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Selective visualization of Golgi apparatus with a simple procedure GolgiSeeing™(Golgi Apparatus Green)

Date：October 16 2023Web Page No：95031

Funakoshi Co.,Ltd.

GolgiSeeing™ is a novel and innovative fluorescent dye for selective Golgi apparatus staining. Compared to conventional ceramide-based Golgi staining reagents, GolgiSeeing™ does not require complicated procedures and suppresses non-specific localization to the endoplasmic reticulum.

GolgiSeeing

GolgiSeeing™ Ceramide-FL
Comparison between GolgiSeeing and conventional dye

Conventional Golgi apparatus staining on live cells and its problems

-What is Golgi apparatus?

The Golgi apparatus is an organelle that plays various roles in physiological functions and is known as a central component of the protein secretory pathway. The Golgi apparatus has unique multiple-layered cisternal membrane structures, with subdivided structures such as cis-Golgi, which is responsible for reciprocal vesicular transport with the endoplasmic reticulum (ER), and trans-Golgi, which is the starting point of the secretory pathway. Since dynamic morphological changes of the Golgi apparatus are essential for secretory function and dysfunction of the Golgi apparatus has been implicated in a number of diseases, it is expected that the Golgi apparatus will be observed by live cell imaging.


-Conventional Golgi apparatus staining methods and its problems

Two major methods have been used to stain the Golgi apparatus in living cells: The first is fluorescent staining using fluorescently labeled ceramide derivatives (hereafter ceramide-FL). Ceramide lipids accumulate in the Golgi apparatus during the metabolic pathway, and ceramide-FLs have been used to visualize the Golgi apparatus. However, 1) The Golgi selectivity of ceramide derivatives is low, 2) Ceramide derivatives also localize to the ER, etc., and 3) Ceramide derivatives have high cytotoxicity and are quickly metabolized intracellularly. The second staining method is to overexpress fluorescent proteins by fusing them to Golgi-specific expressed proteins (such as Giantin, N-acetylgalactosaminyltransferase, etc.). While this method allows visualization of the Golgi apparatus with a high degree of specificity, it requires prior plasmid transfection, making it impossible to immediately observe the Golgi apparatus when necessary. Furthermore, there are concerns overexpression of specific marker genes may affect physiological functions of the Golgi apparatus.


-Novel method; GolgiSeeing™ and its advantages

GolgiSeeing™ is a novel small molecule fluorescent reagent that utilizes a Golgi apparatus-selective localization motif discovered by Dr. Shinya Tsukiji's group at Nagoya Institute of Technology. Unlike conventional ceramide-FLs, it can stain the Golgi apparatus with a simple protocol that requires only 10 minutes of addition to the culture medium. It also shows higher Golgi specificity than ceramide-FLs, allowing Golgi apparatus-focused analysis (Figure 1). The GolgiSeeing™ can visualize the Golgi apparatus of target cells at any desired timing without genetic manipulation and without bias to physiological functions caused by overexpression, allowing the dynamic behavior of the Golgi apparatus to be observed under more physiological conditions.

Category Fluorescent probes Genetically encoded
Staining GolgiSeeing™ Fluorescent-labeled ceramide derivatives (Ceramide-FL) Fluorescent protein-fused Golgi apparatus marker proteins
Golgi apparatus specificity High
(Low ER background)		 Low
(High ER background)		 High
Protocol Easy
(Just addition to medium)		 Complecated
(Require stepwise and temperature-controlled protocol)		 Easy
(Exogenous gene transfection))
Time 10 min >1 hour >half day
(time for protein expression)
Influence on physiological function Low High
(Exogenous ceramide’s toxicity)		 High
(Overexpression of exogenous proteins)


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Features

  • A novel Golgi staining reagent that utilizes S-palmitoyl lipid modification to accumulate fluorescein in the Golgi apparatus.*1
  • Fluorescent characteristics: Ex 480 nm/Em 520 nm*2
  • Staining can be performed simply by adding the reagent to the culture medium. No complicated staining procedures are required unlike conventional fluorescent-labeled ceramide-based Golgi staining reagents, and the visualization can be achieved in a short time.
  • Compared to conventional fluorescent-labeled ceramide-based Golgi staining reagents, nonspecific staining to the endoplasmic reticulum is suppressed.
  • Cytotoxicity is reduced compared to conventional fluorescent-labeled ceramide-based Golgi staining reagents.*3
  • Under the non-wash protocol, GolgiSeeing™ visualizes not only the Golgi apparatus but also the plasma membrane. This property allows simultaneous imaging of the Golgi apparatus and cell morphology (judged by plasma membrane).
  • GolgiSeeing™ is specialized for live cell imaging. Not compatible with both pre-fixed cells staining and post-fix procedures.
  • ＊1 Because of the principle of utilizing the intrinsic S-palmitoylated lipid modification activity within the cell, drugs or stimuli that inhibit S-palmitoylation may interfere with staining. In addition, thiol alkylating agents cannot be used together because they strongly inhibit S-palmitoylation modification.
  • ＊2 GolgiSeeing™ has fluorescein diacetate (FDA), which is quenched by two acetates and emits a slight fluorescence. After hydrolysis of two acetic groups in cells by physiological esterases, fluorescein is exposed and restores strong green fluorescence.
  • ＊3 To prevent nonspecific reagent adsorption, we recommend the use of BSA-containing media for washing procedures. Buffers such as PBS may not remove the reagent sufficiently and may cause adverse effects such as background signals.

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Principle

GolgiSeeing

GolgiSeeing™ (original compound name; mgc3Me-FDA in Ref.1) has a unique structure consisting of fluorescein diacetate (FDA) and N-myristoylated Gly-Cys dipeptide (mgc) linked by a linker, and three amide bonds in the mgc peptide chain are all methylated (Figure left, upper). GolgiSeeing™ functions as a Golgi apparatus-selective fluorescent probe through several processes in the cell. FDA is a modified fluorescein quenched to improve cell membrane permeability, and GolgiSeeing™ emits slight fluorescence before use. After GolgiSeeing™ penetrates the cell membrane and enters the cell, acetyl groups are removed by endogenous esterases, and the green fluorescence of fluorescein is restored (Figure left, middle). The deacetylated form of GolgiSeeing™ transiently localizes mainly to the ER and Golgi apparatus by the effect of N-myristoyl groups and is S-palmitoylation by endogenous S-palmitoylation enzymes. The Golgi apparatus selective localization motif is formed by adding palmitic acid to the Cys side chain by enzymatic action (Figure left, lower). Although the intrinsic palmitoylation modification is known to localize to the Golgi apparatus and the plasma membrane, the effect of the three methyl groups biases the equilibrium toward the Golgi, which allows GolgiSeeing™ to selectively visualize the Golgi apparatus.


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Reference data

Comparison between GolgiSeeing™ and a ceramide-based reagent

GolgiSeeing™ Ceramide-FL
Just addition Just addition Step-wise protocol
GolgiSeeing Just addition Ceramide-FL Just addition Ceramide-FL Step-wise protocol
GolgiSeeing Just addition（Zoom） Ceramide-FL Just addition（Zoom） Ceramide-FL Step-wise protocol（Zoom）

HeLa cells were treated with 10 uM GolgiSeeing™ or 5 uM ceramide-FL (as BSA complex). In the case of GolgiSeeing™, the protocol is a simple addition of GolgiSeeing™into media final 10 uM and incubated for 10 min. After washing cells with 3 mg/ml BSA containing media, fluorescent images were captured by confocal laser microscopy (Ex 488 nm/Em 500-600 nm). On the other hand, ceramide-FL (BSA complex) -staining was performed by two protocols, simple addition or stepwise temperature-controlled protocol. The later protocol cells were incubated with ceramide-FL (BSA complex) for 30 min at 4℃ in HBSS, washed with ice-cold HBSS, and incubated in a fresh culture medium for an additional 30 min at 37℃. Finally, cells were washed with a fresh medium again and observed by confocal laser microscopy. This stepwise protocol requires over 1 hour. The ceramide-FL probe stains not only the Golgi apparatus but also ER structure with high intensity non-specifically. GolgiSeeing™ was able to visualize Golgi apparatus selectively and suppressed non-specific ER staining.


Organelle specificity

HeLa cells were co-stained with GolgiSeeing™ and organelle markers. In the case of the Golgi apparatus marker, overexpression of mCherry-Giantin fusion protein was used. In the cases of ER, mitochondria, and lysosome, each organelle was stained by organelle-specific chemical probes. The fluorescent signal from GolgiSeeing™ is well corresponded with Golgi-marker and weakly overlaps with ER. However, the signals are not matched with mitochondria and lysosomes.

GolgiSeeing™   Organella marker   Merge
Golgi
 GolgiSeeing-Golgi Organella_marker-Golgi Marge-Golgi
ER
 GolgiSeeing-ER Organella_marker-ER Marge-ER
Mitochondria
 GolgiSeeing Mitochondria Organella_marker Mitochondria Marge Mitochondria
Lysosome
 GolgiSeeing Lysosome Organella marker Lysosome Marge Lysosome

Effect of wash-out step on plasma membrane staining

HeLa cells were treated with 10 uM GolgiSeeing™ for 10 min and observed by confocal laser microscopy (Ex 488 nm/Em 500-600 nm) with or without the wash step. For wash-out, 3 mg/ml BSA-containing medium was used as wash buffer. In the case of non-wash observation, the fluorescent signals of GolgiSeeing™ were observed in not only the Golgi apparatus but also the plasma membrane. As the Golgi apparatus is clearly distinguished from the plasma membrane, non-wash staining is useful for simultaneous observation of the Golgi apparatus and cell morphology (the shape of the plasma membrane). The wash-out step dramatically reduced the fluorescent signal from the plasma membrane.

Effect of wash-out step on plasma membrane staining

Various cell staining

Four types of cultured cells (HeLa, COS-7, HEK293, and Jurkat) were treated with 10 uM GolgiSeeing™ for 10 min. After cell wash-out, cells were observed by confocal laser microscopy (Ex 488 nm/ Em 500-600 nm). For all cells tested here, GolgiSeeing™ highly selectively stained the Golgi apparatus.

HeLa COS-7 HEK293 Jurkat
HeLa COS-7 HEK293 Jurkat
HeLa COS-7 HEK293 Jurkat

Cellular toxicity

HeLa cells were seeded into a 12-well plate at 0.5 x 105 cells/well and cultured for 48 hours with/without 10 uM GolgiSeeing™. After 2, 24, and 48 hours, cell numbers were assessed by a cell counter. GolgiSeeing™ shows little effect on cell proliferation.

Cellular toxicity


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Application data

Live cell time-lapse imaging

HeLa cells were seeded on glass bottom dishes and treated with 10 uM GolgiSeeing™ for 10 min. After washing cells with 3 mg/ml BSA-containing media, the cells were observed under live cell condition by confocal microscopy (Ex 488 nm/Em 500-600 nm) for 30 min. Dynamic movement of Golgi apparatus could be observed.


Live cell time-lapse imaging on Brefeldin A-induced collapse of Golgi apparatus

HeLa cells were seeded on glass bottom dishes and treated with 10 uM GolgiSeeing™ for 10 min. After washing cells with 3 mg/ml BSA-containing media, the cells were cultured in brefeldin A (final 1 uM in 0.1% DMSO)-containing medium or 0.1 % DMSO-containing medium as a negative control and observed under live cell condition by confocal microscopy (Ex 488 nm/Em 500-600 nm). In the brefeldin A-treated cells, the fluorescent signal from the Golgi apparatus gradually disappeared.

0 min   6 min   12 min   18 min
GolgiSeeing™
Brefeldin A
 Brefeldin A 0 min Brefeldin A 6 min Brefeldin A 12 min Brefeldin_A-18 min
DMSO
 DMSO 0 min DMSO 6 min DMSO 12 min DMSO 18 min

Live cell time-lapse imaging during cell division

MDCK cells were seeded on a glass bottom dish, incubated with 2.5 uM GolgiSeeing™, and observed for 2 hours by confocal laser microscopy (Ex 488 nm/ Em 500-600 nm) without washing step. As a non-wash procedure, the fluorescent signal of GolgiSeeing™ has observed not only Golgi apparatus but also plasma membrane and cell morphology were easily observed. According to the progression of cell division, the Golgi apparatus disappeared (60 min) and was reconstituted in two daughter cells (90-120 min).

0 min   30 min   60 min   90 min   120 min
GolgiSeeing™
(wash −)
 0 min 30 min 60 min 90 min 120 min

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Original paper

  • Sawada, S., et al., "Palmitoylation-Dependent Small-Molecule Fluorescent Probes for Live-Cell Golgi Imaging", ACS Chem. Biol., 18(5), 1047～1053 (2023). [PMID：37098188]

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Price

[Date : April 25 2025 00:06]

Detail Product Name Product Code Supplier Size Price
GolgiSeeing, Golgi Apparatus Green
DatasheetThis may not be the latest data sheet. 		FDV-0053 FNAFunakoshi Co.,Ltd. 0.1 mg $450

Description GolgiSeeing™ is a novel and innovative fluorescent dye for selective Golgi apparatus staining. Compared to conventional ceramide-based Golgi staining reagents, GolgiSeeing™ does not require complicated procedures and suppresses non-specific localization to the endoplasmic reticulum.
Storage -20°C CAS
Link

[Date : April 25 2025 00:06]

GolgiSeeing, Golgi Apparatus Green

DatasheetThis may not be the latest data sheet.


  • Product Code: FDV-0053
  • Supplier: FNA
  • Size: 0.1mg
  • Price: $450

Description GolgiSeeing™ is a novel and innovative fluorescent dye for selective Golgi apparatus staining. Compared to conventional ceramide-based Golgi staining reagents, GolgiSeeing™ does not require complicated procedures and suppresses non-specific localization to the endoplasmic reticulum.
Storage -20°C CAS
Link

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