Biphasic P-Ser83-HP1 γ is observed during cell cycle progression. (A,B,C) P-Ser83-HP1γ levels vary during the cell cycle. Panoramic view of a growing population of HeLa cells staining with anti-P-Ser83-HP1γ (A, green) demonstrates that the signal for this protein varies in intensity in different cells. Cells were counterstained with DAPI (B, blue) to show DNA and overlay is shown in (C). Three main populations are observed according to the strength of the signal, namely strong, moderate and negligible. Scale bar represents 20 μM. (D,E,F) P-Ser83-HP1γ displays punctate euchromatic localization in G1 phase. Localization of P-Ser83-HP1γ (D, green) was determined in cyclin D-positive cells (E, red), indicative of G1 phase, as shown with arrows and in overlay (F). (G,H,I) Levels of P-Ser83-HP1γ diminish during S phase. Negligible P-Ser83-HP1γ signal (G, green) is found in the majority of cells undergoing S phase (arrows), as determined by EdU positively labeled cells (H, red). Overlay is shown in (I). (J,K,L) P-Ser83-HP1γ levels increase upon G2 entry. Cyclin B-positive cells (K, red), before nuclear envelope breakdown (G2), not only shows the P-Ser83-HP1γ signal (J, green) as a strong punctate pattern in euchromatin, but also with separating centrosomes (L, overlay). Scale bar represents 10 μM for panels (D to L). (M,N,O,P,Q,R) P-Ser83-HP1γ levels persist through mitosis. Cyclin B-positive, prometaphase cell demonstrates an increase in P-Ser83-HP1γ in association with separating centrosomes (M). Metaphase cell shows the P-Ser83-HP1γ remains localized to centrosomes, which are forming the mitotic spindle (N). Early (O) and late (P) anaphase, as well as telophase (Q) cells are shown, where the P-Ser83-HP1γ signal intensity at the centrosomes is decreased as cells prepare to complete cell division. P-Ser83-HP1γ signal within euchromatic regions is again observed during cytokinesis (R). Scale bar represents 5 μM for panels (M to R). DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2´-deoxyuridine; P-Ser83-HP1γ, phosphorylation of HP1γ at serine 83.