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. 2023 Feb 20;31(5):1231–1250. doi: 10.1016/j.ymthe.2023.02.013

Table 1.

Regulators of EV biogenesis

Regulator Cell Intervention EVs studied Effect Reference
Exosomes
Endosome maturation
 RAB5 MCF7 overexpression (RAB5Q79L) Bulk ↓ EV secretion (syndecan, CD63, syntenin, ALIX) Baietti et al.19
mOli-neu + PLP overexpression (RAB5Q79L) Bulk ↓ EV release of PLP Trajkovic et al.20
 RAB7 MCF7 RNAi Bulk ↓ EV secretion (syndecan, CD63, syntenin) Baietti et al.19
MVB formation
ESCRT machinery
 ESCRT-0
 - HRS HeLa-CIITA RNAi CD63 (CD81+/MHCII+) ↓ EV production Colombo et al.21
stimulated mDCs RNAi Bulk ↓ EV production Tamai et al.22
mBMDCs RNAi Bulk ↓ EV production Tamai et al.22
SCC25-H1047R RNAi Bulk ↓ EV production Hoshino et al.23
HeLa RNAi N/A ↑ MVB diameter Edgar et al.24
↓ ILVs per MVB
A431 RNAi N/A ↓ ILVs per MVB area Razi and Futter25
 - STAM1 HeLa-CIITA RNAi CD63 (CD81+/MHCII+) ↓ EV production Colombo et al.21
 ESCRT-I
 - TSG101 HeLa-CIITA RNAi CD63 (CD81+/MHCII+) ↓ EV production Colombo et al.21
HeLa RNAi N/A ↑ MVB diameter Edgar et al.24
↓ ILVs per MVB
A431 RNAi Na ↓ MVB formation Razi and Futter25
 ESCRT-III
 - CHMP6 HeLa GFP-CHMP4B RNAi bulk ↓ EV proteins (CD9, CD63, CD81, syntenin) Larios et al.26
 VPS4B HeLa-CIITA RNAi CD63 (CD81+/MHCII+) ↑ EV production Colombo et al.21
HeLa overexpression (hVPS4E223Q) N/A ↓ ILVs per MVB Sachse et al.27
Tetraspanins
 CD82/CD9 HEK293T overexpression (CD82/CD9) bulk ↑ EV production; EV β-catenin Chairoungdua et al.28
 CD9 HEK293 CD9−/−/- overexpression (CD9YEVM)a bulk ↓ EV protein (CD9) Fordjour et al.12
 CD63 MNT-1 RNAi N/A ↓ ILVs per MVB van Niel et al.29
HEK293 KO bulk ↓ particles per cell Hurwitz et al.30
HEK293 CD63−/− overexpression (CD63Y235A) bulk ↑ EV protein (CD63) Fordjour et al.12
Syndecan-syntenin-ALIX
 Syndecan MCF7 RNAi bulk ↓ EV production Baietti et al.19
 Syntenin MCF7 RNAi bulk ↓ EV production Baietti et al.19
MCF7 Overexpression bulk ↑ EV production Baietti et al.19
 - ARF6 MCF7 RNAi bulk ↓ EV proteins (syntenin, ALIX, CD63, SDC1CTF) Ghossoub et al.31
MCF7 overexpression (ARF6T157N) bulk ↑ EV proteins (syntenin, ALIX, CD63) Ghossoub et al.31
 -- PLD2 MCF7 RNAi bulk ↓ EV proteins (syntenin, ALIX, CD63) Ghossoub et al.31
 ALIX HeLa-CIITA RNAi CD63 (CD81+/MHCII+) ↑ MHCII on EVs Colombo et al.21
MCF7 RNAi bulk ↓ EV production Baietti et al.19
HeLa GFP-CHMP4B overexpression (ALIXΔPRR) bulk ↑ EV proteins (CD9, CD63, CD81) Larios et al.26
HeLa GFP-CHMP4B RNAi bulk ↓ EV proteins (CD9, CD63, CD81, syntenin) Larios et al.26
Ceramide
 nSMase2 mOli-neu + PLP GW4869/spiroepoxide/glutathione/RNAi bulk ↓ EV release of PLP Trajkovic et al.20
HEK293T + CD82 GW4869 bulk ↓ EV proteins (flotillin, β-catenin) Chairoungdua et al.28
SCC25-H1047R GW4869 bulk ↓ EV production Hoshino et al.23
HEK293T GW4869/RNAi bulk ↓ EV production Leidal et al.32
↓ EV proteins (LC3, SAFB, HNRNPK)
HeLa FLAG-RAB31Q65L GW4869 bulk ↓ EV proteins (FLAG, EGFR, FLOT1, FLOT2, CD9, CD81, CD63) Wei et al.33
ATGs
 ATG5 MEF, MDA-MB-231 KO bulk ↓ EV production Guo et al.34
↓ EV proteins (Flotillin2, Tsg101)
 ATG12-ATG3 MEF overexpression (ATG3K243R) bulk ↓ EV proteins (total; ALIX, TSG101, GAPDH) Murrow et al.35
Transport
Cortactin SCC61 RNAi bulk ↓ EV production Sinha et al.36
↓ EV proteins (TSG101, CD63, Flotillin1)
SCC61 Overexpression bulk ↑ EV production Sinha et al.36
RAB GTPases
 RAB11 K562 overexpression (RAB11S25N) bulk ↓ EV proteins (TfR, Lyn, Hsc70) Savina et al.37
↓ EV AChE activity
 RAB35 mOli-neu + PLP overexpression (RAB35N120I), RNAi bulk ↓ EV release of PLP Hsu et al.38
 RAB27A, RAB27B HeLa RNAi bulk ↓ EV production Ostrowski et al.39
↓ EV proteins (HLA-DR, CD63, TSG101, HSC70)
 RAB27A B16-F10,SK-Mel-28 RNAi bulk ↓ EV protein (total) Peinado et al.40
 RAB27A TS/A, 4T1 RNAi bulk ↓ EV proteins (total; ALIX, HSC70, CD63, TSG101) Bobrie et al.41
Fusion
 VAMP7 K562 overexpression (NT-VAMP7) bulk ↓ EV production Fader et al.42
↓ EV AChE activity
 YKT6 HEK293 RNAi bulk ↓ EV proteins (WNT3A, CD81) Gross et al.43
A549 RNAi bulk ↓ EV protein (TSG101) Ruiz-Martinez et al.44
 SNAP23 A549 RNAi bulk ↓ EV production Wei et al.45
HeLa + histamine + CD63-pHluorin RNAi CD63-pHluorin ↓ fusion activity Verweij et al.46
 Syntaxin-4 HeLa + histamine + CD63-pHluorin RNAi CD63-pHluorin ↓ fusion activity Verweij et al.46
 RAL1 4T1 RNAi bulk ↓ EV production Hyenne et al.47
↓ EV proteins (ALIX, CD63, TSG101, HSC70)
 RalA/B 4T1 RNAi bulk ↓ EV production Ghoroghi et al.48
 Calcium K562 monensin bulk ↑ EV protein (TfR, Hsc70) Savina et al.49,50
↑ EV AChE activity
Microvesicles
ARF6 LOXARF6-GTP overexpression (ARF6Q67L) bulk ↑ EV protein (total) Muralidharan-Chari et al.51
RhoA HeLa + EGF overexpression (RhoAF30L) bulk ↑ EV production Li et al.52
↑ GFP release in EVs
ARF1 MDA-MB-231 RNAi bulk ↓ EV protein (total) Schlienger et al.53
↓ EV MMP9 activity
ASM astrocytes imipramine bulk ↓ EV fluorescence Bianco et al.54
N9 r-SMase bulk ↑ EV fluorescence Bianco et al.54
RBCs amitriptyline Annexin V ↓ EV percentage Awojoodu et al.55
ARRDC1 HEK293T overexpression (ARRDC1-GFP) bulk ↑ GFP release in EVs Nabhan et al.56

For each study, the producer cell type and, if relevant, any applied stimuli are mentioned. Moreover, the method of intervention, the investigated EV population, and the impact the intervention had on EV biogenesis are stated. EVs were predominantly studied in bulk and only a few studies looked at specific subpopulations.

Regulators: ALIX, programmed cell death 6-interacting protein; ARF, ADP ribosylation factor; ARRDC1, arrestin-domain-containing protein 1; ASM, acid sphingomyelinase; ATG, autophagy-related protein; CHMP, charged multivesicular body protein; ESCRT, endosomal sorting complexes required for transport; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; nSMase2, neutral sphingomyelinase 2; PLD, phospholipase D; RAB, Ras-associated binding protein; RAL, Ras-like protein; Rho, Ras homologous protein; SNAP, synaptosomal-associated protein; STAM1, signal-transducing adaptor molecule 1; TSG101, tumor susceptibility gene 101; VAMP, vesicle-associated membrane protein; VPS4B, vacuolar protein sorting-associated protein 4B; YKT6, synaptobrevin homolog. Cells: MCF-7, human breast cancer cell line; 4T1, murine breast cancer cell line; A431, human epidermoid squamous carcinoma cell line; B16-F10, murine melanoma cell line; CIITA, major histocompatibility complex class II transactivator; HEK, human embryonic kidney cell line; HeLa, human cervical cancer cell line; K562, human myelogenous leukemia cell line; LOX, human melanoma cell line; mBMDCs, murine bone marrow-derived dendritic cells; MDA-MB-231, human breast cancer cell line; mDCs, murine dendritic cells; MEF, mouse embryonic fibroblasts; mOli-neu, murine oligodendroglial precursor cell line; N9, microglial cell line; RBCs, red blood cells; SCC, squamous cell carcinoma cell line; SK-MEL-28, human melanoma cell line; TS/A, murine mammary adenocarcinoma cell line. Others: ↓, reduction; ↑, increase; AChE, acetylcholinesterase; EGFR, epidermal growth factor receptor; FLOT, flotillin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLA-DR, human leukocyte antigen DR; HNRNPK, heterogeneous nuclear ribonucleoprotein K; HSC70, heat-shock 70 kDa protein; ILV, intraluminal vesicle; KO, knockout; LC3, microtubule-associated protein 1A/1B-light chain 3; Lyn, tyrosine-protein kinase; MHCII, major histocompatibility class II; MMP9, metalloproteinase 9; MV, microvesicle; MVB, multivesicular body; N/A, not applicable; PLP, proteolipid protein; RNAi, RNA interference; r-SMase, recombinant sphingomyelinase; SAFB, scaffold-attachment factor B; SDC1CTF, syndecan-1 cytoplasmic fragment; TfR, transferrin receptor; WNT3A, Wnt family member 3A.

a

CD9 mutant that carries endosome-targeting signal.