Changes in chromosome organization during PHA-activation of resting human lymphocytes measured by cryo-FISH.
During interphase, chromosomes are arranged into territories within a highly organized nuclear space containing several compartments. It is becoming clear that this complex nuclear arrangement is important for gene regulation and therefore expression. The study of chromosome organization in interphase requires high-resolution imaging methods that at the same time allow for flexible labelling strategies and preserve nuclear structure. Tokuyasu cryosections of cells or tissues provide a simple, high-resolution platform for performing immunolabelling and fluorescence in situ hybridization (FISH) on well-preserved samples. Here we show how FISH performed on thin cryosections (cryo-FISH) can be used for the study of chromosome organization at high resolution and in a quantitative manner. We have measured chromosome intermingling, volume and radial position, in resting and activated human lymphocytes, and observed chromosome-specific differences between the two cellular states. These differences are in part related to the nuclear expansion that occurs during activation, but are also likely to be tied to their different transcriptional profiles. Extrapolation of our dataset to the whole genome suggests that activated cells contain a lower amount of chromatin involved in intermingling than resting cells.
Branco
Miguel R
MR
MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Branco
Tiago
T
Ramirez
Francisco
F
Pombo
Ana
A
D002877:Chromosomes, Human / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D016616:Cryoultramicrotomy; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D066298:In Vitro Techniques; D008213:Lymphocyte Activation / Q000187:drug effects / Q000235:genetics; D008214:Lymphocytes / Q000187:drug effects / Q000648:ultrastructure; D018613:Microscopy, Confocal / Q000379:methods; D010835:Phytohemagglutinins / Q000494:pharmacology
D010835:Phytohemagglutinins