The Golgi apparatus structure is composed of flattened membrane-enclosed sacs called cisternae, and associated vesicles. Here we describe a variety of cell-permeant probes that can be used to distinguish the Golgi morphology in both live and fixed cells. Additionally, these probes have applications in lipid metabolism, trafficking studies, or can be used for measuring rates of lipid synthesis.
Golgi apparatus introduction
The Golgi apparatus, also known as the Golgi complex or Golgi, is found in most eukaryotic cells. The Golgi is a flattened membrane-enclosed organelle that consists of multiple compartments and whose function is a continuation from the ER. Macromolecules such as proteins, lipids, and carbohydrates released from the ER are transported to the cis-Golgi compartment of the Golgi complex [1,2]. These macromolecules continue to be transported and processing through the Golgi stack (which contains medial and trans sub-compartments), then travel to the trans-Golgi compartment where they are packaged into vesicles for either secretion from the cell or for use by other organelles within the cell [1,2].
Selection guide for Golgi apparatus stains
| Golgi fluorescent fusion proteins | Ceramide dyes | ||||
|---|---|---|---|---|---|
| CellLight Golgi-GFP, BacMam 2.0 | CellLight Golgi-RFP, BacMam 2.0 | BODIPY FL C5-Ceramide complexed to BSA | BODIPY TR Ceramide complexed to BSA | NBD C6-Ceramide complexed to BSA | |
| Readout | Expression of fluorescent fusion protein | Live cell Golgi staining | |||
| Target | Protein targets Golgi complex | Targets lipids in Golgi complex | |||
| Common filter set | FITC | TRITC | FITC | Texas Red | FITC |
| Labels | GFP | RFP | BODIPY FL | BODIPY TR | NBD |
| Ex/Em (nm) | 488/520 | 555/584 | 504/511 | 587/615 | 466/536 |
| Signal-to-noise ratio |  | |  | |  |
| Photostability | | | | | |
| Multiplexing | Yes | Yes | Yes | Yes | Yes |
| Live cells | Yes | Yes | Yes | Yes | Yes |
| Fixed cells | No | No | No | No | No |
| Fixable | Yes | Yes | Yes | Yes | Yes |
| Platform | Imaging | Imaging | Imaging | Imaging | Imaging |
| Format | 1 mL | 1 mL | 5 mg | 5 mg | 5 mg |
| Cat. No. | C10592 | C10593 | B22650 | B34400 | N22651 |
Golgi fluorescent fusion proteins
CellLight fluorescent fusion proteins are useful for the identification and demarcation of the Golgi complex in live cells and to follow the dynamics of intracellular behavior, such as protein trafficking. CellLight Golgi-GFP (Figure 1) and CellLight Golgi-RFP (Figure 2) are ready-to-use constructs that use a human Golgi-resident enzyme, N-acetylgalactosaminyltransferase-2 (12.6 kDa), to target the Golgi complex.
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 Golgi fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells, or after formaldehyde fixation for colocalization studies.
Learn more about these and other CellLight fluorescent proteins
Figure 1. Live cell imaging following CellLight Golgi-GFP transduction. Gibco human aortic smooth muscle cells (HASMC) were transduced with CellLight Golgi-GFP, CellLight Mitochondria-RFP and Hoechst 33342. Imaging was performed on live cells using a DeltaVision Core microscope and standard DAPI/FITC/TRITC filter sets.
Figure 2. CellLight Golgi-RFP transduced A549 cells. A549 cells were seeded in Nunc 27 mm glass-base dishes and co-transduced with CellLight Mitochondria-GFP and CellLight Golgi-RFP reagents. After incubation overnight, cell nuclei were counter-stained with NucBlue Live cell stain and imaged on an EVOS FL Auto imaging system with a 100X S-Apo oil immersion objective.
Ceramide dyes
Fluorescent ceramides are used extensively to study lipid metabolism and trafficking. They provide selective staining for the Golgi complex in live cells and can also be fixed. NBD dye conjugates are weakly fluorescent in aqueous solvents, and its fluorescence increases in a non-polar environment. The BODIPY dye conjugates (Figures 3 and 4) are brighter and more photostable than the NBD derivative, with less environmental sensitivity. Complexing fluorescent lipids with bovine serum albumin (BSA) eliminates the need for organic solvents to dissolve the lipophilic probe—the BSA-complexed probe can be directly dissolved in water and loaded into live cells.
Fluorescent ceramides can be used in various applications including markers for structural identification of the Golgi complex, outlining boundaries to observe morpho-genetic movements, tracing and tracking of lipid metabolism in cells, and measuring lipid synthesis.
Figure 3. Live NIH 3T3 cells labeled with probes for mitochondria, Golgi, and the nucleus. Mitochondria were labeled with MitoTracker Red FM, Golgi with BODIPY FL ceramide, and the nucleus with Hoechst 33342. The image was deconvolved using Huygens software (Scientific Volume Imaging).
Figure 4. Madin-Darby canine kidney (MDCK) cells labeled for Golgi, lysosomes, and the nucleus. Viable Madin-Darby canine kidney (MDCK) cells sequentially stained with BODIPY FL-ceramide, LysoTracker Red DND-99 and Hoechst 33258. Green fluorescent BODIPY FL-ceramide localized to the Golgi apparatus, red-fluorescent LysoTracker Red stain accumulated in the lysosomes, and the blue-fluorescent Hoechst 33258 dye stained the nuclei. The multiple-exposure image was acquired with bandpass filters appropriate for fluorescein, Texas Red dye, and DAPI.
Learn more about Golgi apparatus stains
References
Resources
Molecular Probes Handbook
BioProbes 70 Journal article
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