Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Summary

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Abstract

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.

Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.

In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Introduction

Fluorescence microscopy, while widely used for labeling and visualizing specific molecules within cells, is inherently limited in resolution by Abbe's diffraction limit of light¹, which restricts the ability to distinguish between objects closer than approximately 200-250 nm. This limitation arises from the wave nature of light and the numerical aperture of the microscope's objective lens, introducing a challenge when imaging subcellular structures. Overcoming these limitations provides better insights into certain biological processes at a nanometric scale.

To surpass the diffraction limit of light, super-re....

Protocol

1. Cell seeding

NOTE: Carry out the next steps under sterile conditions under a laminar flow hood. This method can be applied to any of the cell lines used in the present work (HeLa, MCF7, primary fibroblasts), or to most adherent cell line models commonly used in research^20,24,25.

1. Grow cells in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS) in a T75 flask at 37 °C under a 5% CO₂ atmosphere.
2. Once the cells have reached full confluency, remove the cell culture medium and....

Discussion

The present CuAAC labeling protocol does not include aminoguanidine in the reaction buffer. Since it is aimed at visualizing intracellular glycoconjugates, it is performed on cells that are fixed after the metabolic incorporation step, to avoid any cytotoxicity issue and improve uptake of the catalytic system. The use of aminoguanidine is typically recommended for cell-surface labeling of living cells to prevent side reactions between dehydroascorbate and arginine, histidine, and lysine residues of proteins

Materials

Name	Company	Catalog Number	Comments
(+) Sodium L-ascorbate	Sigma Aldrich	11140
12 well cell culture plate	Corning	3513
Acrylamide	Sigma Aldrich	A8887
Acrylic acid N-hydroxysuccinimide ester	Sigma Aldrich	A8060
Alexa Fluor 488 alkyne	Jena Bioscience	CLK-1277-5
Alexa Fluor 546 goat anti-mouse IgG	Invitrogen	A11003
Amonium persulfate	Sigma Aldrich	9913
Bis-Acrylamide	Sigma Aldrich	146072
BSA	Sigma Aldrich	A7906
BTTAA	Jena Bioscience	CLK-067-100
Centrifugation tube 2 mL	EPPENDORF	30120094
Chloroquine diphosphate salt	Sigma Aldrich	C6628
Conical tube 15 mL	Falcon	352097
cover slips 12 mm #1	epredia	CB00120RA120MNZ0
cover slips 32 mm #1	epredia	CB00320RA140MNZ0
CuSO4	Sigma Aldrich	209198
DMEM high glucose medium	Dutscher	L0104-500
Dulbecco's Phosphate Buffered Saline (PBS)	Dutscher	L0615-500
Fetal Bovine Serum	biowest	S1810-500
Fibroblast 533T	-	-	Collected from healthy individual
FIJI ImageJ 2.9.0	-	-
Gelatin	Bio-RAD	170-6537
Guanidine HCl	Sigma Aldrich	50950
HeLa cells	ATCC	CCL-2
Hoechst 33342	Sigma Aldrich	14533
Imaris 10.2	-	-
K2HPO4	Euromedex	PB0447-B	Anhydrous
LSM 780 Confocal Microscopy	Zeiss	-
MCF7	ATCC	HTB-22
N-acetylmannosamine (ManNAc)	BIOSYNTH	MA05269
NaCl	Carlo Erba	479687
N-azidoacetylmannosamine (ManNAz)	BIOSYNTH	MA46002
Objectif "Plan-Apochromat" 63x/1,4 Oil DIC M27	Zeiss	420782-9900-799
Phosphate Buffered Saline (PBS) 10x	Euromedex	ET330
Proteinase K	Sigma Aldrich	P2308	from Tritirachium album
purified mouse GM130 antibody	BD Bioscience	610822	50 µg
Sodium acrylate	Sigma Aldrich	408220
T75 Flask	Corning	430641
TEMED	Sigma Aldrich	T9281
tris Acetate EDTA (TAE) 10x	Euromedex	EU0202-B
Triton X-100	Sigma Aldrich	X-100
Trypan Blue	Dutscher	702630
Trypsine-EDTA 1x	Dutscher	L0930-100