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. 2008 Feb;14(2):239-51.
doi: 10.1016/j.devcel.2007.12.009.

Cell-type-specific TEV Protease Cleavage Reveals Cohesin Functions in Drosophila Neurons

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Free PMC article

Cell-type-specific TEV Protease Cleavage Reveals Cohesin Functions in Drosophila Neurons

Andrea Pauli et al. Dev Cell. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Outline of the TEV-Cleavage System (A) Schematic of the cohesin complex containing TEV-cleavable Rad21 (green), SMC1 (red), SMC3 (blue), and Scc3/SA (yellow). Cleavage of Rad21 by separase occurs in the flexible linker region. Arrowheads indicate the sites of insertion of TEV-recognition sequences (numbers refer to amino acid positions). (B) Outline of the TEV-cleavage system showing two alternative methods to express TEV in vivo in flies. (a) UAS-TEV is controlled by the UAS/GAL4 system, enabling TEV expression by specific Gal4 driver lines. (b) TEV directly fused to the heat-shock promotor allows for its ubiquitous induction in a time-specific manner. (c) Once expressed, catalytically active TEV protease cleaves Rad21TEV. (C) Representation of the genomic region of the Rad21 locus. The Rad21 gene (CG17436) resides in the centric heterochromatin of chromosome 3L. The exon-intron structure of the Rad21 mRNA is shown in bold. EST-based transcript predictions of neighboring genes are depicted in lighter gray. The EP element GE50159 4 kb upstream of the transcriptional start of Rad21 is represented by a red triangle. The four independently generated imprecise excision mutants of Rad21 lack the chromosomal intervals indicated by solid, red lines. The Rad21 locus is missing in the γ-ray-induced deficiency Def 2-66 (dashed line). The scale bar is 10 kb. (D) Pupal protein extracts were prepared before (t = −0.75 hr) and at different time points after a 45 min heat shock at 37°C (red arrow). Western blot analysis with antibodies against endogenous Rad21 (left panel) or myc (right panel) shows full-length Rad21TEV (arrow) and the C-terminal TEV-cleavage product (arrowhead) as well as gRad21 (asterisk). V5-tagged TEV protease is detected by probing with v5 antibodies (open circle). Actin was used as a loading control. A molecular weight marker (in kDa) is shown on the left.
Figure 2
Figure 2
Cleavage of Rad21TEV during Cycle 14 Causes Precocious Sister-Chromatid Separation and Transient Mitotic Arrest (A) Cycle 14 embryos that survived on Rad21TEV and expressed maternally contributed Gal4 were fixed and double labeled (top rows) with anti-α-tubulin (Tub) and a DNA stain (DNA) or were triple labeled (bottom row) with DNA stain (blue), anti-BubR1 (green), and anti-Cyclin B (red). +TEV indicates the additional presence of the UAS-TEV transgene. The scale bars are 50 μm in the top left panel, 10 μm in the top right panel, and 10 μm in the bottom panel. (Top) Most cells in −TEV embryos have already completed mitosis 14 (arrowhead in whole embryo views). Dividing cells (arrow) during various mitotic stages (pro-, meta-, ana-, telophase) are shown in the high-magnification view. In +TEV embryos, the entire dorsolateral epidermis is arrested in mitosis. (Bottom) In −TEV embryos, high levels of BubR1 and Cyclin B are only observed during metaphase (m), whereas anaphase (a) cells do not stain for BubR1 and Cyclin B. Arrested cells of +TEV embryos are Cyclin B positive and have high levels of BubR1 on separated sister kinetochores. (B) Embryos surviving on Rad21TEV and expressing either only maternal Gal4 (−TEV) or maternal Gal4-driven TEV protease (+TEV) were used for time-lapse imaging. DNA is marked with H2Av-mRFP1; kinetochores are marked with EGFP-Cid. The onset of chromosome condensation was set to zero. Time points are indicated in seconds. Whereas the top two rows represent Z projections, the bottom rows show single confocal sections. The scale bars are 2 μm. (−TEV) Chromosomes congress into a metaphase plate (t = 180), followed by anaphase (t = 210) and telophase (t = 315). (+TEV) Chromosomes fail to congress into a metaphase plate, and sister chromatids separate prematurely (t = 75–105). Note the substantial mitotic delay (t = 630).
Figure 3
Figure 3
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.
Figure 4
Figure 4
Cohesin Is Not Required for the Maintenance of Polytene-Chromosome Morphology (A) Outline of the TEV-cleavage experiment in salivary glands. (B) Western blot analysis of salivary gland extracts prepared either before (t = −0.75 hr) or at various time points after heat shock (red arrow) from GFP-negative larvae. The last lane shows a sample of salivary glands from Rad21TEV-expressing flies that do not contain hs-TEV. Blots were probed with antibodies against myc (detecting full-length transgenic Rad21 [arrow] and the C-terminal TEV-cleavage fragment [arrowhead]) and v5 (detecting TEV protease [open circle]). (C) Representative polytene-chromosome spreads of third-instar larvae that carry hs-TEV and express either transgenic Rad21 (left panel) or Rad21TEV as their only source of Rad21 were prepared before (t = −0.75 hr) and at various time points after heat shock (red arrow). Polytene chromosomes were coimmunostained with antibodies against myc (recognizing Rad21) and CTCF. The morphology of the polytene chromosomes was visualized by DAPI staining (bottom row, higher magnification [2.5×]). All pictures were acquired by using the same acquisition settings. The scale bar is 20 μm.
Figure 5
Figure 5
Cohesin Is Expressed in γ Neurons and Can Be Selectively Destroyed by TEV Cleavage (A) Schematic representation of axonal projections of γ (green) and α/β (red) neurons of wild-type and pruning-defective mutants at three characteristic time points during development. Only the right hemisphere is shown. α′/β′ neurons are omitted from the scheme. In third-instar larva, γ-neuron axons are bundled in the peduncle before they bifurcate to project into the dorsal (d) and medial lobes (m) (filled, green arrowheads). At 18 hr after puparium formation (APF), the dorsal and medial projections from wild-type γ neurons are selectively eliminated (“pruned,” open, green arrowheads). In a pruning mutant, γ-neuron axon projections and dendrites persist (filled, green arrowheads). α/β neurons project into the dorsal and medial lobes. In late pupae/adults, axons of wild-type γ neurons grow out again toward the midline. In a pruning mutant, larval axon projections of γ neurons persist in the dorsal and medial lobes. (B) H24-Gal4 was used to drive expression of v5-tagged nuclear TEV protease and mCD8 in γ neurons of the mushroom body. Third-instar larval brains were immunostained with antibodies against mCD8 (green) and the v5 epitope (red). Images show Z projections of single confocal sections of the right brain hemisphere. The scale bar is 20 μm. (C) H24-Gal4 was used to drive expression of TEV and mCD8 in γ neurons of the mushroom body from flies that expressed endogenous Rad21 (gRad21, top) or Rad21TEV as their sole source of Rad21 (bottom). Brains were stained with antibodies against mCD8 (green) and Rad21 (red). Images show a single confocal section in the plane of γ-neuron cell bodies. Note that there is no overlap between the mCD8 and Rad21 stainings after TEV cleavage in γ neurons from Rad21TEV flies. The scale bars are 20 μm.
Figure 6
Figure 6
TEV Cleavage of Rad21 in γ Neurons Causes a Defect in Pruning (A and B) 201Y-Gal4 was used to drive expression of TEV and mCD8 in γ neurons of the mushroom body from flies that survived on transgenic Rad21 with or without TEV-cleavage sites. The scale bars are 20 μm. (A) Brains were dissected at 18 hr APF and were stained with antibodies against mCD8 (green) and FasII (red). Z projections of single confocal sections of the right brain hemisphere (left three panels). A single FasII-stained slice in the plane of α/β neurons (right panel). Absence/presence of γ-neuron projections (open/filled, green arrowheads), dendrites (green arrow), and α/β neurons (red arrows). In the bottom row, expression of Gal4 was suppressed in muscles by mhc-Gal80 in Rad21TEV flies. (B) Brains of Rad21TEV flies, in which Gal4 expression in muscles was suppressed by mhc-Gal80, were dissected at 18 hr APF and were stained with antibodies against mCD8 (green) and EcR-B1 (red). Images show single confocal sections in the plane of γ-neuron cell bodies. A higher-magnification view (10×) of the white-boxed area is shown on the right.
Figure 7
Figure 7
TEV Cleavage of Rad21 in Cholinergic Neurons Induces Severe Locomotion Defects in Third-Instar Larvae (A) Wandering third-instar larvae expressing TEV under the control of Cha-Gal4 and surviving on transgenic Rad21 with and without TEV sites were tested for motility (Rad21: Cha-Gal4/+; Rad21ex3, Rad21-myc/Rad21ex3, UAS-TEV; Rad21TEV: Cha-Gal4/+; Rad21ex15, Rad21TEV/Rad21ex3, UAS-TEV). Larval movements were tracked and superimposed to a grid. Locomotion was measured by the number of grid squares each larva traveled through. The number of larvae that traveled through the indicated number of squares (1–5, 6–10, etc.) is shown as a percentage of the total number of larvae tested (54 and 48 for strains containing Rad21 and Rad21TEV, respectively). (B) Representative images and temporal projections of movements from larvae that express TEV in cholinergic neurons and survive on either transgenic Rad21 (i and i′) or Rad21TEV (ii–v′) (same genotypes as in [A]). (i)–(v) show the initial position of the larvae. H indicates the position of the head. (i′)–(v′) show the temporal projections of the images taken over a 20 s interval (images taken every 2 s). Note that controls move mostly straight, whereas larvae in which Rad21TEV has been cleaved in cholinergic neurons show frequent episodes of turns, head movement, and backward motion.

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