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Nedd5, a Mammalian Septin, Is a Novel Cytoskeletal Component Interacting with Actin-Based Structures

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Nedd5, a Mammalian Septin, Is a Novel Cytoskeletal Component Interacting with Actin-Based Structures Downloaded from genesdev.cshlp.org on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures Makoto Kinoshita, 1,6 Sharad Kumar, 4 Akira Mizoguchi, 2 Chizuka Ide, 2 Ayae Kinoshita, 3 Tokuko Haraguchi, 5 Yasushi Hiraoka, 5 and Makoto Noda 1 Departments of 1Molecular Oncology, 2Anatomy and Neurobiology, and 3Neurology, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan; 4Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, SA5000, Australia; SKansai Advanced Research Center, Communications Research Laboratory, Kobe 651-24, Japan The mouse Nedd5 gene encodes a 41.5-kD GTPase similar to the Saccharomyces and Drosophila septins essential for cytokinesis. Nedd5 accumulates near the contractile ring from anaphase through telophase, and finally condenses into the midbody. Microinjection of anti-Nedd5 antibody interferes with cytokinesis, giving rise to binucleated cells. In interphase and postmitotic cells, Nedd5 localizes to fibrous or granular structures depending on the growth state of the cell. The Nedd5-containing fibers are disrupted by microinjection of GTP~,S and by Nedd5 mutants lacking GTP-binding activity, implying that GTP hydrolysis is required for its assembly. The Nedd5-containing fibers also appear to physically contact actin bundles and focal adhesion complexes and are disrupted by cytochalasin D, C3 exoenzyme, and serum starvation, suggesting a functional interaction with the actin-based cytoskeletal systems in interphase cells. [Key Words: GTPase; cytokinesis; stress fiber; focal adhesion; neural development] Received March 3, 1997; revised version accepted May 6, 1997. Molecular and genetic studies in yeast and fruit fly have 1996). These data suggest that septin filaments may play revealed the existence of a novel class of cytoskeletal a common role in cytokinesis in both unicellular and components, septins, which constitute an evolutionarily multicellular organisms. conserved GTPase family. Loss-of-function mutants in In an attempt to elucidate molecular mechanisms in- any of the four septin genes in Saccharomyces cerevi- volved in neural development, we isolated a series of siae, CDC3, CDCIO, CDCll, and CDC12, are deficient genes, named Neddl-lO, that are highly expressed in in the M0-nm "neck filaments" that normally localize mouse neuronal precursor cells (NPC) and down-regu- beneath the cleavage furrow between mother and bud lated in adult brain (Kumar et al. 1992). We found that (Byers and Goetsch 1976; for review, see Byers 1981), and one such gene, Nedd5, belongs to the growing family of give rise to multinucleated cells with abnormal bud septins. At least five mammalian septin genes, Diff6 growth at restrictive temperature (Hartwell 1971; for re- (Nottenburg et al. 1990), H5 (Kato 1990, 1992), and view, see Pringle and Hartwell 1981). Localization of Nedd5 (Kumar et al. 1992) in mouse, and hCDCIO (Na- each gene product to the cleavage furrow of the wild-type katsuru et al. 1994) and KIAA0158 (Nagase et al. 1995) in strain has also been demonstrated, establishing that they human, have been isolated so far, but their products re- are the constituent of the bud neck filament (Haarer and main uncharacterized. Here we report on the structure, Pringle 1987; Ford and Pringle 1991; Kim et al. 1991). expression, subcellular localization, and possible func- Similarly, a mutation in a Drosophila septin gene, pnut, tions of the Nedd5 gene and its product in mammalian leads to a lethal phenotype with multinucleated cells in cells. the larval tissues (Neufeld and Rubin 1994). Pnut and another Drosophila septin, Sep 1, colocalize near the con- tractile ring in dividing cells (Neufeld and Rubin 1994; Results Fares et al. 1995). Recently, the three Drosophila septins, Cloning and structure of Nedd5 cDNA Pnut, Sepl, and Sep2, have been purified and their GT- In a previous subtraction screening for developmentally Pase activity and ability to heteropolymerize to form a down-regulated genes in mouse brain, a cDNA fragment 7-nm filament in vitro were demonstrated (Field et al. corresponding to the 3'-terminal 240 bases of Nedd5 mRNA, 4D12, was isolated (Kumar et al. 1992). Because 6Corresponding author. the 4D12 cDNA detects mRNA of -3.5 kb in Northern E-MAIL [email protected]; FAX 81-75-751-4159. blot analyses (see below), this probe was used to screen GENES & DEVELOPMENT 11:1535-1547 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 1535 Downloaded from genesdev.cshlp.org on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Kinoshita et al. mouse cDNA libraries for the full-length cDNA, and a nylation signal (AATAAA) located 26 bases upstream of sequence of 3175 bases was isolated (GenBank accession the poly(A) initiation site. no. D49382). The first in-frame ATG in a good context for mammalian translation initiation (Kozak 1986) is preceded by an in-frame stop codon located 21 bases up- Nedd5 encodes a member of the septin family stream. The open reading frame of 1083 bases encodes a polypeptide of 361 amino acids with a predicted isoelec- A database search revealed that the putative Nedd5 prod- tric point of 6.09 and a molecular mass of 41.5 kD (Fig. uct shares similarities with yeast, fruit fly, and mamma- 1A), which is consistent with the results of transcrip- lian septins (Fig. 1B). The Nedd5 protein contains a set of tion/translation of the cDNA in vitro (data not shown) consensus motifs found in GTPases (Saraste et al. 1990): and immunoblot analysis (see below). The 3' untrans- The G1, G3, and G4 regions are highly conserved in the lated region contains six destabilization signals septin family but are distinct from those found in other (ATTTA) (Wilson and Treisman 1988) and a polyade- GTPase subfamilies (Bourne et al. 1991) (Fig. 1A, C). Sec- ondary structure prediction (Chou and Fasman 1978) in- dicated a possible s-helix near the carboxyl terminus of Neddb. This region also contains an interrupted leucine/ isoleucine repeat at every seventh amino acid residue A MSKQQPTQF INPETPGYVGFANL PNQVHRK$VKKGFEFT~ TL INSL (Fig. 1A), which may form a coiled-coil structure (Lupas FLTDLYPERI I PC~AJ%.EKIERTVO iEASTVEI EERGVKLRL~GDAINCRDC et al. 1991) as suggested in most of the other septins FKTI I SY I DEOFERYLHDESGLNRRHI IDNRVHCCFYF I S PFGHGLKPLDVAFMKAI ~[~'VNIVPVIAKADTLTLKERERLKKRILDE I EEH$ IKIYHLPDAESDEDEDFKEQT (Flescher et al. 1993; Neufeld and Rubin 1994; Fares et al. RLLK~ I PF SWGSNOLIE~GKKVRGRLY~EHNDFLKLRT~. ITHNQ 1995, 1996). DLQEAri~DLHYENFR S ERLKRGG~ I LLEKEAELRRMOEMI ARMOAO * , . , , MOMOMOGGDSDSGALGQHV amino acid number of molecular B identity (%) amino acid mass (kD~ Expression of Nedd5 mRNA Mm Nedd5 100 361 41.5 Hs Nedd5 99 361 41,5 A cDNA probe containing the entire open reading frame Dm Sep 1 74 361 41.1 detected a single band of -3.5 kb in Northern blot analy- Mm Diff6 55 365 41.9 sis with mouse tissues (Fig. 2A). The intensity of this Sc Cdcl0p 41 322 37.0 band gradually decreases during embryonic and postna- Sc Cdcl2p 38 407 46.7 tal brain development. In situ hybridization revealed ~ Dm Sep2 43 419 48.4 Sc Cdcl Ip 37 415 47.6 that Nedd5 was ubiquitously expressed in embryos and ~.~ Mm H5 63 478 54.9 down-regulated in the majority of neuronal populations Dm Pnut 54 539 60.2 in the adult brain (Fig. 2B). Nedd5 mRNA was detected Sc Cdc3p 41 520 60.1 in all adult tissues and cell lines examined, such as PCC4 (mouse embryonal carcinoma), CTLL2 (mouse T- G cell leukemia}, C6 (rat astrocytoma), PC12 (rat pheochro- mocytoma), HeLa, SiHa (human uterine carcinoma), and Mm Nedd5 (32 ) [C<G~'~'I~SGI_~K6TLINSLFLTDLY (29 )VKLRL ~VVDTPGYGDAIN {32 ) Em Sepl (32) ................. ~--V ......... (29) ............ F---M- (32] HT1080 (human fibrosarcoma)(Fig. 2C; data not shown). M~n H5 (30) .... m ..... A t .... ~-~---N ........ (29) -R .... I ..... F---V- (32) Dm Pnut (137) -R .......... A---~ -L ..... M--S-I- (29) -N-T .... ~---F---VD(32 ) Mm Diff6 (20) .... D ..... A ...... - .......... N-- (29) I-VK--L ..... F--S%©(32 ] Dm Sep2 (38) QN--V-NV-CI--T ....... MDT--N-SF. (25) -R-K--IC--V .... Q-- (34} Sc Cdc3p ( 114 ) -N--,-,-,--,,-,--,------,-,, (50 ) ---N-N-I--E-F--FL- (33 ) Sc Cdcl0p (27 ) .... Q-NI----Q .......... T--ASH-I ( 30 ) -R-NIN- I .... F--F-D (33 ) Characterization of an ti-Nedd5 antibodies ScCdcllp (17)-R-IT--V-I -Q--S-R--F--T-CGQQVV(32)--IQ-NII .... F--SLD(33) Sc Cdcl2p (29 ) EE-GT--V-LC ....... T-F--T--Q-V-K (29 ) FE--VN- I .... F--N-g- (32 ) We raised rabbit antisera against Nedd5 using two anti- G4 :XZXD gens: (1) a synthetic peptide, G2, corresponding to resi- Mm Nedd5 DNRVHCCFYFI SPFGHGLKP~IVPVIAKADTLTLK]KRERI/fKRIL ( 161 ) Dm Sepl ---I ................... E---KL-S ........ ----C--K--IL---C--M( 163 } dues K74-T85 conserved between mouse and human Mm H5 ....... L .......... R .... E .... L-QR ..... IL-- ..... PP-VD-K-CK-R (171) Dm Pnut ....... L---A-S .... L---I-C-QSLSD---LI ..... S-I--DD-VHLF--Q- -(234) proteins, and (2) a recombinant polypeptide, N5C, corre- Mm Diff6 -S ..... L ....... RAPA-. RCG-LR-V-E .... I---G---A-MPR-TQA--QK-R (178 ] sponding to a carboxy-terminal portion (R303-V361) of Dm Sep2 -S-I-I-L---C-T ..... S--LVC--KLDS .... I ........ ISKV-LQ-F-AK- I (216} Sc Cdc3p -K-I-A L---E-T--Y ..... LK--QSVYE-C-LI ..... S-I--DE-ILSF--T-M(216] mouse Neddb. The authenticity of each affinity-purified Sc Cdcl 0p -T- - -AIL--LQ-N-KE-SR---EAL-RLTEIA-VI- --G-S ..... D- -TEFREL-Q (125 ] Sc Cdcllp -G ..... L-L-N-T ..... EI--E- IRQLGSL---I---S-S-S--RD-LKI/W--L-M (226 ] antibody was tested by immunoblot analysis using ly- Sc Cdcl2p -L-- -AVL---R-T .....
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