1. FLOW CYTOMETRIC ANALYSIS OF THE CELL CYCLE Jan Bartoš Laboratory of Molecular Cytogenetics and Cytometry Institute of Experimental Botany Olomouc, Czech Republic

2. Introduction to Cell Cycle CELL CYCLE periodic process during cell cycle one cell divides into two daughter cells (new cells can arise only from existing cells during the cell cycle) essential for all organisms (unicellular and multicellular) it is precisely regulated lost of its control could leads to uncontrolled multiplication of cells and cancer

3. Eukaryotic Cell Cycle consist of four phases:  G 1 – preparation of DNA synthesis  S – reduplication of chromosomes  G 2 – preparation of mitosis  M – division of nucleus and cell

4. PRINCIPLE OF FLOW CYTOMETRY Flow cytometry involves the analysis of fluorescence and light scatter properties of particles in flow, moving with respect to the point of measurement

5. Cell Cycle Analysis One of the earliest applications of flow cyto- metry was the analysis of cell cycle position by measurement of cellular DNA. Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions. Recently multiparametric methods were developed, which allow more detailed ana- lysis of the cell cycle.

6. DNA content during the cell cycle (a) Nuclear DNA content doubles from the 2C level to the 4C level during the S phase of the cell cycle. DNA content returns to the 2C level during mitosis (M), when two daughter nuclei are formed. (b) theoretical histogram of nuclear DNA content. a b

7. Histogram of relative nuclear DNA content easy, fast and non- expensive good for determination of cell cycle distribution cannot study cell cycle kinetics G1 or S ?S or G2 ?

8. Detection of DNA synthesis A brief pulse of 5-bromo-2’-deoxyuridine (BrdU) can be used for the detection of cells in S-phase. BrdU is incorporated into newly synthesized DNA in place of thymidine. The incorporated BrdU can be detected with an antibody, identifying those cells that synthesized DNA during the pulse. 1982 – human 1998 – plant

9. Detection of incorporated BrdU 1 2 3 1) Partial denaturation of DNA (heat, acid, enzyme-DNase I) 2) Immunocytochemical detection of incorporated BrdU (1 step or 2 step procedure) 3) Staining of DNA with fluorescent dye (e.g. PI or DAPI) 4) Flow cytometric analysis

10. Detection of S phase (DNA synthesis) using flow cytometry G1 G2 early S late S allow kinetic cell cycle study allow better determination of cell cycle distribution time consuming

11. analysis DNA/BrdU analysis of the cell cycle Cell cycle analysis in Vicia faba root tips. Root tips were incubated with BrdU for 1 hour. Incorporated BrdU was detected via indirect immunofluorescence: imme- diately after the BrdU pulse (a); 1 hour after the pulse (b) and 4 hours after the pulse (c).

12. c.T G2+M = ln[1+f u G2+M (t)] + c.t 0 < t < T G2+M c.(T S +T G2+M ) = ln[1+f lu (t)] + c.t T G2+M < t < T s +T G2+M c.T G2+M = ln[1 – f ld (t)/2] + c.t T G2+M < t < T s +T G2+M T c = ln(2p)/c White et al. 1990 f u G2+M fraction of unlabeled undivided cells f lu fraction of labeled undivided cells f ld fraction of labeled divided cells pfraction of cycling cells Calculation of cell cycle parameters

13. f u G2+M fraction of unlabeled undivided cells f lu fraction of labeled undivided cells f ld fraction of labeled divided cells pfraction of cycling cells f u G2+M f lu f ld Distribution of labelled population (BrdU positive) immediately (a) and 6 hours (b) after the BrdU pulse. Note definition of individual fractions. a b

14. Regulation of eukaryotic cell cycle Leland Hartwell, Tim Hunt and Paul Nurse - Nobel Prize in 2001 for their discoveries of “key regulators of the cell cycle”. mitotic cyclin Mitotic CDK degradation of mitotic cyclin S-phase cyclin S-phase CDK degradation of S-phase cyclin active MPF – enter of mitosis active S-phase CDK – start of DNA replication Proteins involved in the cell cycle control: cyclins CDK – cyclin dependent kinases CKI – cyclin dependent kinase inhibitors phosphatases other proteins (pRB – retinobastoma proteins, APC, transkription factors, etc.) Cell cycle is regulated by both extra- cellular and intracellular signals.

15. Green Fluorescent Protein (GFP) GFP is naturally occurring protein from the jellyfish Aquorea victoria. Wild type GFP fluorescence around 510 nm after excitation with UV or 488 nm laser. Many variants of GFP were developed (including BFP, YFP, EGFP,…) GFP can be used as reporter gene. GFP remain fluorescent after fusion with another protein.

16. Fluorescence of GFP fused with different variants of p34 cdc2 in nuclei isolated from tobacco: wild type p34 cdc2 (a); mutants of p34 cdc2 (b,c); control - nuclei without GFP. Analysis of p34 cdc2 expression in tobacco plant

17. THE LABORATORY http://www.ueb.cas.cz/olomouc1