Cell-Type-Specific TEV Protease Cleavage Reveals Cohesin Functions in Drosophila Neurons

Summary 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 α 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.


Generation of Rad21 alleles
The insertion site of the homozygous viable P{w + , EP}GE50159 line 4 kb upstream of the transcription start site of Rad21 was confirmed by inverse PCR according to a standard protocol. Imprecise excisions were generated by crossing the GE50159 line to flies expressing a stable source of the P-element transposase. Out of 500 excision events, 23 homozygous lethal lines were isolated. 4 independently generated deletions affecting Rad21 were subsequently identified by PCR (Rad21 ex3 , Rad21 ex8 , Rad21 ex15 , Rad21 ex16 ) and confirmed by sequencing DNA fragments spanning the breakpoints.

Generation of flies surviving on transgenic Rad21 TEV
C-terminally 10xmyc-tagged Rad21 was created based on the EST clone LD14219 obtained from the Berkeley Drosophila Genome Project (BDGP). The coding sequence of the 10xmyc-tag was amplified by PCR from the plasmid gthr-myc (Leismann et al., 2000) and cloned as Bst-BI fragment into the unique Bst-BI site in LD14219, located 12 nucleotides upstream of the Rad21 translational stop codon. The sequence encompassing Rad21-myc 10 was excised as a 2790 bp Eco-RI//Kpn-I fragment and cloned into pUAST (Brand and Perrimon, 1993) to obtain pUAS-Rad21myc 10 .
To generate TEV-cleavable versions of Rad21 (Rad21 TEV ), SpeI-restriction sites were introduced into the coding region of Rad21 after amino acids 175,197,271 or 550 by site-directed PCR-mutagenesis, using pUAS-Rad21-myc 10 as template (primer sequences see below). An AvrII/NheI restriction fragment encoding 3 tandem arrays of the TEV-recognition sequence ENLYFQG (kindly provided by Stephan Gruber, for details on the sequence see below) was inserted into the newly generated SpeI-site.
Transgenic lines were produced by standard P-element-mediated germline transformation (Rubin and Spradling, 1983). Transgenes were tested for their ability to rescue the lethality of Rad21 null mutations. Transgenes with TEV-cleavage sites at positions 271 or 550 and a transgene without TEV-sites were functional.

Immunoblotting
For the preparation of embryonic extracts, dechorionated embryos were homogenized in SDS-sample loading buffer 3-6 hours after egg deposition. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane.
Protein extracts from pupae, 3 rd instar larval salivary glands and 3rd instar larvae without salivary glands were prepared after dissection in PBS essentially as described in Experimental Procedures.

Primer and DNA sequences
Restriction enzyme sites are shown in lower case, the start codon ATG in bold, the v5-epitope sequence in italics, the NLS sequence underlined.