Fluorescent Lysosome Probes

Lysosomes are membrane-bound cell organelles, which contain hydrolytic enzymes that breakdown many types of biomolecules. Lysosomes have several important functions: maintaining cellular homeostasis, removing cell waste, autophagy, regulated cell death, cell signaling, plasma membrane repair, cell adhesion, cell migration, and cell-metabolism regulation. Lysosomes are also a major immune defence against pathogens.

Cat. No. Product Name / Activity
7538 NBD-PE
Fluorescent lipid probe, quantitatively measure phospholipidosis
7314 Pepstatin A Janelia Fluor® 526
Fluorogenic green-emitting lysosome tracker and stain
7901 tri-GalNAc-C5-AF647
Fluorescent ASGPR ligand. Application: flow cytometry, fluorescence microscopy

Lysosomal Structure and Function

There are between 50 and 1000 lysosomes distributed throughout the cytoplasm of a typical animal cell, (note that primitive animal cells, fungi and plant cells have a similar although distinct set of organelles called vacuoles, which have a different enzyme make up and different functions to a lysosome). Lysosomes vary in size from 0.1 μm to 1.2 μm, depending on location and cellular function, although the factors regulating lysosome size remain unclear. Lysosomes are formed when a late endosome laden with material from the cell surface fuses with a transport vesicle from the trans-Golgi apparatus.

Lysosomes are comprised of vesicles encapsulated within a plasma membrane layer, containing over 50 membrane proteins that perform many different functions within the cell. Included among these membrane proteins are: the structural protein lysosome-associated membrane protein 1 (LAMP1); soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and RAB GTPases, which are involved in membrane fusion and vesicle trafficking; and LAMP2A, a transporter, which is involved in chaperone-mediated autophagy (CMA).

Lysosomes have a specialized acidic compartment filled with approximately 60 different types of hydrolytic enzymes, including phosphatases, lipases, nucleases and proteases, which break down biological polymers including lipids, nucleic acids, proteins and polysaccharides. Lysosomes also contain a host of enzyme activators, transporters and protective factors.

The enzymes in lysosomes are produced in the rough endoplasmic reticulum, exported to the Golgi apparatus, then trafficked to the lysosome in small vesicles. These small enzyme-laden vesicles are labeled with mannose 6-phosphate, which allows them to be correctly sorted into acidified vesicles producing a fully functional lysosome.

Mature lysosomes have an acidic pH (~4.5 - 5), driven by H+-ATPase (vacuolar-ATPases; v-ATPases), which provides optimum working conditions for hydrolytic enzymes. The internal layer of the mature lysosome has a specialized glycocalyx lining that prevents it from being damaged by the acidic conditions. The cell is protected from the action of any escaped hydrolytic enzymes, because they are much less effective or indeed inactive in the mildly basic environment of the cell's cytosol (pH 7.2).

Lysosome Functions

The lysosome is responsible for breaking down and recycling extracellular material that has been taken up by the cell through endocytosis, pinocytosis and phagocytosis. Lysosomal degradation products are trafficked to the Golgi apparatus for recycling or expelled from the cell through lysosomal exocytosis.

Lysosomes play a key role in autophagy, a process whereby intracellular material, such as damaged organelles or cellular debris, is broken down and recycled. Autophagy is the main pathway for providing cells with nutrients under starvation conditions and for clearing damaged proteins that are too large to be processed by the cell's ubiquitin-proteasome system. (For more information about autophagy visit our autophagy product page, and download or request our autophagy scientific review and autophagy poster. For autophagy detection kits, a range of IHC, ICC and IF antibodies, and a wealth of autophagy resources please visit our sister brand Novus Biologicals).

Lysosomes are dynamic structures that respond to environmental cues and have been shown to be important in nutrient sensing and regulation of cell metabolism. Lysosomes can translate metabolic signals to switch between catabolism and anabolism by regulating lysosomal autophagy and biogenesis. Lysosomes are also able to exchange organelle content, establish membrane contacts and signal with other major organelles.

Lysosome Structures and Functions

Figure 1 Top: Lysosome Degradation and Recycling; Endocytic and Autophagic. Lysosomes are key mediators of endocytic and autophagic degradation, as well as recycling cellular biomolecules.

  • Extracellular material undergoes endocytosis and the newly formed endosome fuses with the lysosome for degradation of the contents. This results in the generation of ATP and production of biomolecules, which may be absent in starvation conditions.
  • The mTOCR1 signaling pathway regulates cellular metabolism through the lysosome, by responding to nutrient levels and affecting lysosomal properties, such as ion channel activity and pH, which influence the catabolic action of the enzymes.
  • The lysosome exports material through lysosomal exocytosis, for bone resorption, plasma membrane repair and as part of the immune response.

Figure 1 Bottom: Lysosome Structure and Function: The lysosome comprises luminal and membrane proteins. The lysosomal lumen contains hydrolytic enzymes, enzyme activators, transport proteins and protective factors. v-ATPases embedded in the lysosome membrane maintain the acidic environment by pumping H+ into the luminal space. Transporters and ion channels are also involved in maintenance of ion homeostasis. LAMPs are key structural proteins that protect the lysosomal membrane from the acidic luminal environment.

The lysosome has a series of solute carriers and lipid and cholesterol transporters that transport nutrients such as glucose and amino acids as well as other degradation by products, across the lysosomal membrane. The lysosome also has a series of SNAREs and tethering factors that mediate lysosome fusion and contact points with other organelles.

Lysosomes can move around the cell through the coupling of scaffold complexes to microtubule tracks.

Types of Lysosomes

There are considered to be two distinct lysosome species: conventional and secretory lysosomes, although this remains contentious. This categorization is largely based on physical and functional properties.

The main function of conventional lysosomes is catabolism. Other lysosome-related organelles, (LROs) including late endosomes, complement conventional lysosome catabolic action. LROs share many typical lysosome characteristics such as internal acidic pH, lysosomal transmembrane proteins and the ability to fuse with phagosomes.

Secretory lysosomes contain additional enzymes compared to conventional lysosomes, which allow them to carry out additional functions such as membrane repair, tissue regeneration, cholesterol homeostasis, apoptosis, cell signaling and immune function.

Lysosomal Regulation

Lysosomal biogenesis and aspects of its functionality including its role in autophagy and lysosomal exocytosis, are regulated by transcription factor EB (TFEB) and the coordinated lysosomal expression and regulation (CLEAR) network. TFEB is also a crucial regulatory element in lysosomal Ca2+ signaling. Both cytosolic and lysosomal pathways regulate TFEB phosphorylation states, which is in turn regulated by mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cell biosynthetic pathways. In the presence of nutrients, mTORC1 promotes anabolism and suppresses catabolic autophagy pathways.

Lysosomal Ca2+ is another important regulating factor for lysosome functions such as plasma membrane repair, endocytic membrane trafficking, and autophagy. Lysosomal Ca2+ channels respond to many types of stimuli including nutrient levels, ATP, change in pH and more, suggesting they play a role in many activities associated with the lysosome action. The three main calcium channels found in lysosomes are: transient receptor potential cation channels of the mucolipin family (TRPML), two-pore channels (TPC) and P2X4 channels. Ca2+ release is required for lysosomal fusion with the plasma membrane, endosomes and autophagosomes. Lysosomal Ca2+ release is also required for contact site formation with the endoplasmic reticulum, which is an important source of calcium for the lysosome.

Lysosomes and Pathogen Entry

Lysosomes are a pivotal defence for cells against bacteria and virus entry. Pathogens can utilize or hijack the cell's endocytic pathways to enter the cell; lysosomes hydrolyze pathogenic biomolecules, thereby inactivating or reducing pathogenic activity within the host cell. Disease states, such as HIV infection, can be worsened by lysosomal dysfunction due to an increased propensity for viral infection.

Lysosomes and Disease

Lysosome dysfunction can take the form of fusion process errors resulting from inefficient or failed lysosomal endocytosis or exocytosis, dysfunctional hydrolytic enzymes, or other dysfunctional proteins that impair or stop its normal functions. When lysosome dysfunction occurs, toxic cellular waste can accumulate causing inflammation, apoptosis and necrosis, as well as causing other cellular signaling abnormalities.

Lysosome dysfunction plays a major role in autoimmune disorders including lupus and rheumatoid arthritis, as well as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis. Lysosomal dysfunction can also disrupt the function of other cellular organelles, including mitochondria and peroxisomes, which can lead to toxic levels of reactive oxygen species (ROS). This is a common pathology in many disease states such as cancer, chronic inflammation and autoimmune diseases.

Lysosomal storage diseases (LSO) are a collection of approximately 50 genetic diseases, which result in accumulation of substrates that can be toxic to the cell. Faulty genes include: β-GCase, which causes Gaucher disease; α-Galactosidase A, which causes Fabry disease; and α-Glucosidase, which causes Pompe disease. For several of these conditions, enzyme replacement therapy has proven very effective at compensating for these dysfunctional enzymes.

LYTACs (LYsosome TArgeting Chimera)- utilizing the Lysosome for Targeted Protein Degradation

LYTAC (LYsosome TArgeting Chimera) molecules represent a new approach to Targeted Protein Degradation (TPD). LYTACs recruit and traffic extracellular target proteins to the lysosome, where they are internalized and degraded.

Like their ubiquitin-proteasome-targeting counterparts, Protein Degraders or PROTAC® molecules, LYTACs are heterobifunctional molecules made up of a recruiting module e.g. an antibody, which targets the protein of interest, and a linker that is joined to a lysosome cell surface receptor ligand, such as the asialoglycoprotein receptor (ASGPR) ligand. The LYTAC forms a ternary complex with the target protein and lysosome receptor, triggering lysosomal endocytosis and subsequent degradation.

tri-GalNAc COOH (Cat. No. 7401) is part of the new LYTAC building block range. It has an asialoglycoprotein receptor ligand with PEG linker and carboxylic acid group, ready for conjugation to a target protein ligand.

Lysosome Stain and Tracker Featured Product: Pepstatin A Janelia Fluor® 526

Pepstatin A Janelia Fluor® 526 (Cat. No. 7314) is a fluorogenic green-emitting lysosomal tracker and stain. Its fluorogenic nature allows researchers to carry out no-wash experiments, enabling easy multicolor, multi-labeling microscopy experiments. Pepstatin A Janelia Fluor® 526 is suitable for live-cell imaging in confocal microscopy and super-resolution microscopy techniques including STED, 3D-SIM and two-color lattice light-sheet microscopy. Pepstatin A Janelia Fluor® 526 is an alternative for Lysotracker.

Spectral data for Pepstatin A Janelia Fluor 526 Lysosome Probes Emission Color Green
λabs 530 nm
λem 549 nm
Extinction Coefficient (ε) 118,000 M-1cm-1
Quantum Yield (φ) 0.87
Application Confocal microscopy, STED, 3D-SIM, lattice light-sheet microscopy
Alternative for LysotrackerTM

Figure 2: Spectral data for Pepstatin A Janelia Fluor® 526




PROTAC® is a registered trademark of Arvinas Operations, Inc., and is used under license.

Lysotracker is a registered trademark of Thermo Fisher.