Lysosomal Function Analysis

Why is Lysosomal Function Important?

The lysosome has been revealed to be an important organelle with a complex role in nutrient sensing and multifaceted signaling. Its importance has been emphasized in research fields as diverse as a neurological disease, cancer, immunity, and senescence.

Recent research reveals that lysosome acidification declines in neurons well before extracellular amyloid deposition, thus lysosomal function is now a hot topic in Alzheimer's disease research.

Hot Topic

Various Functional Pathways in Lysosomes

Lysosomes play a critical role in cellular metabolism and waste management. They are essential for maintaining cellular health and preventing the buildup of cellular waste, which can lead to disease and dysfunction. Understanding the functions of lysosomes is critical for developing treatments for a variety of diseases and disorders. Today, we introduce you to three highlighted articles focusing on lysosomal exocytosis, lysosomal AMPK pathway, and lysosomal pH optimization.
Lysosomal exocytosis in synucleinopathy models Metformin and lysosomal AMPK pathway Lysosomal pH optimum and Parkinson's disease
Lysosomal exocytosis releases pathogenic α-synuclein species from neurons in synucleinopathy models
(Ying Xue Xie, et al., Nature Communications, 13, 4918, 2022)		 Low-dose metformin targets the lysosomal AMPK pathway through PEN2
(Teng Ma, et al., Nature, 603, 159-165, 2022)		 Parkinson’s disease-risk protein TMEM175 is a proton-activated proton channel in lysosomes
(Meiqin Hu, et al., Cell, 185, 2292-2308, 2022)
  • - Pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons.
  • - Neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis
  • - The released aggregates are non-membrane enveloped and seeding-competent
  • - This release is dependent on neuronal activity and cytosolic Ca2+
  • - PEN2 is a binding partner of metformin with a dissociation constant at micromolar levels
  • - Metformin-bound PEN2 forms a complex with ATP6AP1 which leads to the inhibition of v-ATPase and the activation of lysosomal AMPK
  • -  In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects.
  • -  Knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan
  • - TMEM175 is the proton “leak” channel of lysosomes and endosomes
  • - TMEM175 is a highly proton-selective channel that is gated by luminal protons
  • - An endogenous lipid can also activate TMEM175 to trigger lysosomal proton release
  • - TMEM175 sets the lysosomal pH optimum via a classic negative feedback mechanism
Related Technique in This Topic
           Lysosomal function assay Lysosomal pH and mass detection Kit HOT
           Lysosome staining pH-dependent (Red)​ and pH-independent (Green / Deep Red) probes HOT
           Autophagy detection DAPGreen / DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
           Endocytosis detection  ECGreen 
           Endocytic internalization assay AcidSensor Labeling Kit HOT
           Extracellular vesicles labeling ExoSparkler Exosome Membrane Labeling Kit-Green, Red, Deep Red
           Extracellular vesicles Isolation ExoIsolator Exosome Isolation Kit HOT
           Total ROS detection High Sensitive DCFH-DA HOT or Compatible with Immunostaining HOT

 

Learn more about application data with multiple products here

 

 

 


Lysosomal pH Detection Dye: pHLys Red
pHLys Red has a high sensitivity to lysosomal pH change with more precise localization, which allows slight pH changes to be detected in physiological conditions like the early phase of the disease.

 

 

Lysosomal Mass Detection Dye: LysoPrime Green, Deep Red
Dojindo’s LysoPrime Green / Deep Red more precisely detects lysosomal mass due to their unique specificity for lysosomes, and retention independent of lysosomal pH. In addition, the retention time of the dyes in lysosomes has been extended, allowing data to be acquired over time.

 

Recommended for First Time Users
Lysosomal Acidic pH Detection Kit
Two types of dyes and lysosomal pH acidification inhibitors are available as a set. Positive control is included for immediate testing in your experimental system.


- Lysosomal mass detecting dye: LysoPrime Green
- Lysosomal pH detection dye: pHLys Red
- Lysosomal pH acidification inhibitor: Bafilomycin A1

Application data

 

Endocytic vesicles were labeled by ECGreen and the lysosomal mass and pH were detected separately with LysoPrime Deep Red and pHLys Red. Co-staining with ECGreen and Lysosomal dyes showed the inhibition of endocytic vesicle-fusion induced by Bafilmycin A1.

 

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by mtSOX Deep Red - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with mtSOX and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

 

 

Characterization of Each Dye

 

 

 

Product

Unit

Code

LysoPrime Green – High Specificity and pH Resistance

1 tube (35 mm dish x 10)

L261-10

 

3 tubes (35 mm dish x 30)

L261-12

 
       

LysoPrime Deep Red – High Specificity and pH Resistance

1 tube (35 mm dish x 10) 

L264-10

3 tubes (35 mm dish x 30)

L264-12

 
       

pHLys Red – Lysosomal Acidic pH Detection

1 tube (35 mm dish x 10)

L265-10

 

3 tubes (35 mm dish x 30)

L265-12

 
       

Lysosomal Acidic pH Detection Kit

(LysoPrime Green + pHLys Red + Bafilomycin A1)

1 set (35 mm dish x 10)

L266-10

 

 

 

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