Science Note: Exosome / Endocytosis / Phagocytosis

Immune cell senescence is a critical factor in the progression of several diseases. As immune cells age and enter a senescent state, their ability to respond to infection, eliminate senescent cells, and regulate inflammation diminishes. This impaired immune function may contribute to the development and progression of age-related diseases, including cancer, cardiovascular disease, and neurodegenerative disorders. In addition, the accumulation of senescent immune cells in tissues can create a pro-inflammatory environment that further exacerbates disease pathology and impairs tissue function.

Senescence drives immunotherapy resistance by inducing an immunosuppressive tumor microenvironment
Click here for the original article: Damien Maggiorani, et. al., Nature Communications, 2024.

Senescent alveolar macrophages promote early-stage lung tumorigenesis
Click here for the original article: Luis I. Prieto et. al., Cancer Cell, 2023.

Rejuvenating aged microglia by p16ink4a-siRNA-loaded nanoparticles increases amyloid-β clearance in animal models of Alzheimer’s disease
Click here for the original article: Hyo Jung Shin et. al., Molecular Neurodegeneration, 2024.

Point of Interest
- Immunotherapy resistance is associated with reduced CD8 T cell activity in tumors.

- Elimination of senescent cells restores CD8 T cell proliferation and reduces immunotherapy resistance.

- Anti-senescent cell drugs prior to immune checkpoint inhibitors may enhance the effectiveness of cancer immunotherapy.

Point of Interest

- Senescent alveolar macrophages accumulate early in lung cancer neoplasia and suppress cytotoxic T-cell responses.

- Removal of these macrophages reduces adenoma development, indicating their role in promoting tumorigenesis.

- Targeting senescent macrophages may slow lung cancer progression by altering the local microenvironment.

Point of Interest

- Reduced amyloid clearance in Alzheimer's disease is associated with decreased phagocytic ability of older microglia.

- Downregulation of p16ink4a, a cell cycle factor associated with microglial aging, by siRNA increases amyloid clearance by phagocytosis and reverses cognitive deficits in Alzheimer's disease mode.

- Targeting p16ink4a with nanoparticles to rejuvenate microglia offers a potential treatment strategy for Alzheimer's disease.

Phagocytosis assay of labeled apoptotic cells in THP-1 cells

AcidSensor-labeled substances are taken up by cells and their fluorescence increases when they reach acidic organelles such as lysosomes. Taking advantage of this property, we evaluate the phagocytic activity of apoptotic cells by co-culturing AcidSensor-labeled apoptotic cells with Calcein-labeled THP-1 macrophages.  As a result, Calcein (Green) / AcidSensor (Deep red) double-positive cells, indicating THP-1 macrophages phagocytosing apoptotic cells, were observed by flow cytometry (Fig. 1a). Furthermore, when the phagocytosis of THP-1 macrophages was inhibited by Cytochalasin D, the percentage of double-positive cells decreased (Fig. 1b and 1c), confirming that the assay system can accurately evaluate phagocytosis.

A recent report reveals that inhibition of mitochondrial function induces a switch to glycolysis and reduces phagocytosis in cultured microglia, resident macrophages in the central nervous system*. To replicate this result, phagocytosis assays were performed using mitochondria-inhibited THP-1 macrophages. The results show that FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, decreases mitochondrial membrane potential (MT-1, Red) of THP-1 macrophages (Fig. 2) and reduces phagocytosis (Fig. 3).

*Lauren H. Fairley, et al., PNAS (2023)

Products in Use
① AcidSensor Labeling Kit – Endocytic Internalization Assay [code: A558]
② -Cellstain- Calcein-AM solution [code: C396]
③ MT-1 MitoMP Detection Kit [code: MT13]

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Endocytosis induction refers to the initiation of the endocytic process, where specific signals trigger the cell to internalize extracellular substances. This induction often involves membrane proteins that can act as receptors to recognize and bind external ligands, initiating the formation of endocytic vesicles. The interaction between ligands and membrane proteins not only triggers endocytosis, but also ensures that the internalized cargo is selectively processed. Thus, membrane proteins are integral to both the initiation and specificity of endocytosis, influencing cellular uptake and signaling pathways.

Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation
Click here for the original article: Ryouhei Tsutsumi, et. al., Nature Communications, 2024.

Endocytosis blocks the vesicular secretion of exosome marker proteins
Click here for the original article: Yiwei Ai et. al., Science Advances, 2024.

Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis
Click here for the original article: David Paul et. al., Nature Communications, 2023.

Point of Interest
- Platelet-derived growth factor(PDGF) enhances cellular glucose uptake via receptor endocytosis and independently of known mechanisms, resulting in a near doubling of glucose uptake by the cells.

- PDGF receptor (PDGFR) co-endocytoses with subset of glucose transporter 1 (GLUT1/SLC2A1) upon PDGF-stimulation.

- The PDGFR/GLUT1-containing endosomes have multiple glycolytic enzymes and localize to adjacent mitochondria.

- The glucose-loaded endosomes generated by growth factors deliver glucose to the glycolytic machinery in proximity to mitochondria.

Point of Interest
- Endocytosis inhibition induces plasma membrane accumulation and vesicular secretion of CD63.

- High expression of CD63, CD81 or CD9 inhibits its own endocytosis and induces its plasma membrane accumulation and vesicular secretion.

- Induction of endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, causes their destruction in the lysosome.

- Vesicular secretion of exosome marker proteins occurs primarily through an endocytosis-independent pathway.

Point of Interest
- Aggregation of protein ectodomains by cross-linking antibodies ability triggers specific and fast endocytosis of the receptor, independent of clathrin and dynamin.

- Upon aggregation, even canonical clathrin-dependent cargoes are redirected to the aggregation-dependent endocytosis (ADE) pathway.

- ADE is an actin-driven process that morphologically resembles macropinocytosis.

- ADE clears stress-induced receptor aggregates and facilitates their lysosomal degradation to maintain cell surface proteostasis.

Clear visualization of intracellular vesicular trafficking

Wortmannin is known to inhibit endosomal recycling and lysosomal translocation, leading to endosomal enlargement.
These changes induced by Wortmannin were confirmed by co-staining with ECGreen (green) and the following indicators.

①Eary endosome: Rab5-RFP (red)
② Recycling endosome: Fluorescent labeled Transferin (red)
③ Late endosome: Rab5-RFP (red)
④ Lysosome: Lamp1-RFP (red)

As a result, it was confirmed that ECGreen (green) co-localizes only with enlarged early endosomes and recycling endosomes (Fig. ① and ②), but not with late endosomes or Lysosomes (Fig. ③ and ④), supporting Wortmannin's effect. ECGreen can visualize changes in the intracellular vesicular trafficking system and endosome shape.

Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 – 560 nm
Early endosomes (Rab5-RFP, red): Ex. 561 nm / Em. 560 – 620 nm
Recycling endosome (Transferrin-Alexa fluor 488 conjugate, red: pseudo-color): Ex. 488 nm / Em. 500 – 550 nm
Late endosomes (Rab7-RFP, red): Ex. 561 nm / Em. 560 – 620 nm
Lysosomes (Lamp1-RFP, red): Ex. 561 nm / Em. 560 – 620 nm

[Experimental Procedure]
(1) Prepare HeLa cells in 8 wells of μ-Slide and incubate overnight.
(2) After washing with HBSS, 200 µl of Wortmannin (final concentration: 100 nmol/l) prepared in 10% FBS-containing MEM medium was added.
(3) Incubate at 37°C for 30 minutes
(4) 200 µl of ECGreen (diluted 1,000-fold) prepared in 10% FBS-containing MEM medium without removing the supernatant
(5) Incubate at 37°C for 30 minutes
(6) Wash the cells twice with HBSS and add MEM medium containing 10% FBS.
(7) Observation with a confocal laser microscope

Phagocytosis assay of labeled apoptotic cells in THP-1 cells

AcidSensor-labeled substances are taken up by cells and their fluorescence increases when they reach acidic organelles such as lysosomes. Taking advantage of this property, we evaluate the phagocytic activity of apoptotic cells by co-culturing AcidSensor-labeled apoptotic cells with Calcein-labeled THP-1 macrophages.  As a result, Calcein (Green) / AcidSensor (Deep red) double-positive cells, indicating THP-1 macrophages phagocytosing apoptotic cells, were observed by flow cytometry (Fig. 1a). Furthermore, when the phagocytosis of THP-1 macrophages was inhibited by Cytochalasin D, the percentage of double-positive cells decreased (Fig. 1b and 1c), confirming that the assay system can accurately evaluate phagocytosis.

A recent report reveals that inhibition of mitochondrial function induces a switch to glycolysis and reduces phagocytosis in cultured microglia, resident macrophages in the central nervous system*. To replicate this result, phagocytosis assays were performed using mitochondria-inhibited THP-1 macrophages. The results show that FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, decreases mitochondrial membrane potential (MT-1, Red) of THP-1 macrophages (Fig. 2) and reduces phagocytosis (Fig. 3).

*Lauren H. Fairley, et al., PNAS (2023)

Products in Use
① AcidSensor Labeling Kit – Endocytic Internalization Assay [code: A558]
② -Cellstain- Calcein-AM solution [code: C396]
③ MT-1 MitoMP Detection Kit [code: MT13]

[Experimental Procedure]

Preparation of AcidSensor-labeled apoptotic cells (day before assay)

1. Add 10 μl of DMSO to NH₂-Reactive AcidSensor and dissolve. 5 μl NH₂-Reactive AcidSensor solution was added to 5 ml HBSS to make the Working solution (1000-fold dilution).
2. Wash Jurkat cells twice with HBSS.
3. Jurkat cells (5×10⁷ cells) were transferred to the tube and the supernatant was removed after centrifugation.
4. Add the Working solution to the tube with Jurkat cells (5×10⁷ cells) and suspend.
5. Incubate at 37°C for 30 minutes for labeling with AcidSensor.
6. After labeling, the cells were washed twice with HBSS.
7. AcidSensor-labeled Jurkat cells were suspended in RPMI medium with 10% FBS added with 0.5 μM staurosporine.
8. Apoptosis was induced by o/n culture in an incubator (37°C, 5% CO₂) for 18 hours.
9. After induction of apoptosis, the cells were washed with culture medium and used for the phagocytosis assay.

Phagocytosis assay using THP-1 macrophages

1. To differentiate THP-1 cells into macrophages, THP-1 cells were seeded into 6 well plates at 1x10⁶ cells/well and incubated with 100 nM PMA for 3 days in an incubator.
2. After washing THP-1 macrophages twice with HBSS, Calcein-AM (Code: C396, 0.5 μg/ml) and MT-1 (Code: MT13, 1000-fold dilution) included HBSS solution was added to the wells and incubated in the incubator for 30 minutes.
3. Wash twice with a culture medium.
4. The cells were incubated with 10 μM Cytochalasin D for 1 hour or 5 μM FCCP for 30 minutes in the incubator.
5. After twice washing with culture medium, AcidSensor-labeled apoptotic cells (3x10⁶ cells/well) were added to the well with or without Cytochalasin D or FCCP. The cells were incubated in the incubator for 4 hours.
6. Wash twice with HBSS.
7. Add 500 μl Imaging buffer solution (Code: MT13) and collect THP-1 macrophages from plates with a cell scraper.
8.Cell suspensions were analyzed by flow cytometry.

Membrane trafficking involves vital processes such as phagocytosis, endocytosis, and exocytosis, each of which plays a critical role in cellular homeostasis. Phagocytosis and endocytosis allow cells to ingest external particles and fluids, facilitating nutrient uptake and the removal of pathogens or debris, which is critical for cellular health and immune response. Exocytosis, on the other hand, allows cells to expel waste products and secrete signaling molecules that are essential for communication and functional coordination within tissues. These coordinated processes ensure that the cell maintains a balanced internal environment, adapts to changing external conditions, and maintains cellular integrity and function. 

CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma
Click here for the original article: Dan Chen, et. al., Immunity, 2023.

Stress granules plug and stabilize damaged endolysosomal membranes
Click here for the original article: Claudio Bussi et. al., Nature, 2024.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion
Click here for the original article: Di-Ao Liu et. al., Nature Communications, 2023.

Point of Interest
- αCTLA-4 therapy promotes the induction of protective CD4⁺ Th1 cells against mesenchymal glioblastomas.

- IFNγ, derived from Th1 cells, stimulates the induction of MHC-II+ microglia, which sustains Th1-mediated anti-glioma immunity.

- Th1 cells enhance microglial phagocytosis of gliomas through AXL-MER receptor signaling.

- The presence of microglia and MHC-II expression are associated with protective outcomes in human glioblastoma.

Point of Interest
- Stress granule formation and membrane stabilization allow efficient repair of damaged endolysosomes.

- This process requires both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms.  

- Blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, which exploits endomembrane damage to survive in the host.

Point of Interest
- A sequential conversion of phosphatidylinositol 3-phosphate (PI(3)P) to phosphatidylinositol 4-phosphate (PI(4)P) catalyzed by myotubularin 1.

- Phosphatidylinositol 4-kinase type IIα on the surface of MVEs mediates the recruitment of the exocyst complex.

- The exocyst targets MVEs to the plasma membrane for exosome secretion.

- Disruption of PI(4)P generation or exocyst function blocked exosomal secretion of programmed death ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes.

Phagocytosis assay of labeled apoptotic cells in THP-1 cells

AcidSensor-labeled substances are taken up by cells and their fluorescence increases when they reach acidic organelles such as lysosomes. Taking advantage of this property, we evaluate the phagocytic activity of apoptotic cells by co-culturing AcidSensor-labeled apoptotic cells with Calcein-labeled THP-1 macrophages.  As a result, Calcein (Green) / AcidSensor (Deep red) double-positive cells, indicating THP-1 macrophages phagocytosing apoptotic cells, were observed by flow cytometry (Fig. 1a). Furthermore, when the phagocytosis of THP-1 macrophages was inhibited by Cytochalasin D, the percentage of double-positive cells decreased (Fig. 1b and 1c), confirming that the assay system can accurately evaluate phagocytosis.

A recent report reveals that inhibition of mitochondrial function induces a switch to glycolysis and reduces phagocytosis in cultured microglia, resident macrophages in the central nervous system*. To replicate this result, phagocytosis assays were performed using mitochondria-inhibited THP-1 macrophages. The results show that FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, decreases mitochondrial membrane potential (MT-1, Red) of THP-1 macrophages (Fig. 2) and reduces phagocytosis (Fig. 3).

*Lauren H. Fairley, et al., PNAS (2023)

Products in Use
① AcidSensor Labeling Kit – Endocytic Internalization Assay [code: A558]
② -Cellstain- Calcein-AM solution [code: C396]
③ MT-1 MitoMP Detection Kit [code: MT13]

[Experimental Procedure]

Preparation of AcidSensor-labeled apoptotic cells (day before assay)

1. Add 10 μl of DMSO to NH₂-Reactive AcidSensor and dissolve. 5 μl NH₂-Reactive AcidSensor solution was added to 5 ml HBSS to make the Working solution (1000-fold dilution).
2. Wash Jurkat cells twice with HBSS.
3. Jurkat cells (5×10⁷ cells) were transferred to the tube and the supernatant was removed after centrifugation.
4. Add the Working solution to the tube with Jurkat cells (5×10⁷ cells) and suspend.
5. Incubate at 37°C for 30 minutes for labeling with AcidSensor.
6. After labeling, the cells were washed twice with HBSS.
7. AcidSensor-labeled Jurkat cells were suspended in RPMI medium with 10% FBS added with 0.5 μM staurosporine.
8. Apoptosis was induced by o/n culture in an incubator (37°C, 5% CO₂) for 18 hours.
9. After induction of apoptosis, the cells were washed with culture medium and used for the phagocytosis assay.

Phagocytosis assay using THP-1 macrophages

1. To differentiate THP-1 cells into macrophages, THP-1 cells were seeded into 6 well plates at 1x10⁶ cells/well and incubated with 100 nM PMA for 3 days in an incubator.
2. After washing THP-1 macrophages twice with HBSS, Calcein-AM (Code: C396, 0.5 μg/ml) and MT-1 (Code: MT13, 1000-fold dilution) included HBSS solution was added to the wells and incubated in the incubator for 30 minutes.
3. Wash twice with a culture medium.
4. The cells were incubated with 10 μM Cytochalasin D for 1 hour or 5 μM FCCP for 30 minutes in the incubator.
5. After twice washing with culture medium, AcidSensor-labeled apoptotic cells (3x10⁶ cells/well) were added to the well with or without Cytochalasin D or FCCP. The cells were incubated in the incubator for 4 hours.
6. Wash twice with HBSS.
7. Add 500 μl Imaging buffer solution (Code: MT13) and collect THP-1 macrophages from plates with a cell scraper.
8.Cell suspensions were analyzed by flow cytometry.

Scientists have discovered that Microglia respond to amyloid-beta through the activation of signaling mechanisms mediated by TREM2, involving Syk and DAP10. In mice expressing the Alzheimer’s disease-associated human TREM2R47H allele, antibody-mediated activation of Syk rescues microglial activation.

TREM2 drives microglia response to amyloid-β via SYK-dependent and -independent pathways
Click here for the original article: Shoutang Wang, et. al., Cell, 2022.

Point of Interest
- Microglia must activate SYK to limit Aβ pathology.
- SYK maintains microglial fitness through the PI3K-AKT-GSK-3β-mTOR pathway.
- TREM2 activates microglia through SYK and SYK-independent, DAP10-dependent pathways.
- Direct activation of SYK by anti-CLEC7A rescues TREM2-depleted microglia.

Related Techniques

Related Applications

Phagocytosis assay of labeled apoptotic cells in THP-1 cells

[Experimental Procedure]

Preparation of AcidSensor-labeled apoptotic cells (day before assay)

1. Add 10 μl of DMSO to NH₂-Reactive AcidSensor and dissolve. 5 μl NH₂-Reactive AcidSensor solution was added to 5 ml HBSS to make the Working solution (1000-fold dilution).
2. Wash Jurkat cells twice with HBSS.
3. Jurkat cells (5×10⁷ cells) were transferred to the tube and the supernatant was removed after centrifugation.
4. Add the Working solution to the tube with Jurkat cells (5×10⁷ cells) and suspend.
5. Incubate at 37°C for 30 minutes for labeling with AcidSensor.
6. After labeling, the cells were washed twice with HBSS.
7. AcidSensor-labeled Jurkat cells were suspended in RPMI medium with 10% FBS added with 0.5 μM staurosporine.
8. Apoptosis was induced by o/n culture in an incubator (37°C, 5% CO₂) for 18 hours.
9. After induction of apoptosis, the cells were washed with culture medium and used for the phagocytosis assay.

Phagocytosis assay using THP-1 macrophages

1. To differentiate THP-1 cells into macrophages, THP-1 cells were seeded into 6 well plates at 1x10⁶ cells/well and incubated with 100 nM PMA for 3 days in an incubator.
2. After washing THP-1 macrophages twice with HBSS, Calcein-AM (Code: C396, 0.5 μg/ml) and MT-1 (Code: MT13, 1000-fold dilution) included HBSS solution was added to the wells and incubated in the incubator for 30 minutes.
3. Wash twice with a culture medium.
4. The cells were incubated with 10 μM Cytochalasin D for 1 hour or 5 μM FCCP for 30 minutes in the incubator.
5. After twice washing with culture medium, AcidSensor-labeled apoptotic cells (3x10⁶ cells/well) were added to the well with or without Cytochalasin D or FCCP. The cells were incubated in the incubator for 4 hours.
6. Wash twice with HBSS.
7. Add 500 μl Imaging buffer solution (Code: MT13) and collect THP-1 macrophages from plates with a cell scraper.
8. Cell suspensions were analyzed by flow cytometry.

Scientists have discovered that the mitochondrial translocator protein (TSPO) and hexokinase-2 play key roles in controlling microglial metabolism and phagocytosis. Microglia lacking TSPO resembled dysfunctional microglia observed in aging and Alzheimer's disease, and this could be partially reversed by blocking hexokinase-2 binding to mitochondria. They conclude that targeting mitochondrial hexokinase-2 binding may provide an immunotherapeutic approach to inhibit glycolytic metabolic reprogramming and promote microglial phagocytosis in Alzheimer's disease.

Mitochondrial control of microglial phagocytosis by the translocator protein and hexokinase 2 in Alzheimer’s disease
Click here for the original article: Lauren H. Fairley, et. al., PNAS, 2023.

Point of Interest
- The translocator protein (TSPO) is pivotal for respiratory metabolism and energy supply in microglia.
- Hexokinase-2 (HK) affects glycolytic metabolism and phagocytosis through its interaction with mitochondria.
- Mitochondrial HK influences glycolysis and inflammation, and its displacement improves phagocytosis in TSPO-deficient microglia.
- Alzheimer’s beta-amyloid drastically stimulated mitochondrial HK recruitment in cultured microglia, which may contribute to microglial dysfunction in Alzheimer’s disease.

Related Techniques

Related Applications

Phagocytosis assay of labeled apoptotic cells in THP-1 cells

[Experimental Procedure]

Preparation of AcidSensor-labeled apoptotic cells (day before assay)

1. Add 10 μl of DMSO to NH₂-Reactive AcidSensor and dissolve. 5 μl NH₂-Reactive AcidSensor solution was added to 5 ml HBSS to make the Working solution (1000-fold dilution).
2. Wash Jurkat cells twice with HBSS.
3. Jurkat cells (5×10⁷ cells) were transferred to the tube and the supernatant was removed after centrifugation.
4. Add the Working solution to the tube with Jurkat cells (5×10⁷ cells) and suspend.
5. Incubate at 37°C for 30 minutes for labeling with AcidSensor.
6. After labeling, the cells were washed twice with HBSS.
7. AcidSensor-labeled Jurkat cells were suspended in RPMI medium with 10% FBS added with 0.5 μM staurosporine.
8. Apoptosis was induced by o/n culture in an incubator (37°C, 5% CO₂) for 18 hours.
9. After induction of apoptosis, the cells were washed with culture medium and used for the phagocytosis assay.

Phagocytosis assay using THP-1 macrophages

1. To differentiate THP-1 cells into macrophages, THP-1 cells were seeded into 6 well plates at 1x10⁶ cells/well and incubated with 100 nM PMA for 3 days in an incubator.
2. After washing THP-1 macrophages twice with HBSS, Calcein-AM (Code: C396, 0.5 μg/ml) and MT-1 (Code: MT13, 1000-fold dilution) included HBSS solution was added to the wells and incubated in the incubator for 30 minutes.
3. Wash twice with a culture medium.
4. The cells were incubated with 10 μM Cytochalasin D for 1 hour or 5 μM FCCP for 30 minutes in the incubator.
5. After twice washing with culture medium, AcidSensor-labeled apoptotic cells (3x10⁶ cells/well) were added to the well with or without Cytochalasin D or FCCP. The cells were incubated in the incubator for 4 hours.
6. Wash twice with HBSS.
7. Add 500 μl Imaging buffer solution (Code: MT13) and collect THP-1 macrophages from plates with a cell scraper.
8. Cell suspensions were analyzed by flow cytometry.

Tumour extracellular vesicles and particles induce liver metabolic dysfunction

Click here for the original article: Gang Wang, et al., Nature, 2023

Uptake of tumor-derived microparticles induces metabolic reprogramming of macrophages in the early metastatic lung

Click here for the original article: Kelly Kersten, et al., Cell Reports, 2023

Delivery of low-density lipoprotein from endocytic carriers to mitochondria supports steroidogenesis

Click here for the original article: Yu-Xia Zhou, et al., Nature Cell Biology, 2023

- All subpopulations of tumor-derived extracellular vesicles and particles (EVPs) could dysregulate liver function.

- The fatty acid cargo of tumor EVPs induced secretion of tumor necrosis factor (TNF) by Kupffer cells, promoting fatty liver formation.

- Kupffer cell ablation or TNF blockade significantly reduced tumor-induced fatty liver formation.

- Ingestion of tumor-derived material leads to the phenotypic reprogramming of macrophages.

- The reprogramming of macrophages influences their patrolling behavior in response to tumor cells.

- ZsGreen+ macrophages demonstrate elevated mitochondrial metabolism, characterized by oxidative phosphorylation (OXPHOS).

- mTORC1 is essential for enhanced oxidative phosphorylation (OXPHOS) and ATP production in ZsGreen+ macrophages.

- PLD6 promotes the entrance of LDL and LDLR into the mitochondria.

- The fusogenic lipid phosphatidic acid generated by PLD6 facilitates the membrane fusion of LDLR vesicles with the mitochondria.

- This intracellular transport pathway of LDL–LDLR bypasses the lysosomes and delivers cholesterol to the mitochondria for steroidogenesis.

Visualization of EVs uptake via endocytic pathway

Observation of temperature-dependent endocytosis changes in floating cells

Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation
 Click here for the original article: Aya O Satoh, et. al., Cell Rep., 42, 112229 (2023)

   Point of Interest
   - VDAC2, a protein located on the outer membrane of mitochondria, interacts with PI3K, serving as a link between endosomes and mitochondria.
   - This interaction facilitates clathrin-independent endocytosis.
   - The binding of VDAC2 and PI3K triggers the maturation of endosomes that are connected to mitochondria.
   - Besides its structural role in facilitating this association, VDAC2 also has a functional role in promoting endosome maturation at these contact points.

Fatty acid starvation induced by uptake inhibitor evoke reprogramming of cellular metabolism 

Mitochondrial fatty acid β-oxidation and oxidative phosphorylation (OXPHOS) are crucial biochemical processes that metabolize fats and sugars to produce ATP, the cell's primary energy source. In this section, we underscored the significance of fatty acid starvation and energy pathways, with an emphasis on the fatty acid uptake inhibitor, FATP2. Here are the key findings from our experiments conducted on HeLa cells:

・Inhibition of fatty acid uptake results in reduced cell proliferation, though it does not lead to cell death. This was determined through the use of a Cell Counting Kit-8 and Fatty Acid Uptake Kit (Image 1).

・Fatty acid starvation shifts cellular metabolism from OXPHOS to glycolysis, as indicated by the Glycolysis/JC-1 MitoMP Assay Kit. (Image 2)

・When fatty acid uptake is inhibited, a compensatory increase in glucose and glutamine uptake occurs to preserve cell viability, as observed using the Glucose Assay Kit and Glutamine Assay Kit. (Image 3)

Products in Use
for Fatty Acid Uptake Assay
  ① Fatty Acid Uptake Assay Kit

for Cell Proliferation/Cytotoxicity Assay
  ② Cell Counting Kit-8

for Glycolysis Assay and Mitochondrial Membrane Potential Detection
  ③ Glycolysis/JC-1 MitoMP Assay Kit

for Glucose and Glutamine Consumption Assay
  ④ Glucose Assay Kit

  ⑤ Glutamine Assay Kit

• - EVs and their cargo miRNAs mediate adipose tissue-brain inter-organ communication are used in this study
• - Adipose tissue-derived EVs induce cognitive impairment associated with insulin resistance
• - miR-9-3p in adipose tissue-derived EVs mediates synaptic damage
• - Targeting adipose tissue-derived EVs or miRNAs prevents cognitive impairment in diabetes

• - The common view is that T lymphocytes activate telomerase to delay senescence
• - Upon contact with T cells, antigen-presenting cells (APCs) degraded shelterin to donate telomeres and then transferred in EVs at the immunological synapse
• - Antigen-specific populations of T cells decide aging fate, and it is based on telomere vesicle transfer upon initial contact with APCs
• - These telomere-acquiring T cells are protected from senescence, conferring long-lasting immune protection

• - Small EVs (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals brought an improvement in several parameters usually altered with aging
• - ADSC-sEVs induced pro-regenerative effects and a decrease in oxidative stress, inflammation, and senescence markers in muscle and kidney
• - Predicted epigenetic age was lower in tissues of old mice treated with ADSC-sEVs and their metabolome changed to a youth-like pattern
• - The microRNAs contained in sEVs might be responsible for the observed effects