Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy.
Single-molecule localisation microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal 'snapshots' of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination and protein labelling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analysing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Srinjan
S
Basu
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Yi Lei
YL
Tan
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Edward J R
EJ
Taylor
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Ernest D
ED
Laue
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK. sl591@cam.ac.uk.
Steven F
SF
Lee
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008322:Mammals / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*
D002843:Chromatin; D011506:Proteins
Chromatin; Diffusion coefficient; Fluorescence imaging; Jump distance; Mean squared displacement; PALM; Residence time; SPIM; SPT; STORM; Super-resolution microscopy