Limits...
Characterization of vertebrate cohesin complexes and their regulation in prophase.

Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM - J. Cell Biol. (2000)

Bottom Line: SA1 is also a subunit of 14S cohesin in Xenopus.The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase.These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

ABSTRACT
In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Show MeSH

Related in: MedlinePlus

The APC is not required for the mitotic dissociation of cohesins from chromatin. (A) CDC27 immunoblot showing Xenopus interphase extract before and after depletion with either control or CDC27 antibodies. (B) APC-depleted (left) and control-depleted (right) Xenopus interphase extracts were incubated with sperm nuclei for 30 min and then nondegradable cyclin B Δ90 was added to trigger entry into mitosis. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S35CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of 35S-labeled CDC25 and of endogenous cyclin B. Data from two different experiments are shown. In experiment 2, the degree of APC depletion and the cell cycle behavior of the extracts were the same as in experiment 1 (data not shown).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169443&req=5

Figure 8: The APC is not required for the mitotic dissociation of cohesins from chromatin. (A) CDC27 immunoblot showing Xenopus interphase extract before and after depletion with either control or CDC27 antibodies. (B) APC-depleted (left) and control-depleted (right) Xenopus interphase extracts were incubated with sperm nuclei for 30 min and then nondegradable cyclin B Δ90 was added to trigger entry into mitosis. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S35CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of 35S-labeled CDC25 and of endogenous cyclin B. Data from two different experiments are shown. In experiment 2, the degree of APC depletion and the cell cycle behavior of the extracts were the same as in experiment 1 (data not shown).

Mentions: To further test this hypothesis we immunodepleted the APC from Xenopus interphase extracts using antibodies to its subunit CDC27. Immunoblotting experiments indicated that at least 95% of the APC was removed from the extracts by these antibodies (Fig. 8 A). APC could also not be detected in protein extracts from demembranated sperm nuclei that were added as a chromatin source (data not shown), ruling out that the extract was supplemented with APC via this source. Upon mitotic activation, the depleted extracts were still able to phosphorylate CDC25 but could not degrade cyclin B, whereas cyclin B proteolysis occurred in extracts depleted with nonspecific control antibodies (Fig. 8 B). Importantly, the dissociation of cohesin subunits from chromatin occurred normally in the APC-depleted extracts (Fig. 8 B), demonstrating that the APC pathway is not required for this event.


Characterization of vertebrate cohesin complexes and their regulation in prophase.

Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM - J. Cell Biol. (2000)

The APC is not required for the mitotic dissociation of cohesins from chromatin. (A) CDC27 immunoblot showing Xenopus interphase extract before and after depletion with either control or CDC27 antibodies. (B) APC-depleted (left) and control-depleted (right) Xenopus interphase extracts were incubated with sperm nuclei for 30 min and then nondegradable cyclin B Δ90 was added to trigger entry into mitosis. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S35CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of 35S-labeled CDC25 and of endogenous cyclin B. Data from two different experiments are shown. In experiment 2, the degree of APC depletion and the cell cycle behavior of the extracts were the same as in experiment 1 (data not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169443&req=5

Figure 8: The APC is not required for the mitotic dissociation of cohesins from chromatin. (A) CDC27 immunoblot showing Xenopus interphase extract before and after depletion with either control or CDC27 antibodies. (B) APC-depleted (left) and control-depleted (right) Xenopus interphase extracts were incubated with sperm nuclei for 30 min and then nondegradable cyclin B Δ90 was added to trigger entry into mitosis. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S35CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of 35S-labeled CDC25 and of endogenous cyclin B. Data from two different experiments are shown. In experiment 2, the degree of APC depletion and the cell cycle behavior of the extracts were the same as in experiment 1 (data not shown).
Mentions: To further test this hypothesis we immunodepleted the APC from Xenopus interphase extracts using antibodies to its subunit CDC27. Immunoblotting experiments indicated that at least 95% of the APC was removed from the extracts by these antibodies (Fig. 8 A). APC could also not be detected in protein extracts from demembranated sperm nuclei that were added as a chromatin source (data not shown), ruling out that the extract was supplemented with APC via this source. Upon mitotic activation, the depleted extracts were still able to phosphorylate CDC25 but could not degrade cyclin B, whereas cyclin B proteolysis occurred in extracts depleted with nonspecific control antibodies (Fig. 8 B). Importantly, the dissociation of cohesin subunits from chromatin occurred normally in the APC-depleted extracts (Fig. 8 B), demonstrating that the APC pathway is not required for this event.

Bottom Line: SA1 is also a subunit of 14S cohesin in Xenopus.The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase.These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

ABSTRACT
In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Show MeSH
Related in: MedlinePlus