Endocytosis and Pinocytosis

Endocytosis is a process by which cells internalize non-particulate materials such as proteins or polysaccharides by engulfing them. This process is important for metabolism and cell signaling. Fluorescent proteins and organic dyes are useful indicators for staining vesicle walls or vesicle contents. Because the vesicle pH changes during the endocytic process, pH indicators are useful to monitor stages in the pathway.

Fluorescent proteins

Fluorescent proteins can be used to specifically label components in the endocytic pathway such as early endosomes, late endosomes, or lysosomes. Using BacMam technology, the specific structures are targeted within the cell by transgenic expression of RFP or GFP fusion proteins. Endocytic structures can be monitored in live cells, or the cells can be fixed for additional immunohistochemical analysis.

BacMam technology is highly efficient, and transiently transduced cells typically express fusion protein for about five days, though in slowly dividing cells, such as some primary cell types, expression has been demonstrated for up to two weeks.

See selection guide for fluorescent proteins

Microscopic image HeLa cells expressing fluorescent proteins

HeLa cells were co-transduced with CellLight Early Endosomes-GFP and CellLight Late Endosomes-RFP and and incubated overnight.

Dextran conjugates

Tracking internalization of fluorescent dextrans is a routine method for analyzing fluid-phase endocytosis. pHrodo dyes provide the most complete solution by allowing discrimination of stages in the endocytosis pathway from early endosome to lysosome formation with no quench or wash required.

pHrodo dyes are essentially non-fluorescent at neutral pH and exhibit increasing signal with a red or green readout respectively as the pH decreases. The increase in fluorescent signal can be used to monitor progression in the endocytic pathway.

Alexa Fluor conjugates can be used to monitor internalization by using either quenching or washing steps.

See selection guide for dextran conjugates

Microscopic image of mouse cells with fluorescent dextrans and nuclear staining

Mouse monocyte/macrophage cells (MMM cells, ATCC) incubated with pHrodo Red dextran 10,000 MW in Live Cell Imaging Solution and counterstained with NucBlue Live ReadyProbes Reagent .

LysoTracker dyes

LysoTracker probes specifically label acidic organelles with organic dyes and without using a transgenic protein system. Their stable fluorescent signal covers a range of wavelengths but does not distinguish between lysosomes and other acidic compartments.

LysoTracker probes can be multiplexed with other dyes or proteins. LysoTracker Deep Red, in particular, multiplexes well with both RFP and GFP.

Both membrane proteins and LysoTracker dyes serve as effective tools for tracking lysosome behavior. Use the selection guides to determine multiplexing options and fixability.

See selection guide for LysoTracker dyes

Microscopic image of a cell stained with LysoTracker Deep Red

Co-localization using LysoTracker Deep Red.

Membrane stains

An additional tool to track endocytosis uses membrane probes to monitor vesicle formation by visualizing changes in membrane morphology.

Water-soluble FM dyes are lipophilic, nontoxic to cells and virtually nonfluorescent in aqueous medium. They insert into the outer leaflet of the cell membrane and become intensely fluorescent in a range of colors. FM dyes will stain the plasma membrane and become internalized in endocytic vesicles. The non-specific staining of cell surface membranes can be washed off prior to imaging.

FM 1-43 is particularly used to investigate the mechanisms of activity-dependent vesicle cycling especially in actively firing neurons.

See selection guide for membrane stains

Fluorescent cell image via staining from membrane and nucleus stains

Bovine pulmonary artery endothelial cells stained with the reactive oxygen species indicator, 6-carboxy-2´,7´-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) and FM 5-95 and Hoechst 33342.

The red-fluorescent FM 5-95 appears to stain both the plasma membrane and early endosomes; the green-fluorescent, oxidized carboxydichlorofluorescein localizes to the cytoplasm; and the blue-fluorescent Hoechst 33342 dye stains the nucleus.