The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.

Ana Katrina Estandarte,Ian Robinson,Stanley Botchway,Mohammed Yusuf,Christophe Lynch

doi:10.1038/srep31417

Abstract

Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.

Highlights

Visualization of distinct subchromosomal regions can be achieved through so-called banding, a characteristic striped appearance that results from the differential staining along the length of a chromosome
Calf thymus (CT) and micrococcus luteus (ML) DNA were used for this part of the study because of their different base pair compositions (42% GC for CT DNA and 72% GC for ML DNA)
We employed Fluorescence lifetime imaging microscopy (FLIM) to study chromatin condensation across different human cell lines and cell cycle phases, finding that fluorescence lifetime is sensitive to variations in chromatin compaction

Summary

Introduction

Visualization of distinct subchromosomal regions can be achieved through so-called banding, a characteristic striped appearance that results from the differential staining along the length of a chromosome. FLIM has several applications across both physical and life sciences It is widely used for investigations at Förster resonance energy transfer (FRET) lengths, typically from 1–10 nm, between a fluorophore and another molecule to probe distances and study interactions between molecules (e.g., homo- and hetero-dimerizations and protein-protein interactions[11,12]). A major advantage of FLIM is that it is not explicitly dependent upon molecular concentrations of the fluorophore unlike intensity-based fluorescence imaging[10]. FLIM is used to investigate the different chromosome substructures at the nanometer length scales by the variations in the DAPI (4′-6-diamidino-2-phenylindole) excited state lifetime loaded into human metaphase chromosomes and interphase nuclei. Other binding modes of DAPI are observed at high DAPI loadings such as external binding through electrostatic interactions with the phosphate groups of the DNA and intercalation between GC base pairs[19,20,21,22]

Methods

Results

Conclusion