Lysed cell models and isolated chromosomes for the study of kinetochore/centromere biochemistry in vitro

Abstract

The centromere or kinetochore functions in both chromosome movement and in regulation of progression through mitosis. It appears likely that the signaling pathways involved are keenly dependent on solid phase cytoskeletal and karyoskeletal scaffolds that may mediate important physical signals such as tension. Understanding these pathways will be greatly aided by reconstructing the signaling in lysed cell models. Here we present approaches to the in vitro study of signaling pathways in mitotic cells, particularly those involved in protein phosphorylation changes at kinetochores that may control cell cycle progression in M phase.

Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

“…To pursue the idea that PP6 is a mitotic regulator important for spindle formation, stage 2 of the screen was focused on the key kinase regulators of spindle formation from the centrosome Aurora A and Plk1 ( Llamazares et al, 1991 ; Glover et al, 1995 ; Lane and Nigg, 1996 ; Roghi et al, 1998 ). If the T-loop phosphatase acting on either kinase is absent, the phosphorylated form will be stabilized under conditions in which dephosphorylation is normally favored over phosphorylation ( Ahonen et al, 2005 ; Daum and Gorbsky, 2006 ). This can be simply achieved by cell lysis on ice for 2 h in the absence of phosphatase inhibitors (outlined in Fig.…”

Loss of p38γ MAPK induces pleiotropic mitotic defects and massive cell death

“…We conclude that the antibody against phosphorylated p38 also recognizes the p38 isoform in mitotic cells and that the p38 does not interact with the p38 isoform. To investigate which kinases are involved in p38 MAPK activation in early mitosis, we used the lysed cell assay (Ahonen et al, 2005;Daum and Gorbsky, 2006). In this assay, cells growing on coverglasses are extracted with a detergent and incubated in a buffer without the phosphatase inhibitor microcystin-LR (MC) or PhosSTOP phosphatase inhibitor cocktail allowing removal of the soluble cytoplasm and dephosphorylation of phosphoepitopes at kinetochores and other subcellular structures by the active phosphatases.…”

“…Silencing of p38 alone was not sufficient to abolish the phoshoepitope signals, suggesting that p38 is not the only active p38 isoform present at these subcellular foci (unpublished data). Using the lyzed cell model (Ahonen et al, 2005;Daum and Gorbsky, 2006), we tested the conditions that are required for creating phosphorylated p38 in mitotic cells. First, kinetochore and spindlepole-bound kinases were able to re-phosphorylate the epitope pointing to the presence of immediate upstream kinases of p38 MAPK at these subcellular locations, or TAB1-dependent autophosphorylation mechanism of p38 MAPKs (Ge et al, 2002;Salvador et al, 2005).…”