Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence

Summary

Laminopathy disorders often lead to changes in the nuclear envelope, which may cause nuclear blebbing and leakage. This study presents an immunofluorescence method to visualize the nuclear lamina along with double-stranded DNA (dsDNA), providing a means to assess nuclear structure and integrity in mammalian cells.

Abstract

The nuclear lamina is a network of filaments underlying the nuclear membrane, composed of lamins and lamin-associated proteins. It plays critical roles in nuclear architecture, nuclear pore positioning, gene expression regulation, chromatin organization, DNA replication, and DNA repair. Mutations in genes involved in the expression or post-translational processing of lamin proteins result in genetic disorders known as laminopathies. Specifically, mutations in the LMNA or ZMPSTE24 genes can lead to the accumulation of incompletely processed forms of lamin A that retain farnesyl and methyl groups, which are absent in fully processed lamin A. These incompletely processed lamin A proteins localize to the inner nuclear membrane instead of the nuclear lamina, where mature lamin A resides. Mislocalized lamin proteins profoundly disrupt nuclear function and structure, often resulting in nuclear blebbing. In severe cases, nuclear rupture can occur, causing a loss of compartmentalization and leakage of genomic DNA into the cytosol. Abnormal nuclear structure and compartmentalization loss can be identified through indirect immunofluorescence (IF) on fixed cells. This study outlines such a method, employing specific antibodies against a lamin protein and double-stranded DNA (dsDNA) to simultaneously visualize the nuclear envelope and DNA. This approach enables a rapid assessment of nuclear structural integrity and the potential leakage of nuclear DNA into the cytosol.

Introduction

The nuclear lamina is a network of filaments that underlies the nuclear membrane and is made up of proteins called lamins. The nuclear lamina plays essential roles in nuclear architecture, positioning of nuclear pores, gene expression regulation, chromatin organization, DNA replication, and DNA repair^1,2,3. Mutations in genes that play a role in the expression of the lamin proteins lead to genetic disorders called laminopathies³.

Restrictive dermopathy (RD) is a severe laminopathy disorder predominantly caused by compound he....

Protocol

Details of the reagents and the equipment used are listed in the Table of Materials.

1. Preparation of materials

1. Autoclave at least ten 22 mm x 22 mm square glass coverslips and forceps in advance of plating cells onto the coverslips.

2. Preparation of the solutions

1. Supplemented media: Prepare 500 mL of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL streptomycin.
2. Antibody dilution buffer (ADB): Dissolve 2 g of bovine serum albumin (BSA) and 1 g of Fish gelatin i....

Results

This study introduces an IF method for visualizing the nuclear lamina in conjunction with double-stranded DNA (dsDNA). Once IF is performed, the images captured can be examined for signs of nuclear blebbing and DNA leakage.

Figure 2: Nuclear blebbing in HeLa CT and ZMPSTE24<.......

Discussion

The protocol presented contains several critical steps, the most important being the treatment of the fixed cells with primary and secondary antibodies. Ensuring the use of a good quality primary antibody against the target of interest with a correct corresponding secondary antibody with fluorophores within the range of the microscope used will yield optimal results¹⁹. The quality of the antibody can greatly impact the results of this technique, as faulty antibodies can interact with nonspecific t.......

Materials

Name	Company	Catalog Number	Comments
22 mm Square Glass Coverslips	Propper Manufacturing Company	M8710	Any size or shape can be used as long as they can be fixed to a standard microscope slide.
36.5% Formaldehyde	Sigma Aldrich	F8775	Fixation Reagent.
75 cm 2 Flasks	Corning	430725U	For cell culture.
Bovine Serum Albinum (BSA)	Fisher Scientific	9048-46-8	Antibody dilution buffer.
DMEM with 1 g/L glucose, L-glutamine & sodium pyruvate	Corning	10-014-CV	For cell culture.
Ethanol (200 Proof)	Decon Laboratories	2701	For dehydration of samples before mounting, diluted to make multiple concentrations.
EVOS FL Digital Inverted Fluorescence Microscope	Fisher Scientific	12-563-460	Imaging.
Fish Gelatin	Sigma Aldrich	G7041	Antibody dilution buffer.
Fluoromount-G	Southern Biotech	0100-01	Mounting medium to prevent photobleaching.
Parafilm (4 in)	Fisher Scientific	13-374-12	Any size can be substituted, as long as the coverslips being used can fit.
Penicillin-Streptomycin	Gibco	15-140-122	For cell culture.
Phosphate Buffered Saline (PBS)	N/A	N/A	Made and sterilized in the lab for tissue culture and solutions.
Premium Fetal Bovine Serum (FBS)	Atlanta Biologicals	S11150	For cell culture.
Superfrost Premium Microscope Slides	Fisher Scientific	12-544-7	Any standard microscope slides can be used.
Tissue Culture Treated 6-well Flat Bottom Plates	Falcon	353046	For cell culture.
TritonX-100	Thermofisher Scientific	A16046.AP	For washing.
Trypsin-EDTA (0.5%)	Gibco	15-400-054	For cell culture.
Tween 20	Fisher Scientific	9005-64-5	Cell Permeation reagent.