into biological fluids, including the blood,
CSF and urine, where they move to distant sites (endocrine signaling). In addition to being phagocytosed,
MV may interact with a target cell through several mechanisms, including (1) fusion with its PM and
release of cargo (such as proteins, mRNA and miRNA) into the cytosol, (2) docking onto a specific surface
receptor with initiation of signal transduction and (3) endocytosis and subsequent fusion with endosome
membranes. The horizontal transfer of oncogenic and procoagulant activities to target cells via
SV/ectosomes is associated with flotillin-1, a protein marker for lipid rafts. Tetraspanins may serve as
target cell selection markers for exosomes. Intercellular signaling occurs via exocytosis, proteolytic
cleavage, juxtacrine signaling and gap junctions. It has been long recognized that MV released in vitro and
in vivo may traffic signals among cells. In 1982, we determined that SV mediate juxtacrine communication
among hematopoietic cells when we reported that an erythroid-directed growth factor (burst-promoting
activity; BPA) was present in the pellet after centrifugation at 40,000 x g, 30 min of serum-free medium
conditioned (CM) by resting lymphocytes (later identified as B-cells). Freeze-fracture EM showed the
pellet to contain numerous MV that were heterogeneous in size (0.1-0.4 u) and texture, many of which
contained pits representing integral membrane proteins. Subcellular fractionation showed BPA to localize
to the PM. Activity was enriched in MV, compared to that in the PM. MV contained 2 of the 4 major
glycoproteins present in PMs, consistent with a PM origin of MV with the exclusion of selected proteins.
Thus, MV are constituitively and preferentially exfoliated/shed (SV) from the cell surface. SV that were
centrifuged to equilibrium on continuous dextran gradients prepared in medium of different tonicities
behaved as osmometers and layered at p=1.030, consistent with their being sealed and spherical with an
9 | P a g e
