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Cell-type-specific TEV protease cleavage reveals cohesin functions in Drosophila neurons.

Pauli A, Althoff F, Oliveira RA, Heidmann S, Schuldiner O, Lehner CF, Dickson BJ, Nasmyth K - Dev. Cell (2008)

Bottom Line: Crucially, cleavage in postmitotic neurons is lethal.In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion.These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

ABSTRACT
Cohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the alpha kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

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Cohesin Binds to Distinct Regions on Polytene Chromosomes(A) Polytene chromosomes of wild-type flies (w1118) were stained with Rad21 antibodies (green) and DAPI (DNA, red). The lower panel shows a higher-magnification view (2.5×). The strongly DAPI-stained heterochromatic chromocenter (arrow) is devoid of Rad21 staining. The scale bars are 20 μm.(B) Polytene chromosomes from flies expressing myc-tagged Rad21TEV in addition to endogenous Rad21 were coimmunostained with antibodies against Rad21 (green) and myc (red). DNA was visualized with DAPI (blue). In the right two frames, part of one chromosome arm is shown at higher magnification with split Rad21- and myc channels. The scale bars are 20 μm in the left four frames and 10 μm in the right two frames.
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fig3: Cohesin Binds to Distinct Regions on Polytene Chromosomes(A) Polytene chromosomes of wild-type flies (w1118) were stained with Rad21 antibodies (green) and DAPI (DNA, red). The lower panel shows a higher-magnification view (2.5×). The strongly DAPI-stained heterochromatic chromocenter (arrow) is devoid of Rad21 staining. The scale bars are 20 μm.(B) Polytene chromosomes from flies expressing myc-tagged Rad21TEV in addition to endogenous Rad21 were coimmunostained with antibodies against Rad21 (green) and myc (red). DNA was visualized with DAPI (blue). In the right two frames, part of one chromosome arm is shown at higher magnification with split Rad21- and myc channels. The scale bars are 20 μm in the left four frames and 10 μm in the right two frames.

Mentions: Immunostaining of wild-type polytene-chromosome squashes showed that Rad21, detected with a Rad21-specific antibody, localizes mainly to interband regions (Figure 3A), as has been suggested in previous reports (Dorsett et al., 2005; Gause et al., 2007; Markov et al., 2003). Several lines of evidence imply that these bands genuinely correspond to cohesin. First, coimmunostainings showed that myc-tagged Rad21TEV is bound to the same chromosomal regions as endogenous Rad21 (Figure 3B). Second, cohesin's other three subunits (SMC1, SMC3, and SA/Scc3) colocalize with Rad21 on polytene-chromosome squashes (Figure S3A). Third, staining by myc-, Rad21-, and SMC1-specific antibodies is greatly reduced after TEV-induced cleavage of Rad21TEV in flies in which this is the only form of Rad21 (Figure S3B). The fact that SMC proteins are also released implies that TEV cleavage of Rad21 releases the entire cohesin complex from chromosomes. Cohesin did not colocalize with known interband-specific proteins (Z4, BEAF32, Jil1, MSL2, CTCF), and its distribution differed significantly from numerous other proteins whose localization on polytene chromosomes has been well documented (PolII, Rpb3, HSF, trx, Pc, Su[Hw], CP190, Mod[mdg4]) (Figures S4A and S4B and data not shown). The cohesin holocomplex appears to be bound to distinct but as yet undefined regions of polytene chromosomes.


Cell-type-specific TEV protease cleavage reveals cohesin functions in Drosophila neurons.

Pauli A, Althoff F, Oliveira RA, Heidmann S, Schuldiner O, Lehner CF, Dickson BJ, Nasmyth K - Dev. Cell (2008)

Cohesin Binds to Distinct Regions on Polytene Chromosomes(A) Polytene chromosomes of wild-type flies (w1118) were stained with Rad21 antibodies (green) and DAPI (DNA, red). The lower panel shows a higher-magnification view (2.5×). The strongly DAPI-stained heterochromatic chromocenter (arrow) is devoid of Rad21 staining. The scale bars are 20 μm.(B) Polytene chromosomes from flies expressing myc-tagged Rad21TEV in addition to endogenous Rad21 were coimmunostained with antibodies against Rad21 (green) and myc (red). DNA was visualized with DAPI (blue). In the right two frames, part of one chromosome arm is shown at higher magnification with split Rad21- and myc channels. The scale bars are 20 μm in the left four frames and 10 μm in the right two frames.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2258333&req=5

fig3: Cohesin Binds to Distinct Regions on Polytene Chromosomes(A) Polytene chromosomes of wild-type flies (w1118) were stained with Rad21 antibodies (green) and DAPI (DNA, red). The lower panel shows a higher-magnification view (2.5×). The strongly DAPI-stained heterochromatic chromocenter (arrow) is devoid of Rad21 staining. The scale bars are 20 μm.(B) Polytene chromosomes from flies expressing myc-tagged Rad21TEV in addition to endogenous Rad21 were coimmunostained with antibodies against Rad21 (green) and myc (red). DNA was visualized with DAPI (blue). In the right two frames, part of one chromosome arm is shown at higher magnification with split Rad21- and myc channels. The scale bars are 20 μm in the left four frames and 10 μm in the right two frames.
Mentions: Immunostaining of wild-type polytene-chromosome squashes showed that Rad21, detected with a Rad21-specific antibody, localizes mainly to interband regions (Figure 3A), as has been suggested in previous reports (Dorsett et al., 2005; Gause et al., 2007; Markov et al., 2003). Several lines of evidence imply that these bands genuinely correspond to cohesin. First, coimmunostainings showed that myc-tagged Rad21TEV is bound to the same chromosomal regions as endogenous Rad21 (Figure 3B). Second, cohesin's other three subunits (SMC1, SMC3, and SA/Scc3) colocalize with Rad21 on polytene-chromosome squashes (Figure S3A). Third, staining by myc-, Rad21-, and SMC1-specific antibodies is greatly reduced after TEV-induced cleavage of Rad21TEV in flies in which this is the only form of Rad21 (Figure S3B). The fact that SMC proteins are also released implies that TEV cleavage of Rad21 releases the entire cohesin complex from chromosomes. Cohesin did not colocalize with known interband-specific proteins (Z4, BEAF32, Jil1, MSL2, CTCF), and its distribution differed significantly from numerous other proteins whose localization on polytene chromosomes has been well documented (PolII, Rpb3, HSF, trx, Pc, Su[Hw], CP190, Mod[mdg4]) (Figures S4A and S4B and data not shown). The cohesin holocomplex appears to be bound to distinct but as yet undefined regions of polytene chromosomes.

Bottom Line: Crucially, cleavage in postmitotic neurons is lethal.In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion.These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

ABSTRACT
Cohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the alpha kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

Show MeSH
Related in: MedlinePlus