EVs

Question 1

What is the ESCRT machinery for? What purpose

Answer 1

Biogenesis, trafficking and release of EVs (vesicle formation and cargo sorting)

Question 2

How do recipient cells incorporate EVs?

Answer 2

• receptor/kigand interaction
• endocytosis
• phagocytosis
• micropinocytosis
• fusion with cell membrane

Question 3

Explain endocytosis of recipient cells of EVs?

Answer 3

• 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

Question 4

Biogenesis of microvesicles?

Answer 4

Released via outward budding of the cell membrane (100-1000nm)

Question 5

Explain what endocytosis is (for recipient cell uptake of EVs)

Answer 5

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.

Question 6

Mechanism of biogenesis/cargo sorting of exosomes (EXTRACELLULAR, lots of DEETS)

Answer 6

1. 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
2. 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.

Question 7

What other EV cargo sorting/biogenesis mechanisms are there? (EXTRACELLULAR)

Answer 7

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)

Question 8

Basic mechanism of cargo selection of EVs? (EXTRACELLULAR, BASIC CONCEPT)

Answer 8

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).

Question 9

What about mechanism of sorting INTRACELLULARLY? (RNA)

Answer 9

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).

Question 10

Is there DNA in small EVs?

Answer 10

Small EVs can contain fragmented nuclear DNA, but mechanisms for DNA recruitment to these vesicles are largely unknown.

Question 11

Overall, what is EV cargo sorting regulated by? Many factors. Summary

Answer 11

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.

Question 12

What does ESCRT stand for?

Answer 12

Endosomal sorting complex responsible for transport

Question 13

Tell me more about ESCRT functions?

Answer 13

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

Question 14

What about the role of lipids in EVs?

Answer 14

While exosomes originating from cholesterol-rich MVBs are determined for secretion, a low cholesterol level directs MVBs to lysosomal degradation

Question 15

What kind of proteins are there in EVs?

Answer 15

cytokines, growth factors, antigen presentation related proteins, heat-shock proteins, tetraspanins, cell-adhesion related proteins

Question 16

EVs are rich in what kind of lipids?

Answer 16

cholesterol, ceramide (most abundant lipid)

Question 17

How do EVs deliver their cargo?

Answer 17

clathrin-dep or indep endocytosis, phagocytosis, micropinocytosis and/or endosomal membrane fusion

Question 18

When ESCRT pathway is used, are they kept inside the EV and then brought into the cell?

Answer 18

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.

Question 19

Do all proteins require ubiquitylation to be sorted into exosomes?

Answer 19

no

Question 20

What kind of proteins are in EVs as markers?

Answer 20

ESCRT machinery-associated proteins (such as TSG101, HRS, and ALIX), which are involved in MVB synthesis, and tetraspanins (such as CD9, CD63, and CD81)

Question 21

What are the two sub-classes of EVs released by neutrophils, and how do they differ in function?

Answer 21

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.

Question 22

PMN-EVs can contain what?

Answer 22

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

Question 23

Explain phagocytosis in EV recipient cell uptake

Answer 23

A specialized form of endocytosis primarily used by immune cells (like macrophages) to engulf and digest large particles

Question 24

Explain macropinocytosis

Answer 24

A form of endocytosis that involves the nonspecific uptake of large volumes of extracellular fluid

Question 25

Explain how some EVs can directly fuse with the cell membrane

Answer 25

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

Question 26

Exosomes produced by which cells relevant to my project?

Answer 26

Neural cells, astrocytes, microglia, immune cells, trophoblasts

Question 27

Aside from cytokines, what else could be in cargo in CA? (in preeclampsia)

Answer 27

-miRNA (impaired trophoblast invasion - preeclampsia)
- antiangiogenic factors (preclampsia)

Question 28

Which miRNA in LPS induced neutrophil exosomes?

Answer 28

miRNA-122, reduced occcludin expression, increased permeability of BBB