Current Biology 18, 1426–1431, September 23, 2008 ª2008 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2008.08.061

ReportA Role for Very-Long-Chain Fatty Acidsin Furrow Ingression during Cytokinesisin Drosophila Spermatocytes

Edith Szafer-Glusman,1 Maria Grazia Giansanti,2

Ryuichi Nishihama,3 Benjamin Bolival,1 John Pringle,3

Maurizio Gatti,2 and Margaret T. Fuller1,3,*1Department of Developmental BiologyStanford University School of MedicineStanford, California 94305-53292Istituto Pasteur-Fondazione Cenci Bolognetti andIstituto di Biologia e Patologia Molecolari del CNRDipartimento di Genetica e Biologia MolecolareUniversita di Roma ‘‘La Sapienza’’P.A. Moro 500185 RomaItaly3Department of GeneticsStanford University School of MedicineStanford, California 94305-5120

Summary

Cell shape and membrane remodeling rely on regulated in-

teractions between the lipid bilayer and cytoskeletal arraysat the cell cortex. During cytokinesis, animal cells build an

actomyosin ring anchored to the plasma membrane at theequatorial cortex. Ring constriction coupled to plasma-

membrane ingression separates the two daughter cells [1].Plasma-membrane lipids influence membrane biophysical

properties such as membrane curvature and elasticity and

play an active role in cell function [2], and specialized mem-brane domains are emerging as important factors in regulat-

ing assembly and rearrangement of the cytoskeleton [3].Here, we show that mutations in the gene bond [4], which

encodes a Drosophila member of the family of Elovl proteinsthat mediate elongation of very-long-chain fatty acids [5],

block or dramatically slow cleavage-furrow ingressionduring early telophase in dividing spermatocytes. In bond

mutant cells at late stages of division, the contractile ring fre-quently detaches from the cortex and constricts or collapses

to one side of the cell, and the cleavage furrow regresses.Our findings implicate very-long-chain fatty acids or their

derivative complex lipids in allowing supple membranedeformation and the stable connection of cortical contractile

components to the plasma membrane during cell division.

Results and Discussion

bond Mutants Implicate Very-Long-Chain Fatty Acidsin Cytokinesis

Loss of function of the Drosophila bond gene caused failure ofcytokinesis in dividing spermatocytes. Several alleles of bondwere identified in a screen for ethylmethane sulphonate (EMS)-induced male-sterile mutations that cause defects in cytokine-sis during male meiosis [4]. Round spermatids from bond mu-tant males commonly display two to four nuclei associatedwith an abnormally large mitochondrial derivative ([4], Figures

*Correspondence: mtfuller@standford.edu

1A and 1B), indicating cytokinesis defects during the meioticdivisions [4]. We mapped the bond locus to a small polytenechromosome region (Supplemental Experimental Procedures,available online) containing five genes (FlyBase) (Figure 1D).Sequencing of five bond alleles revealed mutations in the an-notated gene CG6921 (Figures 1E and 1F). A 7.5 kb wild-typegenomic fragment including CG6921 but not neighboringgenes (Figure 1D) rescued the cytokinesis defects of bondmutants (Figure 1C), confirming CG6921 as bond.

bond encodes a member of the Elovl family of enzymes in-volved in elongation of very-long-chain fatty acids, which areconserved from yeast to mammals (Figure 1E). Pair-wise Blastanalysis (NCBI bl2seq) revealed that the predicted Bond pro-tein has 28% and 29% amino acid identity, respectively, tothe Saccharomyces cerevisiae Elovl proteins Elo2p andElo3p and 38% and 47% amino acid identity, respectively,to human Elovl4 and Elovl7. Bond shares sequence motifswith other Elovl proteins, including five to seven predictedtransmembrane domains [5, 6] (Kyte-Doolittle analysis), theessential HXXHH motif conserved in the Elovl family [5, 6],and a C-terminal sequence similar to the KXKXX motif ofsome transmembrane proteins resident in the endoplasmicreticulum. Elovl proteins are enzymes that participate in theelongation of fatty acids with acyl chains longer than 18 carbonatoms [5]. These very-long-chain fatty acids are most com-monly found in sphingolipids and are essential for sphingolipidformation [5] and function [7].

To determine whether Bond is a functional Elovl protein, wetested whether the Drosophila gene can substitute for Elovlgenes. In S. cerevisiae, simultaneous loss of ELO2 and ELO3causes lethality [8]. Overexpression of mammalian or Arabi-dopsis very-long-chain fatty-acid elongases had previouslybeen shown to restore viability of an elo2D elo3D double mu-tant and to generate very-long-chain fatty acids in yeast cells[9, 10]. Overexpression of Bond in yeast also restored theviability of an elo2D elo3D double mutant (Figure 1G), indicat-ing that Bond indeed has elongase activity and can function asan Elovl enzyme in yeast.

Expression of bond Is Cell-Type Specific

Bond is one of 20 predicted Elovl proteins in the Drosophilagenome, on the basis of tBlastn analysis (Figure 2A). Three ofthe corresponding genes were previously described as havingrestricted expression patterns [11–13], suggesting tissue-specific expression for different elovl genes. Analysis of bondexpression by in situ hybridization with two different probesthat distinguish bond from other elovl homologs revealedbond transcripts in spermatocytes, but not in spermatogonia(Figure 2B). This expression pattern is consistent with the cyto-kinesis defect observed in bond mutants: Multinucleate bondspermatids contained a maximum of four nuclei, indicating fail-ure of cytokinesis in the two meiotic divisions but not in the pre-ceding mitotic divisions. Two other elovl homologs, CG17821and CG31141, are expressed in the adult testis (FlyAtlas,[14]). We confirmed testis expression of CG31141 andCG17821 by RT-PCR and identified CG17821 expression inspermatocytes by in situ hybridization; this gene, however,failed to show Elovl activity in yeast (not shown).

Very-Long-Chain Fatty Acids in Cytokinesis1427

bond transcripts were also detected in specific cell types inembryos and ovaries. In embryos, bond transcripts localizedto groups of cells that participate in the formation of steroid-

Figure 1. Loss of Function of bond, a Conserved Elovl Gene,

Causes Cytokinesis Defects in Drosophila Spermatocytes

(A–C) Phase-contrast images of Drosophila round sperma-

tids showing nuclei (arrowheads) and mitochondria deriva-

tives (arrows). (A) Normal mononucleate spermatids of

bondZ3437/TM6 flies. (B) Homozygous mutant bondZ3437

spermatids with four nuclei and a single enlarged mitochon-

drial derivative. (C) Mononucleate spermatids of bondZ3437/

bondZ3437 rescued with a wild-type bond transgene. The

scale bar represents 20 mm.

(D) Map of the bond (CG6921) genomic region with the 7.5 kb

rescue fragment.

(E) ClustalW alignment of Bond to yeast Elo2p and Elo3p and

human ELOVL4 with the five most strongly predicted trans-

membrane domains and the C-terminal putative ER-reten-

tion sequence underlined. (Box) Conserved HXXHH motif;

(asterisks) sites of mutations in bond alleles.

(F) Molecular defects in bond alleles. Amino acid positions

are relative to the predicted start of translation. Allele

strengths are based on the frequencies of multinucleate

round spermatids in homozygotes.

(G) Rescue of a yeast elo2D elo3D strain by bond or the Ara-

bidopsis At2g16280 ORF, but not by the empty expression

vector (see Supplemental Experimental Procedures).

derived molecules, such as oenocytes(Figure 2C) and corpus alatum (data not shown;Flybase/BDGP, http://www.fruitfly.org/cgi-bin/ex/insitu.pl). In ovaries, bond transcripts weredetected in stage 9–10 egg chambers(Figure 2D). Mutants harboring loss-of-functionbond alleles (Figure 1F) were viable and devel-oped to adulthood with no overt morphologicaldefects, but were male- and female-sterile. It ispossible that other elovl family members can pro-vide enzymatic function in tissues other than sper-matocytes.

Bond Is Required for Successful Cleavage-Furrow Ingression

Analysis of living spermatocytes revealed that lossof Bond function dramatically affects cleavage-furrow ingression. In wild-type spermatocytes,a GFP-tagged myosin regulatory light chain(Sqh-GFP, [15]) assembled into a ring at the equa-tor of dividing cells (n = 10) in early anaphase(Figure 3A, 0 min; Movies S1 and S2), then immedi-ately initiated constriction accompanied by mem-brane deformation into a furrow (Figure 3A, 5–25min; Movies S1 and S2). The myosin regulatorylight chain, a subunit of myosin, can localize tothe cleavage furrow independently of F-actin [16].

We analyzed cell division in 15 spermatocytesfrom flies transheterozygous for the two strong al-leles, bondZ5274 and bondZ3437, and 33 spermato-cytes from bondZ3437 homozygotes. The homozy-gotes and transheterozygotes displayed similarphenotypes, indicating that the defects observedwere not due to secondary mutations. Seeminglynormal Sqh-GFP rings assembled during earlyanaphase in the bond mutants. However, con-striction of the Sqh-GFP rings was minimal or

substantially slowed in 10 of the 15 bondZ5274/bondZ3437 andin 21 of the 33 bondZ3437 homozygous spermatocytes (Figures3B and 3C; Movies S3–S6), and these cells ultimately failed to

Current Biology Vol 18 No 181428

complete cytokinesis during the period of observation. Mea-surements of Sqh-GFP ring diameter over time indicated thatthe rate of ring constriction in the bond cells that went on tofail cytokinesis was slower than in the wild-type (Figure 3Dand Figure S1). Sqh-GFP rings remained poorly constrictedin these cells for 15–27 min after the rings first appeared (Fig-ures 3B and 3C; Figure S1). By this time, wild-type rings hadconstricted almost to the size of the stable ring canals that re-main to connect sister germ cells after cytokinesis (Figure 3A,20 min; Figure S1).

Bond function also appeared to be required for long-termstable attachment of the contractile machinery to the cell cor-tex. Late in cytokinesis, after furrow ingression had stalled forsome time, the plasma membrane detached from the contrac-tile ring in bond mutants, and the furrow regressed rapidly (Fig-ures 3B and 3C, arrowheads; Figure S1). At exactly the sametime, the poorly constricted Sqh-GFP rings constricted orcollapsed rapidly and directionally from the site of membranedetachment toward the opposite side of the cell, where thering remained attached to the cell cortex (Figures 3B and 3C, ar-rows; Figure 3D; Figure S1). The rapid constriction of the Sqh-GFP ring after detachment from the membrane indicates that atleast in some cells, the actomyosin ring retained the capacity toconstrict in bond mutants. The slow rate of furrow ingressionobserved in bond spermatocytes could be due to defects inthe ability of the membrane to deform and bend inward underthe contractile force of the actomyosin ring. Very-long-chainfatty acids present in phosphoinositides have been implicatedin the stability of high membrane curvature [17], and sphingoli-pid- and cholesterol-rich domains at the plasma membrane ap-pear to modulate membrane deformability [18]. Thus, it is

Figure 2. bond Is Expressed in Spermatocytes

(A) Phylogenetic tree [31] of the Elovl proteins from

S. cerevisiae, human, and D. melanogaster.

(B–D) In situ hybridization. (B) Expression of bond mRNA

in spermatocytes (arrow) but not in spermatogonia at the

apical region of the testis (arrowhead). (C) Embryonic

expression in oenocytes (arrowhead) and in a segment

of the intestine (arrow). (D) Expression in the ovary in

stage 9–10 egg chambers. The scale bar represents

100 mm.

possible that very-long-chain fatty acids thatdepend on Bond can facilitate membrane cur-vature during cleavage-furrow ingression.

Previous analyses failed to detect F-actinrings in most bond mutant spermatocytes,which also displayed poorly constricted anillinrings [4]. However, examination of fixed mu-tant spermatocytes with an improved F-actinstaining technique revealed the presence ofF-actin rings in 39 of 69 bond spermatocytesin telophase. These actin rings were substan-tially thinner than the constricted F-actin ringsof wild-type cells and were extended or onlypartially constricted (Figures 4A and 4B), con-sistent with the defect in cleavage-furrow in-gression observed in live bond mutant cells.

Mutations in bond Affect Organization of

the Central SpindlePrevious studies showed that telophase bondspermatocytes exhibit severely defective

central spindles [4]. However, it was not clear when and howthe defects in microtubule organization arose in bond sper-matocytes. We analyzed the central spindles in living sper-matocytes from wild-type and bondZ6275 mutants expressingEGFP-tagged b-tubulin. In agreement with previous observa-tions [19], we found that in the wild-type, a population of micro-tubules extended from the poles to contact the cell cortex atthe future site of cleavage-furrow ingression in early anaphase(Figure 4C, 0–15 min, arrows; Movie S7; n = 8). As these micro-tubules began to bundle, the cell elongated (Figure 4C, 10–20min) and the microtubule array of the central spindle becamevisible in the cell midzone (Figure 4C, 15–25 min). With ingres-sion of the furrow, the pole-to-equatorial-cortex microtubulebundles compacted together with the internal microtubulearray to form a tight telophase central spindle (Figure 4C, 25–35 min).

In bond mutant spermatocytes (n = 8), microtubules fromthe pole also contacted the equatorial cortex and bundledthere in early anaphase (Figure 4D, 5–10 min, arrows; MovieS8). However, the internal microtubules failed to form an or-ganized central-spindle array (Figure 4D, 10–20 min). Thecleavage furrow ingressed only minimally and stalled(Figure 4D, 20–25 min). Eventually, the microtubule bundlesthat contacted the equatorial cortex became disorganizedand the cleavage furrow regressed, as seen also withSqh-GFP (see above). Examination of living cells expressingPavarotti-YFP, a marker of microtubule plus ends, indicatedthat internal microtubule arrays initially formed in early ana-phase of bond mutants but were destabilized and lostduring the failure of furrow ingression (see SupplementalResults).

Figure 3. Cleavage Furrows Fail to Ingress and Contractile Rings Eventually Detach from Plasma Membranes in bond Spermatocytes

(A–C) Frames from time-lapse sequences. Phase-contrast (upper panels) and Sqh-GFP (lower panels) images of spermatocytes undergoing cytokinesis in

bondZ3437/TM6 (A), bondZ3437/bondZ5274 (B), and bondZ3437/ bondZ3437 (C) flies. Time 0 is the time of earliest Sqh-GFP detection at the cell equator. Arrows

indicate detached Sqh-GFP rings; the arrowheads indicate plasma membranes (outlined in white). The scale bars represent 10 mm.

(D) Dynamics of plasma-membrane ingression and contractile-ring contraction during cytokinesis in wild-type and bond spermatocytes, with diameters of

Sqh-GFP rings and cleavage furrows (measured from the phase-contrast images) plotted over time. (Solid circles) Sqh-GFP ring and furrow diameters in

a typical wild-type spermatocyte; ring and furrow diameters were identical throughout cytokinesis in these cells. (Open circles) Sqh-GFP rings; (dots)

furrows in bond cells. (Left) Cell in (B); (right) cell in (C).

Very-Long-Chain Fatty Acids in Cytokinesis1429

Observation of living cells indicated that mutant spermato-cytes did not elongate properly during anaphase and telo-phase. Whereas wild-type cells increased in pole-to-polelength by w51% during this period, bond mutant cells elon-gated by only w24% (Figure S3). This difference becameapparent in early anaphase B, when wild-type cells initiatedfurrow ingression accompanied by rapid elongation. Themaximum elongation of bond mutant cells was comparableto that achieved by wild-type cells at the initiation of furrowingression.

The behavior of b-tubulin and Pavarotti in bond spermato-cytes indicates that Bond function is not required for theformation and behavior of the pole-to-equatorial-cortex micro-tubules, which are thought to determine the site of contractile-ring assembly and cleavage-furrow ingression (reviewed in[20]). However, Bond function is required for formation of a ro-bust central spindle, suggesting that its activity is necessaryfor the stabilization of midzone microtubules during anaphaseand telophase. There are several non-mutually-exclusiveexplanations for the abnormal central-spindle assembly inbond mutants. It is possible that changes in membrane fatty-acid composition due to loss of Bond function affect elementsof the contractile apparatus that normally stabilize midzonemicrotubules to ensure the formation of a robust and well-or-ganized central spindle [21, 22]. It is also possible that side-to-side compression of midzone microtubule bundles by in-gression of the cleavage furrow in wild-type cells may facilitatethe formation of crossbridges that stabilize the dense array ofthe telophase central spindle. In bond mutants, the slow cleav-age-furrow ingression may delay formation of those cross-bridges, allowing disassembly of microtubules from their mi-nus ends. Finally, proper organization of the central spindlemay depend on the capacity of the cell to elongate along thespindle axis, which appears compromised in bond cells(Figure 4D and Figure S3).

Role of Bond in Spermatocyte Cytokinesis

The phenotype of bond spermatocytes differed from those ofspermatocytes mutant in genes thought to affect membrane-

Figure 4. Actomyosin Rings and Central Spin-

dles Are Abnormal in bond Mutants

(A and B) bondZ3437/TM6 (A) and bondZ6275/

bondZ3734 (B) telophase spermatocytes stained

for actin (red), tubulin (green), and DNA (blue).

Arrowheads indicate actin rings.

(C and D) Living bondZ6275/TM6 (C) and

bondZ6275/bondZ6275 (D) spermatocytes express-

ing EGFP-tagged b-tubulin. Arrows indicate bun-

dled astral microtubules that contact the equato-

rial membrane. The scale bars represent 10 mm.

trafficking events, such as loss of func-tion of Arf6 [23], Rab11 [24], or the phos-phatidylinositol-transfer protein Vibra-tor/Giotto [25, 26]. In arf6 and rab11mutant spermatocytes, initial rates ofcleavage-furrow ingression were com-parable to the wild-type for more than15 min, and telophase central spindlesformed transiently. When cytokinesisfailed in arf6 mutants, the furrow re-gressed slowly, and the ring of corticalSqh-GFP expanded with it, returning to

a large diameter [23]. In contrast, the rate of cleavage-furrowingression was slow almost from the outset in bond spermato-cytes, telophase central spindles failed to organize, and inmost cells the Sqh-GFP ring eventually detached from the cor-tex and rapidly collapsed, rather than accompanying the re-gressing cleavage furrow. The striking difference betweenthe bond and arf6 phenotypes indicates that bond may con-tribute to cytokinesis thorough a role different from the sup-port of membrane expansion mediated by membrane traffick-ing [27].

Our observations indicate that very-long-chain fatty acidsdependent on Bond function are required for successful cleav-age-furrow ingression during cell division in spermatocytes.Specialized very-long-chain fatty acids dependent on Bondmay be required to allow the plasma membrane to deformfor supple invagination during cleavage-furrow ingression.The biophysical properties of very long acyl chains, proposedto stabilize highly curved membranes [17], are consistent witha role in membrane deformation during furrow ingression.Membrane composition that depends on bond may also berequired to mediate proper membrane-contractile ring in-teractions. Growing evidence from studies of T cell activationsuggests that specialized membrane domains rich in sphingo-lipids and cholesterol regulate cytoskeletal-membrane inter-actions [3]. Sphingolipids have also been implicated in the ac-tivation of the Rho-Dia pathway that controls the tubulin andactin cytoskeletons [28]. Thus, it is possible that bond-depen-dent lipids may participate in specialized membrane domainsthat promote actin-ring formation and stability at the cortex.Although we were unable to detect concentration of sphingo-lipids or cholesterol at the equator of dividing Drosophila sper-matocytes or cultured S2 cells (data not shown), similar lipidshave been found to localize at the cytokinesis furrows offission yeast and sea urchin eggs [29, 30]. Consistent withthese observations, our findings indicate that very-long-chainfatty acids that depend on bond, and their derived lipids, maycoordinate membrane deformation with the contractile activityof the actomyosin ring, which leads to successful furrowingression during cytokinesis in Drosophila spermatocytes.

Current Biology Vol 18 No 181430

Very-Long-Chain Fatty Acids in Cytokinesis1431

Supplemental Data

Supplemental Data include Supplemental Experimental Procedures, Sup-

plemental Results, three figures, and twelve movies and can be found

with this article online at http://www.current-biology.com/cgi/content/full/

18/18/1426/DC1/.

Acknowledgments

We thank Charles Zuker, Gotha Goshima, David Glover, and Roger Karess

for providing fly stocks and reagents, Theresa Dunn for sharing yeast stocks

and plasmids, and Carmen Robinett for comments on the manuscript. This

work was funded by American Heart Association fellowship 0525061Y to

E.G., National Institutes of Health Grants GM31006 to J.R.P. and

GM62276 to M.T.F., and funds from the Reed-Hodgson Professorship to

M.T.F.

Received: April 23, 2008

Revised: July 26, 2008

Accepted: August 5, 2008

Published online: September 18, 2008

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