Endosome structures are intracellular sorting organelles involved in trafficking proteins within the cell. Here we describe fluorescent fusion proteins that can be used to identify early and late endosomes in live cells.
Endosome introduction
Endosomes are intracellular sorting organelles that transport subcellular components such as proteins and lipids from the Golgi [1]. Proteins can also be internalized through the plasma membrane in a process known as endocytosis, pinocytosis, and phagocytosis [1]. Learn more about these processes here. Endosomes are classified as early or late based on their stage following the internalization of cellular components. They are also distinguished by their morphology and Rab marker. Early endosomes are commonly formed near the plasma membrane and are distinguished by the Rab5A marker. Late endosomes mature from early endosomes and are distinguished by the Rab7A marker.
Selection guide for endosome stains
| Early endosome stains | Late endosome stains | |||
|---|---|---|---|---|
| Name | ||||
| Readout |
Stable signal from fluorescent protein targeted to the specific stage of endocytosis
| |||
| Range |
Early endosome
|
Late endosome
| ||
| Common filter set |
FITC
|
TRITC
|
FITC
|
TRITC
|
| Labels |
GFP
|
RFP
|
GFP
|
RFP
|
| Ex/Em (nm) |
488/510
|
555/584
|
488/510
|
555/584
|
| Signal-to-noise ratio |  |  | | |
| Photostability | | | | |
| Multiplexing |
Yes
| |||
| Live cells |
Yes
| |||
| Fixed cells |
No
| |||
| Fixable |
Yes
| |||
| Format |
1 mL
|
1 mL
|
1 mL
|
1 mL
|
| Cat. No. | ||||
Early and late endosome fluorescent proteins
CellLight fluorescent fusion proteins are ready-to-use constructs that label endosomes in live cells to follow the dynamics of intracellular behavior. CellLight Early Endosome labels use the Rab5a construct fused to emGFP (Figure 1) or TagRFP (Figure 2) while CellLight Late Endosome labels use the Rab7a construct fused to emGFP (Figure 3) or TagRFP (Figure 4).
Introducing CellLight fluorescent fusion proteins involves a simple transfection step using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These endosome fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells, or after formaldehyde fixation for colocalization studies.
Early endosome images
Figure 2. Early Endosomes-RFP and mitochondria-GFP. HeLa cells were transduced with CellLight Endosomes-RFP and Mitochondria-GFP, then and co-stained Hoechst 33342. Imaging was performed on live cells using a DeltaVision Core microscope and standard DAPI/FITC/TRITC filter sets.
Late endosome images
Figure 3. Late endosome-GFP and Golgi-RFP. A549 cells were co-transduced with CellLight Late Endosomes-GFP and CellLight Golgi-RFP then incubated overnight. Following staining with MitoTracker Deep Red, cells were imaged on a Zeiss LSM confocal microscope.
Figure 4. Late endosome-RFP co-localization with LysoTracker Deep Red. HeLa cells expressing CellLight Tubulin-GFP and CellLight Late Endsome-RFP was loaded with 50 nM LysoTracker Deep Red and Hoechst 33342. Images were pseudo colored as Hoechst 33342 (magenta), LysoTracker Deep Red (blue), CellLight Tubulin-GFP (green), and CellLight Late Endsome-RFP (red).
Resources
Molecular Probes Handbook
BioProbes 70 Journal article
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