Current Biology 18, 1426–1431, September 23, 2008 ª2008 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2008.08.061
Report
A Role for Very-Long-Chain Fatty Acids
in Furrow Ingression during Cytokinesis
in 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 Biology
Stanford University School of Medicine
Stanford, California 94305-5329
2Istituto Pasteur-Fondazione Cenci Bolognetti and
Istituto di Biologia e Patologia Molecolari del CNR
Dipartimento di Genetica e Biologia Molecolare
Università di Roma ‘‘La Sapienza’’
P.A. Moro 5
00185 Roma
Italy
3Department of Genetics
Stanford University School of Medicine
Stanford, California 94305-5120
Summary
Cell shape and membrane remodeling rely on regulated interactions between the lipid bilayer and cytoskeletal arrays
at the cell cortex. During cytokinesis, animal cells build an
actomyosin ring anchored to the plasma membrane at the
equatorial cortex. Ring constriction coupled to plasmamembrane 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 membrane domains are emerging as important factors in regulating 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 proteins
that mediate elongation of very-long-chain fatty acids [5],
block or dramatically slow cleavage-furrow ingression
during early telophase in dividing spermatocytes. In bond
mutant cells at late stages of division, the contractile ring frequently 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 membrane
deformation 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 Acids
in Cytokinesis
Loss of function of the Drosophila bond gene caused failure of
cytokinesis in dividing spermatocytes. Several alleles of bond
were identified in a screen for ethylmethane sulphonate (EMS)induced male-sterile mutations that cause defects in cytokinesis during male meiosis [4]. Round spermatids from bond mutant males commonly display two to four nuclei associated
with an abnormally large mitochondrial derivative ([4], Figures
*Correspondence: [email protected]
1A and 1B), indicating cytokinesis defects during the meiotic
divisions [4]. We mapped the bond locus to a small polytene
chromosome region (Supplemental Experimental Procedures,
available online) containing five genes (FlyBase) (Figure 1D).
Sequencing of five bond alleles revealed mutations in the annotated gene CG6921 (Figures 1E and 1F). A 7.5 kb wild-type
genomic fragment including CG6921 but not neighboring
genes (Figure 1D) rescued the cytokinesis defects of bond
mutants (Figure 1C), confirming CG6921 as bond.
bond encodes a member of the Elovl family of enzymes involved in elongation of very-long-chain fatty acids, which are
conserved from yeast to mammals (Figure 1E). Pair-wise Blast
analysis (NCBI bl2seq) revealed that the predicted Bond protein has 28% and 29% amino acid identity, respectively, to
the Saccharomyces cerevisiae Elovl proteins Elo2p and
Elo3p and 38% and 47% amino acid identity, respectively,
to human Elovl4 and Elovl7. Bond shares sequence motifs
with other Elovl proteins, including five to seven predicted
transmembrane domains [5, 6] (Kyte-Doolittle analysis), the
essential HXXHH motif conserved in the Elovl family [5, 6],
and a C-terminal sequence similar to the KXKXX motif of
some transmembrane proteins resident in the endoplasmic
reticulum. Elovl proteins are enzymes that participate in the
elongation of fatty acids with acyl chains longer than 18 carbon
atoms [5]. These very-long-chain fatty acids are most commonly found in sphingolipids and are essential for sphingolipid
formation [5] and function [7].
To determine whether Bond is a functional Elovl protein, we
tested whether the Drosophila gene can substitute for Elovl
genes. In S. cerevisiae, simultaneous loss of ELO2 and ELO3
causes lethality [8]. Overexpression of mammalian or Arabidopsis very-long-chain fatty-acid elongases had previously
been shown to restore viability of an elo2D elo3D double mutant and to generate very-long-chain fatty acids in yeast cells
[9, 10]. Overexpression of Bond in yeast also restored the
viability of an elo2D elo3D double mutant (Figure 1G), indicating that Bond indeed has elongase activity and can function as
an Elovl enzyme in yeast.
Expression of bond Is Cell-Type Specific
Bond is one of 20 predicted Elovl proteins in the Drosophila
genome, on the basis of tBlastn analysis (Figure 2A). Three of
the corresponding genes were previously described as having
restricted expression patterns [11–13], suggesting tissuespecific expression for different elovl genes. Analysis of bond
expression by in situ hybridization with two different probes
that distinguish bond from other elovl homologs revealed
bond transcripts in spermatocytes, but not in spermatogonia
(Figure 2B). This expression pattern is consistent with the cytokinesis defect observed in bond mutants: Multinucleate bond
spermatids contained a maximum of four nuclei, indicating failure of cytokinesis in the two meiotic divisions but not in the preceding mitotic divisions. Two other elovl homologs, CG17821
and CG31141, are expressed in the adult testis (FlyAtlas,
[14]). We confirmed testis expression of CG31141 and
CG17821 by RT-PCR and identified CG17821 expression in
spermatocytes by in situ hybridization; this gene, however,
failed to show Elovl activity in yeast (not shown).
Very-Long-Chain Fatty Acids in Cytokinesis
1427
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 spermatids showing nuclei (arrowheads) and mitochondria derivatives (arrows). (A) Normal mononucleate spermatids of
bondZ3437/TM6 flies. (B) Homozygous mutant bondZ3437
spermatids with four nuclei and a single enlarged mitochondrial 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 transmembrane domains and the C-terminal putative ER-retention 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 Arabidopsis 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 were
detected in stage 9–10 egg chambers
(Figure 2D). Mutants harboring loss-of-function
bond alleles (Figure 1F) were viable and developed to adulthood with no overt morphological
defects, but were male- and female-sterile. It is
possible that other elovl family members can provide enzymatic function in tissues other than spermatocytes.
bond transcripts were also detected in specific cell types in
embryos and ovaries. In embryos, bond transcripts localized
to groups of cells that participate in the formation of steroid-
Bond Is Required for Successful CleavageFurrow Ingression
Analysis of living spermatocytes revealed that loss
of Bond function dramatically affects cleavagefurrow ingression. In wild-type spermatocytes,
a GFP-tagged myosin regulatory light chain
(Sqh-GFP, [15]) assembled into a ring at the equator of dividing cells (n = 10) in early anaphase
(Figure 3A, 0 min; Movies S1 and S2), then immediately initiated constriction accompanied by membrane deformation into a furrow (Figure 3A, 5–25
min; Movies S1 and S2). The myosin regulatory
light chain, a subunit of myosin, can localize to
the cleavage furrow independently of F-actin [16].
We analyzed cell division in 15 spermatocytes
from flies transheterozygous for the two strong alleles, bondZ5274 and bondZ3437, and 33 spermatocytes from bondZ3437 homozygotes. The homozygotes and transheterozygotes displayed similar
phenotypes, indicating that the defects observed
were not due to secondary mutations. Seemingly
normal Sqh-GFP rings assembled during early
anaphase in the bond mutants. However, constriction of the Sqh-GFP rings was minimal or
substantially slowed in 10 of the 15 bondZ5274/bondZ3437 and
in 21 of the 33 bondZ3437 homozygous spermatocytes (Figures
3B and 3C; Movies S3–S6), and these cells ultimately failed to
Current Biology Vol 18 No 18
1428
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 that
depend on Bond can facilitate membrane curvature during cleavage-furrow ingression.
Previous analyses failed to detect F-actin
rings in most bond mutant spermatocytes,
which also displayed poorly constricted anillin
rings [4]. However, examination of fixed mutant spermatocytes with an improved F-actin
staining technique revealed the presence of
F-actin rings in 39 of 69 bond spermatocytes
in telophase. These actin rings were substantially thinner than the constricted F-actin rings
of wild-type cells and were extended or only
partially constricted (Figures 4A and 4B), consistent with the defect in cleavage-furrow ingression observed in live bond mutant cells.
complete cytokinesis during the period of observation. Measurements of Sqh-GFP ring diameter over time indicated that
the rate of ring constriction in the bond cells that went on to
fail cytokinesis was slower than in the wild-type (Figure 3D
and Figure S1). Sqh-GFP rings remained poorly constricted
in these cells for 15–27 min after the rings first appeared (Figures 3B and 3C; Figure S1). By this time, wild-type rings had
constricted almost to the size of the stable ring canals that remain to connect sister germ cells after cytokinesis (Figure 3A,
20 min; Figure S1).
Bond function also appeared to be required for long-term
stable attachment of the contractile machinery to the cell cortex. Late in cytokinesis, after furrow ingression had stalled for
some time, the plasma membrane detached from the contractile ring in bond mutants, and the furrow regressed rapidly (Figures 3B and 3C, arrowheads; Figure S1). At exactly the same
time, the poorly constricted Sqh-GFP rings constricted or
collapsed rapidly and directionally from the site of membrane
detachment toward the opposite side of the cell, where the
ring remained attached to the cell cortex (Figures 3B and 3C, arrows; Figure 3D; Figure S1). The rapid constriction of the SqhGFP ring after detachment from the membrane indicates that at
least in some cells, the actomyosin ring retained the capacity to
constrict in bond mutants. The slow rate of furrow ingression
observed in bond spermatocytes could be due to defects in
the ability of the membrane to deform and bend inward under
the contractile force of the actomyosin ring. Very-long-chain
fatty acids present in phosphoinositides have been implicated
in the stability of high membrane curvature [17], and sphingolipid- and cholesterol-rich domains at the plasma membrane appear to modulate membrane deformability [18]. Thus, it is
Mutations in bond Affect Organization of
the Central Spindle
Previous studies showed that telophase bond
spermatocytes exhibit severely defective
central spindles [4]. However, it was not clear when and how
the defects in microtubule organization arose in bond spermatocytes. We analyzed the central spindles in living spermatocytes from wild-type and bondZ6275 mutants expressing
EGFP-tagged b-tubulin. In agreement with previous observations [19], we found that in the wild-type, a population of microtubules extended from the poles to contact the cell cortex at
the future site of cleavage-furrow ingression in early anaphase
(Figure 4C, 0–15 min, arrows; Movie S7; n = 8). As these microtubules began to bundle, the cell elongated (Figure 4C, 10–20
min) and the microtubule array of the central spindle became
visible in the cell midzone (Figure 4C, 15–25 min). With ingression of the furrow, the pole-to-equatorial-cortex microtubule
bundles compacted together with the internal microtubule
array to form a tight telophase central spindle (Figure 4C, 25–
35 min).
In bond mutant spermatocytes (n = 8), microtubules from
the pole also contacted the equatorial cortex and bundled
there in early anaphase (Figure 4D, 5–10 min, arrows; Movie
S8). However, the internal microtubules failed to form an organized central-spindle array (Figure 4D, 10–20 min). The
cleavage furrow ingressed only minimally and stalled
(Figure 4D, 20–25 min). Eventually, the microtubule bundles
that contacted the equatorial cortex became disorganized
and the cleavage furrow regressed, as seen also with
Sqh-GFP (see above). Examination of living cells expressing
Pavarotti-YFP, a marker of microtubule plus ends, indicated
that internal microtubule arrays initially formed in early anaphase of bond mutants but were destabilized and lost
during the failure of furrow ingression (see Supplemental
Results).
Very-Long-Chain Fatty Acids in Cytokinesis
1429
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).
Current Biology Vol 18 No 18
1430
Figure 4. Actomyosin Rings and Central Spindles 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 expressing EGFP-tagged b-tubulin. Arrows indicate bundled astral microtubules that contact the equatorial membrane. The scale bars represent 10 mm.
Observation of living cells indicated that mutant spermatocytes did not elongate properly during anaphase and telophase. Whereas wild-type cells increased in pole-to-pole
length by w51% during this period, bond mutant cells elongated by only w24% (Figure S3). This difference became
apparent in early anaphase B, when wild-type cells initiated
furrow ingression accompanied by rapid elongation. The
maximum elongation of bond mutant cells was comparable
to that achieved by wild-type cells at the initiation of furrow
ingression.
The behavior of b-tubulin and Pavarotti in bond spermatocytes indicates that Bond function is not required for the
formation and behavior of the pole-to-equatorial-cortex microtubules, which are thought to determine the site of contractilering assembly and cleavage-furrow ingression (reviewed in
[20]). However, Bond function is required for formation of a robust central spindle, suggesting that its activity is necessary
for the stabilization of midzone microtubules during anaphase
and telophase. There are several non-mutually-exclusive
explanations for the abnormal central-spindle assembly in
bond mutants. It is possible that changes in membrane fattyacid composition due to loss of Bond function affect elements
of the contractile apparatus that normally stabilize midzone
microtubules to ensure the formation of a robust and well-organized central spindle [21, 22]. It is also possible that sideto-side compression of midzone microtubule bundles by ingression of the cleavage furrow in wild-type cells may facilitate
the formation of crossbridges that stabilize the dense array of
the telophase central spindle. In bond mutants, the slow cleavage-furrow ingression may delay formation of those crossbridges, allowing disassembly of microtubules from their minus ends. Finally, proper organization of the central spindle
may depend on the capacity of the cell to elongate along the
spindle 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 of
spermatocytes mutant in genes thought to affect membrane-
trafficking events, such as loss of function of Arf6 [23], Rab11 [24], or the phosphatidylinositol-transfer protein Vibrator/Giotto [25, 26]. In arf6 and rab11
mutant spermatocytes, initial rates of
cleavage-furrow ingression were comparable to the wild-type for more than
15 min, and telophase central spindles
formed transiently. When cytokinesis
failed in arf6 mutants, the furrow regressed slowly, and the ring of cortical
Sqh-GFP expanded with it, returning to
a large diameter [23]. In contrast, the rate of cleavage-furrow
ingression was slow almost from the outset in bond spermatocytes, telophase central spindles failed to organize, and in
most cells the Sqh-GFP ring eventually detached from the cortex and rapidly collapsed, rather than accompanying the regressing cleavage furrow. The striking difference between
the bond and arf6 phenotypes indicates that bond may contribute to cytokinesis thorough a role different from the support of membrane expansion mediated by membrane trafficking [27].
Our observations indicate that very-long-chain fatty acids
dependent on Bond function are required for successful cleavage-furrow ingression during cell division in spermatocytes.
Specialized very-long-chain fatty acids dependent on Bond
may be required to allow the plasma membrane to deform
for supple invagination during cleavage-furrow ingression.
The biophysical properties of very long acyl chains, proposed
to stabilize highly curved membranes [17], are consistent with
a role in membrane deformation during furrow ingression.
Membrane composition that depends on bond may also be
required to mediate proper membrane-contractile ring interactions. Growing evidence from studies of T cell activation
suggests that specialized membrane domains rich in sphingolipids and cholesterol regulate cytoskeletal-membrane interactions [3]. Sphingolipids have also been implicated in the activation of the Rho-Dia pathway that controls the tubulin and
actin cytoskeletons [28]. Thus, it is possible that bond-dependent lipids may participate in specialized membrane domains
that promote actin-ring formation and stability at the cortex.
Although we were unable to detect concentration of sphingolipids or cholesterol at the equator of dividing Drosophila spermatocytes or cultured S2 cells (data not shown), similar lipids
have been found to localize at the cytokinesis furrows of
fission yeast and sea urchin eggs [29, 30]. Consistent with
these observations, our findings indicate that very-long-chain
fatty acids that depend on bond, and their derived lipids, may
coordinate membrane deformation with the contractile activity
of the actomyosin ring, which leads to successful furrow
ingression during cytokinesis in Drosophila spermatocytes.
Very-Long-Chain Fatty Acids in Cytokinesis
1431
Supplemental Data
Supplemental Data include Supplemental Experimental Procedures, Supplemental 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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