EVs Flashcards
What is the ESCRT machinery for? What purpose
Biogenesis, trafficking and release of EVs (vesicle formation and cargo sorting)
How do recipient cells incorporate EVs?
- receptor/kigand interaction
- endocytosis
- phagocytosis
- micropinocytosis
- fusion with cell membrane
Explain endocytosis of recipient cells of EVs?
- most freq mechanism of exosome uptake
- clathrin dep or indep (cholesterol-rich, rigid regions of the plasma membrane (lipid rafts) serve as platforms for clustering and internalizing specific receptors or molecules, allowing cells to selectively take up materials
Biogenesis of microvesicles?
Released via outward budding of the cell membrane (100-1000nm)
Explain what endocytosis is (for recipient cell uptake of EVs)
Small vesicles form by invaginating the plasma membrane, which encloses the target cargo
clathrin-dependent (involving a protein coat) and clathrin-independent (such as caveolin-dependent) mechanisms.
Mechanism of biogenesis/cargo sorting of exosomes (EXTRACELLULAR, lots of DEETS)
- ESCRT-0 (HRS–STAM) gathers tagged proteins (ubiquitylated cargo).
It brings in clathrin to help form intraluminal vesicles (ILVs) inside the endosome.
A complex that cuts the membrane to release the vesicle.
ESCRT proteins also recruit enzymes that remove tags (ubiquitin) from the cargo - ALIX-Syntenin Pathway:
This pathway can form vesicles without using the early ESCRT parts.
ALIX acts like a connector that interacts with different lipids and proteins.
It helps syntenin capture specific cargo for the vesicles.
What other EV cargo sorting/biogenesis mechanisms are there? (EXTRACELLULAR)
Lipids (rich in cholesterol), tetraspanin (at the cell membrane, they interact with integrins and other proteins to create structured areas called tetraspanin-enriched microdomains (TEMs). Classic tetraspanins, like CD63, CD81, and CD9)
Basic mechanism of cargo selection of EVs? (EXTRACELLULAR, BASIC CONCEPT)
Selecting cargo (specifically transmembrane receptors) for inclusion in extracellular vesicles (like exosomes and ectosomes) is primarily achieved through ubiquitylation (the tagging of proteins with ubiquitin) and the subsequent recognition of these tagged proteins by ESCRT components (the proteins involved in the endosomal sorting complexes required for transport).
What about mechanism of sorting INTRACELLULARLY? (RNA)
Not well understood
RNA binding proteins: act as adaptors bw RNA cargo and at EV formation sites
Specific RBPs recognize certain sequences in miRNAs to help load them into small EVs (e.g., hnRNPA2B1).
Is there DNA in small EVs?
Small EVs can contain fragmented nuclear DNA, but mechanisms for DNA recruitment to these vesicles are largely unknown.
Overall, what is EV cargo sorting regulated by? Many factors. Summary
Ubiquitylation: ubiquitination marks proteins for removal, ESCRT pathways facilitates their sorting and degradation in a highly organized manner
RNA-Binding Proteins (RBPs): RBPs act as adaptors, facilitating the sorting of RNAs and other cytoplasmic cargoes into EVs at biogenesis sites.
Tetraspanins: These membrane proteins help organize microdomains on the cell surface, promoting the sorting and incorporation of associated proteins into EVs. They are transmembrane, more on outer
Cell Signaling: The cell’s signaling state and type can influence the RNA content and sorting mechanisms for cargoes in EVs.
Cellular Stress: Conditions like senescence and DNA damage can upregulate specific cargoes, particularly in cancer cells, impacting their presence in EVs.
Metabolic Regulation: EV cargo sorting is influenced by the cell’s metabolic state, including factors like fatty acids, autophagy, oxidative stress, and senescence.
Specific Mechanisms: Transmembrane cargoes are recruited through various pathways (e.g., syntenin–ALIX, tetraspanins, ceramide formation).
RNA Sorting: RNAs may be sorted based on their location at ER membrane contact sites with EV biogenesis membranes, but the mechanisms for their transport to vesicular lumens are still unclear.
Cargo Location: The positioning of cargo on the plasma membrane or endosome can affect whether it is incorporated into ectosomes or exosomes.
What does ESCRT stand for?
Endosomal sorting complex responsible for transport
Tell me more about ESCRT functions?
Sorting ubiquinated proteins into vesicles, best studied pathway
Consists of 4 multi-protein complexes
Involved in both exosome and ecotosome biogenesis
It helps regulate membrane dynamics, such as inward budding and vesicle scission
What about the role of lipids in EVs?
While exosomes originating from cholesterol-rich MVBs are determined for secretion, a low cholesterol level directs MVBs to lysosomal degradation
What kind of proteins are there in EVs?
cytokines, growth factors, antigen presentation related proteins, heat-shock proteins, tetraspanins, cell-adhesion related proteins
EVs are rich in what kind of lipids?
cholesterol, ceramide (most abundant lipid)
How do EVs deliver their cargo?
clathrin-dep or indep endocytosis, phagocytosis, micropinocytosis and/or endosomal membrane fusion
When ESCRT pathway is used, are they kept inside the EV and then brought into the cell?
Although these ESCRT subunits are released into the cytosol for recycling, some ESCRT components and accessory proteins, such as TSG101, HRS, and ALIX, remain in exosomes.
Do all proteins require ubiquitylation to be sorted into exosomes?
no
What kind of proteins are in EVs as markers?
ESCRT machinery-associated proteins (such as TSG101, HRS, and ALIX), which are involved in MVB synthesis, and tetraspanins (such as CD9, CD63, and CD81)
What are the two sub-classes of EVs released by neutrophils, and how do they differ in function?
Neutrophil-derived trails (NDTRs): Formed by integrin-mediated interactions during neutrophil migration in response to vascular forces.
Neutrophil-derived microvesicles (NDMVs): Released by membrane blebbing after neutrophil activation, dependent on the PI3K pathway.
NDTRs have pro-inflammatory functions, while NDMVs are anti-inflammatory.
PMN-EVs can contain what?
adhesion molecules (integrins, selectins), Fc and complement receptors, neutrophil granule-associated molecules (e.g., MPO, elastase, and CD63), neutrophil cytosolic proteins (S100A8), and micro RNAs through which exo/ectosomes can influence other cells
Explain phagocytosis in EV recipient cell uptake
A specialized form of endocytosis primarily used by immune cells (like macrophages) to engulf and digest large particles
Explain macropinocytosis
A form of endocytosis that involves the nonspecific uptake of large volumes of extracellular fluid
Explain how some EVs can directly fuse with the cell membrane
The lipid bilayer of the vesicle fuses with the target cell’s membrane, releasing the vesicle’s contents into the cytosol without forming an endosome or other vesicle
Exosomes produced by which cells relevant to my project?
Neural cells, astrocytes, microglia, immune cells, trophoblasts
Aside from cytokines, what else could be in cargo in CA? (in preeclampsia)
-miRNA (impaired trophoblast invasion - preeclampsia)
- antiangiogenic factors (preclampsia)
Which miRNA in LPS induced neutrophil exosomes?
miRNA-122, reduced occcludin expression, increased permeability of BBB
explain TEM of EVs?
white (or light-colored) structures against a dark background
electrons dont penetrate the membrane bc it is lipid - less electron dense - so electrons pass thru
he uranyl acetate stain surrounds the EVs, but due to the lipid bilayer membrane, the stain doesn’t penetrate deeply into the vesicles. In summary, when the electron beam passes through a sample stained with uranyl acetate, the dense regions (e.g., the outer membrane of EVs) scatter electrons, creating a dark outline, while the inner, unstained regions of the EVs appear lighter or transparent in the TEM image
what is acute phase response signaling
immediat response to inflammation , production of acute pharsre response proteins - c reactive protein
what is ferroptosis signaling
iron overload cell death lipd peroxidation, cell membranes become damaged by ROS
