2323
Journal of Cell Science 112, 2323-2334 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0470
The role of anillin in meiotic cytokinesis of Drosophila males
Maria Grazia Giansanti1, Silvia Bonaccorsi1,2 and Maurizio Gatti1,2,*
1Istituto
Pasteur-Fondazione Cenci Bolognetti and 2Centro di Genetica Evoluzionistica del CNR, Dipartimento di Genetica e
Biologia Molecolare, Universita’ di Roma ‘La Sapienza’, P.le A. Moro 5, 00185 Roma, Italy
*Author for correspondence
Accepted 11 May; published on WWW 24 June 1999
SUMMARY
Anillin is a 190 kDa actin-binding protein that concentrates
in the leading edges of furrow canals during Drosophila
cellularization and in the cleavage furrow of both somatic
and meiotic cells. We analyzed anillin behavior during D.
melanogaster spermatogenesis, and focused on the
relationships between this protein and the F-actin enriched
structures. In meiotic anaphases anillin concentrates in a
narrow band around the cell equator. Cytological analysis
of wild-type meiosis and examination of mutants defective
in contractile ring assembly (chickadee and KLP3A),
revealed that the formation of the anillin cortical band
occurs before, and does not require the assembly of the Factin based contractile ring. However, once the acto-myosin
ring is assembled, the anillin band precisely colocalizes
with this cytokinetic structure, accompanying its
contraction throughout anaphase and telophase. In
chickadee and KLP3A mutant ana-telophases the cortical
anillin band fails to constrict, indicating that its contraction
is normally driven by the cytokinetic ring. These findings,
coupled with the analysis of anillin behavior in twinstar
mutants, suggested a model on the role of anillin during
cytokinesis. During anaphase anillin would concentrate in
the cleavage furrow before the assembly of the contractile
ring, binding the equatorial cortex, perhaps through its
carboxy-terminal pleckstrin homology (PH) domain.
Anillin would then interact with the actin filaments of the
acto-myosin ring through its actin-binding domain,
anchoring the contractile ring to the plasma membrane
throughout cytokinesis.
INTRODUCTION
Drosophila males four gonial divisions generate 16 primary
spermatocytes which remain connected by cytoplasmic bridges
called the ring canals (henceforth abbreviated as RCs). These
cells, engulfed in a cyst formed by two specialized cyst cells,
undergo a dramatic growth phase that results in a 25-fold
increase in nuclear volume. Mature spermatocytes enter
meiosis synchronously and produce 64 spermatids which also
remain connected by RCs (Lindsley and Tokuyasu, 1980;
Fuller, 1993; Hime et al., 1996). Each spermatid consists of a
nucleus and a mitochondrial derivative called the nebenkern.
During both meiotic divisions mitochondria associate
lengthwise along the central spindle and are equally partitioned
between the two daughter cells when cytokinesis occurs. Thus,
newly formed ‘onion stage’ spermatids have spherical
nebenkerns of constant dimensions, each associated with a
nucleus of similar shape and size (Lindsley and Tokuyasu,
1980; Fuller, 1993; Cenci et al., 1994). Failures of meiotic
cytokinesis can be easily detected because they result in
spermatids containing abnormally large nebenkerns associated
with two or four normally-sized nuclei (Fuller, 1993; Castrillon
and Wasserman, 1994; Williams et al., 1995; Gunsalus et al.,
1995; Carmena et al., 1998; Giansanti et al., 1998).
Examination of male meiotic and postmeiotic stages not
only provides a way to assess failures in cytokinesis but may
also reveal the primary defects that impair the cytokinetic
process. Due to the relatively large size of primary
In animal cells cytokinesis is accomplished by the contraction
of a ring-shaped cellular structure containing actin and myosin
II filaments. This structure is anchored to the plasma
membrane at the equator of the dividing cell and constricts it
like a purse string, leading to the separation of the two daughter
cells (reviewed by Fishkind and Wang, 1995; Glotzer, 1997;
Goldberg et al., 1998). Although this ‘contractile ring’ model
is the commonly accepted mechanism for cytokinesis, there are
several questions that remain to be answered. For example,
little is known about the structural and regulatory proteins that
compose the contractile ring in addition to actin and myosin
II, and about the molecules that mediate the anchoring of the
ring to the cell cortex. Moreover, the molecular mechanisms
underlying the assembly, contraction and disassembly of
contractile ring are still poorly understood. Finally, although
there is evidence that the spindle dictates the positioning and
timing of ring assembly, the molecules involved in these
signaling pathways have not been identified.
Drosophila melanogaster male meiosis provides an
excellent model system for the genetic, molecular and
cytological analysis of cytokinesis (Cenci et al., 1994;
Williams et al., 1995; Gunsalus et al., 1995; Giansanti et al.,
1996; Hime et al., 1996; Basu et al., 1998; Carmena et al.,
1998; Giansanti et al., 1998; Herrmann et al., 1998). In
Key words: Anillin, Cytokinesis, Contractile ring, Ring canal,
Fusome, Drosophila, Spermatogenesis
2324 M. G. Giansanti, S. Bonaccorsi and M. Gatti
spermatocytes, meiotic spindles are very prominent and can be
readily detected by tubulin immunostaining. In wild-type
males both meiotic divisions exhibit conspicuous central
spindles which are pinched in the middle during telophase
(Cenci et al., 1994). These cells also exhibit an actin-based
contractile ring around the central spindle midzone where
pinching occurs (Gunsalus et al., 1995; Hime et al., 1996;
Giansanti et al., 1998).
Several mutations have been identified which disrupt
meiotic cytokinesis and affect specific cellular structures
involved in this process. For example, mutations in KLP3A, a
gene encoding a kinesin-like protein that accumulates at the
central spindle midzone, disrupt the central spindle formation
and suppress contractile ring assembly (Williams et al., 1995;
Giansanti et al., 1996, 1998). Similar meiotic phenotypes are
produced by mutations in chickadee (chic) and diaphanous
(dia) (Giansanti et al., 1996, 1998). chic encodes a Drosophila
profilin, a low molecular mass protein that regulates actin
polymerization (Cooley et al., 1992); the Dia polypeptide is
homologous to the limb deformity gene product of mouse, to
the BNI1 protein of Saccharomyces cerevisiae and to the Cdc
12 protein of Schizosaccharomyces pombe (Castrillon and
Wasserman, 1994; Chang et al., 1996). The Cdc 12 protein
interacts with profilin through its prolin-rich domain and is
required for both actin ring formation and cytokinesis (Chang
et al., 1997). These findings have suggested a cooperative
interaction between the central spindle microtubules and
elements of the cortical actin cytoskeleton involved in the
assembly of the contractile ring (Giansanti et al., 1996, 1998).
A simultaneous absence of the central spindle and the
contractile ring, accompanied by a failure in cytokinesis, has
been also observed in embryonic cells of mutants in the
pavarotti (pav) locus. pav encodes a kinesin-like protein (PAVKLP) which forms a complex with the POLO kinase and
concentrates in the central spindle midzone during telophase
(Adams et al., 1998). These findings indicate that the
interdependence of the central spindle and the contractile ring
may be a common feature of both meiotic and somatic cells.
Interestingly, meiotic cells of polo males also exhibit defects
in both the central spindle and the contractile ring, resulting in
frequent disruptions of cytokinesis (Carmena et al., 1998).
Because the PAV-KLP and POLO proteins appear to be
mutually dependent for their correct localization, the
cytological phenotype observed in polo meiosis may be a
consequence of the incorrect localization of either protein
(Carmena et al., 1998).
While KLP3A, chic, dia and polo are required for contractile
ring assembly, twinstar (tsr) is primarily involved in the
disassembly of this structure (Gunsalus et al., 1995). tsr
encodes a polypeptide homologous to the cofilins, a class of
proteins that can sever and depolymerize actin filaments in
vitro (reviewed by Moon and Drubin, 1995). In tsr mutants
contractile rings are initially morphologically regular and
undergo a normal contraction. However, at the end of each
meiotic division, they become abnormally shaped, fail to
disassemble and form large and persistent F actin aggregates
which interfere with the proper execution of cytokinesis
(Gunsalus et al., 1995).
Another protein that is likely to play an important role in
meiotic cytokinesis of Drosophila males is anillin. Anillin is a
190 kDa protein isolated by actin filament cromatography of
Drosophila embryo extracts (Miller et al., 1989). Molecular
analysis of the anillin gene showed that its product is not
homologous to known polypeptides. However, in vitro studies
revealed that anillin can bundle actin filaments through a
novel actin-binding domain. Indirect immunofluorescence
experiments using antibodies to anillin showed that this protein
concentrates in the leading edges of furrow canals during
cellularization and in the cleavage furrows of a variety of
Drosophila cell types, including tissue culture cells, gonial
cells of both sexes and male meiotic cells (Field and Alberts,
1995; Hime et al., 1996; de Cuevas and Spradling, 1998). In
addition, anillin accumulates in the walls of RCs
interconnecting developing germ line cells of both sexes, and
in somatic RCs that link ovarian follicle cells (Field and
Alberts, 1995; Hime et al., 1996; de Cuevas and Spradling,
1998).
In the present paper we have analyzed anillin behavior
during Drosophila spermatogenesis, with a particular focus on
its role during meiotic cytokinesis. Moreover, we have
examined by immunofluorescence the relationships between
anillin and actin in mutants with altered actin behavior, such
as KLP3A, chic and tsr. The results of our analyses indicate
that anillin concentrates in the cleavage furrow during gonial
mitoses and meiotic divisions, and in the RCs that interconnect
germ cells. Anillin is not always associated with F-actin but
exhibits a dynamic pattern of F-actin binding. Our data are
consistent with the hypothesis that anillin concentrates in
specialized cortical sites with an unknown mechanism,
mediating the anchoring of F-actin to these sites at particular
times of the cell cycle. Our studies on the relationships between
anillin and F-actin have also provided detailed information on
the organization and development of the male fusome.
MATERIALS AND METHODS
Drosophila stocks
Immunocytological analyses of wild-type spermatogenesis were made
using an Oregon-R stock that has been maintained in our laboratory
for about 30 years. Observations on KLP3A mutants were performed
using the KLP3Ae4 allele which carries a deficiency that removes 82
amino acids at the COOH terminus of the KLP3A protein (Williams
et al., 1995). To obtain KLP3Ae4 males, KLP3Ae4/FM7 virgin females
were mated with FM7 males and their progeny scored for Malpighian
tubule coloration (Williams et al., 1995).
Analyses on chic and tsr were performed using the chic R1, chic35A
(Giansanti et al., 1998) and tsr1 mutant alleles (Gunsalus et al., 1995).
These mutations were all induced by single P element mutagenesis
and were kept over the compound balancer ST, kindly provided by Dr
Garcia-Bellido, Madrid. ST is a translocation between the second
chromosome balancer SM6a and the third chromosome balancer
TM6b, that carries the body-shape marker Tubby (Tb) and the
dominant marker Curly (Cy). Homozygous mutant larvae and pupae
were distinguished from their heterozygous sibs for their non-Tubby
phenotype.
The flies were reared on standard Drosophila medium at 25+1°C.
Fixation procedures
Dissection, fixation and staining, if not otherwise specified, were
performed at room temperature. Cytological preparations were made
with testes from third instar larvae or young pupae, using three
different procedures. In two of these procedures, testes were dissected
in testis isolation buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-
The role of anillin in cytokinesis 2325
Fig. 1. Fusome development during Drosophila spermatogenesis. The merged images were obtained by overlapping the anillin and F-actin
signals. (A) A 4-cell gonial cyst with 3 RCs traversed by the fusome. The anillin not associated with RCs is concentrated in the nucleus but
excluded from the area occupied by the chromatin. Note that the cyst cell nuclei (arrows) contain less anillin than the spermatogonial nuclei.
(B) An 8-cell gonial cyst with a growing fusome. The fusome elements associated with the newly formed RCs are not yet connected with the
central fusome. Anillin has just begun to enter the nuclei and forms a thin layer around the chromatin. The arrow points to the two cyst cells
which are very close to each other. (C) A cyst containing 16 young primary spermatocytes connected by 15 RCs. Seven of these RCs are
traversed by a preexisting fusome, while the 8 newly formed RCs exhibit actin-enriched, growing fusome elements. The anillin not associated
with RCs has a cytoplasmic localization. The cyst cells cannot be unambiguously recognized. (D) A partial cyst containing 4 primary
spermatocytes in the S3 stage and 3 ring canals connected by the fusome. Anillin is concentrated in the nucleus and excluded from the
nucleolus. Bar, 5 µm.
HCl, pH 6.8), gently squashed in 2 µl of the same buffer under a
20×20 coverslip, and frozen in liquid nitrogen. After the removal of
the coverslip, testis preparations were fixed according to either of the
following protocols: (1) For simultaneous immunostaining with antiα-tubulin and anti-anillin antibodies, the slides were fixed by cold
methanol and acetone according to the method of Cenci et al. (1994).
(2) For F-actin staining with phalloidin the slides were fixed with
3.7% formaldeyde according to the method of Gunsalus et al. (1995).
For simultaneous immunostaining with anti-α-tubulin and anti-anillin
antibodies, we also used the following procedure (protocol 3). Testes
were dissected in saline (0.7% NaCl in distilled water) and fixed in
3.7% formaldehyde in PBS for 30 minutes. They were rinsed for 30
seconds in 45% acetic acid and transferred into a drop (4 µl) of 60%
acetic acid placed on a 20×20 coverslip. Testes were kept in 60%
acetic acid for 2-3 minutes and then squashed applying a moderate
pressure. Slides were frozen in liquid nitrogen and, after removal of
the coverslip, immersed in ethanol at −20°C for 15 minutes. Slides
were then incubated in PBT (PBS containing 0.1% Triton-X) for 10
minutes, washed 2 times (5 minutes each) in PBS and air dried.
Staining procedures
Double and triple stainings were always performed in the following
2326 M. G. Giansanti, S. Bonaccorsi and M. Gatti
order: immunostaining, phalloidin staining and Hoechst 33258
staining. Before immunostaining, fixed preparations were incubated
with 1% BSA (Sigma) in PBS for 45 minutes. All subsequent
incubations with antibodies were performed in a humid chamber in
the dark.
For anillin localization two different rabbit antisera were used as
primary antibodies, one raised against amino acids 1-371 and the other
against amino acids 401-828 (Field and Alberts, 1995), both diluted
1:300 in 1% BSA in PBT. After overnight incubation at 4°C, the slides
were washed twice in PBT and once in PBS, for a total of 15 minutes.
The primary antibodies were then detected by incubation for one
hour with either tetramethylrhodamine isothiocyanate- (TRITC)
conjugated anti-rabbit IgG (Cappel), diluted 1:50 in PBT, or with
fluorescein isothiocyanate- (FITC) conjugated anti-rabbit IgG
(Cappel), diluted 1:50 in PBT. Regardless of the protocol used for
fixation, these two antisera produced identical immunostaining
patterns for all cell types in Drosophila spermatogenesis.
For simultaneous visualization of anillin and microtubules, testis
preparations were first immunostained with anti-anillin antibodies as
described above; they were then treated for 45 minutes with a
monoclonal anti-α-tubulin antibody (Amersham; Blose et al., 1982),
diluted 1:50 in PBS. After two washes in PBS (5 minutes each), the
slides were incubated with the secondary antibody (sheep anti-mouse
IgG F(ab′)2 fragment conjugated with 5(6)-carboxy-fluorescein-Nhydroxysuccinimide ester (FLUOS), Boehringer), diluted 1:10 in
PBS.
For actin staining, testis preparations were incubated with
rhodamine-labelled phalloidin (Molecular Probes) dissolved in PBS
(100 units/µl) for 1 hour and 30 minutes at 37°C in the dark, and then
rinsed for 2 minutes in PBS. To prepare the phalloidin solution, an
aliquot of a stock solution of rhodamine-labelled phalloidin in
methanol (300 units in 1.5 ml of methanol) was vacuum dried and
resuspended in the appropriate volume of PBS.
After immunostaining, or immunostaining plus actin staining, testis
preparations were air dried and stained with Hoechst 33258 according
to the method of Bonaccorsi et al. (1988).
Microscopy and image analysis
All preparations were examined with a Zeiss Axioplan microscope
equipped with an HBO 50W mercury lamp (Osram) for
epifluorescence, and with a cooled charge-coupled device (CCD;
Photometrics). Hoechst 33258, FLUOS or FITC, and TRITC
fluorescence were detected using the 0.1 (BP 365/11, FT 395, LP
397), 10 (BP 450/490, FT 510, LP 515/565) and 15 (BP 546, FT 580,
LP 590) Zeiss filter sets, respectively. Gray-scale digital images were
collected separately using the IP Lab Spectrum software, converted to
Photoshop 2.5 format (Adobe), pseudocoloured and merged. We also
recorded the phase-contrast images underlying the various types of
immunostained preparations. The combined examination of these
images with those produced by fluorescent stainings enabled us to
unambiguously recognize the various stages into which Drosophila
spermatogenesis has been subdivided (Cenci et al., 1994).
RESULTS
Anillin localization in premeiotic stages
To analyze anillin localization in spermatogonia we examined
cysts containing 2, 4 or 8 cells. The Hoechst 33258 staining
pattern (Cenci et al., 1994) and the developmental stage of
the fusome (see below) permitted unambiguous distinction
between cysts containing cells in early interphase and those
with late interphase cells. In late interphase spermatogonia
anillin is localized primarily within the nucleus but it is
excluded from the chromatin. Because in these cells
chromatin occupies most of the nuclear space, anillin is
usually concentrated in a narrow layer at the periphery of the
nucleus (Fig. 1A). During gonial divisions anillin is released
in the cytoplasm and concentrates in the cleavage furrow at
anaphase and telophase (not shown). At the end of each
gonial division the two daughter cells remain connected by a
RC which is a derivative of the contractile ring (Hime et al.,
1996). A fraction of the anillin accumulated in the cleavage
furrow remains in the RC, while another, larger fraction of
this protein becomes diffuse in the cytoplasm. Thus, in
recently divided, early interphase gonial cells anillin has a
predominant cytoplasmic localization (not shown). However,
as the cell cycle proceeds, it progressively migrates into the
nucleus (Fig. 1B).
Anillin also has a cytoplasmic localization in very young
spermatocytes (Fig. 1C) but, here again, it rapidly concentrates
into the nucleus as these cells enter their growth phase. During
spermatocyte growth anillin remains confined to the nucleus
and to the RCs (Figs 1D and 2). In early stages of spermatocyte
growth (stages S1-S4; see Cenci et al., 1994, for stage
nomenclature) anillin tends to be excluded from the nucleolus
(Fig. 2). However, in mature spermatocytes (stage S5) anillin
progressively concentrates in this structure, but is released
again into the nucleoplasm when the nucleolus breaks down
during the S6 stage (Figs 2 and 3).
Previous studies have shown that anillin consistently
decorates the male RCs. These structures develop from arrested
contractile rings after a specialized type of cytokinesis in which
the closing of the invaginating plasma membrane is incomplete
(Hime et al., 1996). In each cyst the number of RCs is therefore
equal to the number of germ line cells contained in the cyst
minus one. Thus, gonial cysts of 2, 4, and 8 cells and primary
spermatocyte cysts of 16 cells contain 1, 3, 7 and 15 RCs,
respectively (Hime et al., 1996; Figs 1 and 2). Interestingly,
once formed, these RCs persist without substantial
morphological changes throughout spermatogenesis (Hime et
al., 1996; see below).
F-actin staining by phalloidin and anillin immunostaining
revealed that RCs are often traversed by a ribbon-like structure
containing F-actin. This structure, which has been already
identified as the male fusome (Gunsalus et al., 1995; Hime et
al., 1996), exhibits a characteristic developmental pattern (Fig.
1). In 2-, 4- and 8-cell cysts, when spermatogonia are about to
enter mitotic division, all the RCs present in any given cyst are
traversed by the fusome. At the end of cell division, the newly
formed RCs are very similar but not identical to contractile
rings (see also below). Whereas in contractile rings F-actin and
anillin colocalize precisely, in new RCs F-actin tends to be
slightly dislocated with respect to anillin. In particular, in some
cases we can see small actin-rich masses protruding from one
or both sides of the anillin rings. As cells proceed through the
cell cycle, these actin ‘plugs’ progressively extend, and
eventually join to the preexisting fusome. For example, a 4-cell
gonial cyst, before entering mitosis, contains 3 RCs connected
by the fusome (Fig. 1A). After completion of cell division, the
8 early interphase cells contained in the cyst exhibit 3 RCs
traversed by the fusome and 4 newly formed RCs (Fig. 1B).
As cell cycle progresses these 4 RCs emanate new fusome
elements which eventually become connected to the
preexisting fusome.
The 16-cell primary spermatocyte cysts exhibit a similar
The role of anillin in cytokinesis 2327
pattern of fusome development. In very young spermatocytes
(stages S0 and S1) the fusome connects 7 RCs, while the 8
new RCs are not associated with this structure (Fig. 1C). Here
again, as spermatocyte growth proceeds, these 8 RCs become
traversed by newly formed actin-rich fusome elements which
progressively conjoin to the preexisting ones, forming a
continuous branched structure. By the S3 stage, in most cysts
all the 15 RCs are connected by the fusome (Fig. 1D). The
primary spermatocyte fusome persists till the S4 stage and
then gradually disassembles. In mature spermatocytes at the
S5 stage the fusome regresses and breaks down into pieces
(Fig. 4).
Anillin behavior in meiotic and postmeiotic stages
In the M1a and M1b stages, when chromatin has already
attained a high degree of condensation and two prominent
asters are present, anillin is still concentrated in the nucleus
(Fig. 3). In the M2 prometaphase stage, when the laminbased nuclear envelope dissolves (Cenci et al., 1994; WhiteCooper et al., 1993; Eberhart and Wasserman, 1995), anillin
diffuses in the cytoplasm (Fig. 3). During metaphase (stage
M3) and early anaphase I (stage M4a) anillin remains diffuse
within the cell (Fig. 5A,D). However, at mid-anaphase (stage
M4b) anillin concentrates in a narrow circumferential band
around the equator of the cell (Figs 5B,E, 6A). Simultaneous
staining with phalloidin and anti-anillin antibodies revealed
that the anillin band appears earlier than the actin ring (Fig.
6). Anillin accumulation at the cleavage furrow is clearly
visible when the cell is in mid-anaphase and the central
spindle has just begun to assemble (stage M4b; Figs 5B,E,
6A). The F-actin ring becomes first detectable when the
dividing cell is further elongated and the central spindle
is fully formed (Fig. 6B). Remarkably, since its first
appearance, the actin ring colocalizes with the anillin
cortical band. These two structures remain strictly associated
till the completion of acto-myosin ring contraction (stage
M5; Figs 5C,F, 6C).
Interestingly, in preparations fixed after testis squashing and
freezing (protocols 1 and 2; see Materials and Methods), the
anillin band encircling mid-anaphase cells (stage M4b) is often
interrupted and/or dislocated from the cell equator (Figs 5B,E,
6A). However, if preparations are fixed before squashing and
freezing (protocol 3; see Materials and Methods), the anillin
band of M4b cells is usually continuous and consistently
located at the cell equator (Fig. 7A,B). Regardless of the type
of fixation, when anillin becomes associated with the actin ring
it is almost invariably organized in a continuous structure
which encircles the central spindle midzone (Fig. 6B). The
most obvious interpretation of these findings is that the
discontinuity and mislocation of the anillin band observed in
M4b cells fixed according to either protocol 1 or 2, is due to
the rupture of the plasma membrane with which anillin is
associated, during the squashing/freezing procedure. M4b cells
treated with formaldheyde before squashing (protocol 3) are
hardened by the fixation, and are thus likely to be much less
subject to rupturing than cells squashed and frozen before
fixation. Similarly, when the anillin band is tightly associated
with the acto-myosin ring, it is likely to be resistant to damage
caused by squashing and freezing.
At the end of the first meiotic division the contractile ring
reorganizes into a RC. Here again, only a fraction of the anillin
initially accumulated in the cleavage furrow remains in the RC
(Fig. 5). Most of the anillin associated with the cytokinetic
apparatus, during the isovolumetric contraction of the
actomyosin ring, diffuses in the cytoplasm.
During telophase I the two daughter nuclei reform a nuclear
envelope, becoming sharply demarcated from the cytoplasm.
This nuclear/cytoplasmic demarcation persists through the
short interphase between the first and the second meiotic
division (stage M6) and early prometaphase II (stage M7), but
it disappears when cells enter late prometaphase II (stage M8).
Despite the presence of a regular nuclear envelope, telophase
I cells and secondary spermatocytes at the M6-M7 stages do
not accumulate anillin into the nucleus; in these cells the anillin
not associated with RCs is dispersed in the cytoplasm (Fig.
5G). In meiotic prometaphase, metaphase and early anaphase
II (stages M8-M10a) anillin remains dispersed in the
cytoplasm; it concentrates in a cortical band at the equator of
the cell during mid-anaphase (stage M10b). Here again, the
anillin band appears earlier than the actin ring which first
becomes detectable in stage M10c. In subsequent meiotic
stages anillin behaves just as in the first meiotic division and
eventually becomes a component of the newly formed RCs
(Fig. 5).
In postmeiotic stages the overall amount of anillin detectable
by immunofluorescence appears to be substantially reduced
with respect to the previous stages of germ cell differentiation.
Little or no anillin appears to be present either in the cytoplasm
or in the spermatid nucleus (Fig. 8). However, anillin remains
associated with the spermatid RCs which appear to contain the
same amount of anillin as those of premeiotic and meiotic
stages (Fig. 8).
At the end of the second meiotic division spermatid cysts
contain 63 RCs. While a few of these RCs are associated with
persisting fragments of the spermatocyte fusome, most of them
have the typical appearance of newly formed RCs (i.e. they
contain a small plug of actin enriched material, as shown
in Fig. 1). As spermatogenesis proceeds, the spermatid RCs
become progressively traversed by actin-rich ribbon-like
structures that eventually merge in a highly branched fusome
(not shown). By the onion stage (T4 according to Cenci et al.,
1994) most, if not all, the spermatid RCs are connected by the
fusome.
Anillin behavior in mutants affecting cytokinesis
To obtain further insight into the interactions between anillin
and F-actin during male meiosis, we immunostained with antianillin antibodies KLP3A, chic and tsr mutant testes. As
mentioned previously, mutations in KLP3A and chic suppress
the formation of both the central spindle and the contractile
ring (Williams et al., 1995; Giansanti et al., 1996, 1998). In
late telophase, mutants in tsr exhibit morphologically
misshaped contractile rings that fail to disassemble at the end
of each meiotic division. These abnormal actin rings grow
slightly larger than the normal cytokinetic structures, and
eventually become compact masses of F-actin (Gunsalus et al.,
1995).
KLP3A and chic mutant testes exhibit similar anillin
immunostaining patterns. In both mutants anillin cycling from
the nucleus to the cytoplasm is not altered. Moreover, during
the M4b stage, anillin accumulates normally at the equator
of the dividing cells forming a circumferential band
2328 M. G. Giansanti, S. Bonaccorsi and M. Gatti
Fig. 2. Anillin localization in primary spermatocytes. (A) A complete
cyst containing 16 primary spermatocytes in the S2 stage. Anillin is
already concentrated within the nuclei and excluded from the
nucleolus; note the intense staining of the 15 RCs (arrows).
(B) Partial cyst with two primary spermatocytes in the S5 stage; in
these cells most anillin has migrated into the nucleolus. Bar, 5 µm.
indistinguishable from that seen in wild type (Fig. 7). However,
this anillin band fails to contract properly because most KLP3A
(69/70) and chic (83/89) telophases fixed according to protocol
3 (see Materials and Methods) exhibit an unconstricted anillin
ring. In telophases fixed with either protocol 1 or 2 this ring is
often discontinuous and/or displaced from the equator of the
dividing cell (Fig. 8C). We have previously shown that about
90% of KLP3A telophases and 95% of chicR1 telophases
fail to assemble an acto-myosin ring. Thus, the present
observations indicate that the persistence of the anillin ring
throughout telophase does not require the presence of the
contractile ring. Yet, the acto-myosin ring appears to be
necessary for proper contraction of the anillin band. Most
likely, the discontinuity and the frequent displacement of the
anillin ring in KLP3A and chic telophases fixed with either
protocol 1 or 2 are not direct consequences of these mutations.
As mentioned above, we believe that these abnormalities are
artifacts reflecting the lack of interaction between anillin and
the contractile ring. In the absence of an acto-myosin ring,
anillin would be associated only with the plasma membrane,
Fig. 3. Anillin localization during the M1-M2 stages of male
meiosis. (A,D) phase contrast; (B,E) microtubule immunostaining
(green), and chromatin staining with Hoechst 33258 (blue);
(C,F) anillin immunostaining (red). (A-C) Partial M1 cyst containing
two nuclei, each surrounded by parafusorial and astral membranes
(A), and associated with prominent meiotic asters (B). Note that
anillin is uniformly concentrated within the nuclei (C). (D-F) Partial
M2 cyst. Nuclear-cytoplasmic demarcation has begun to disappear
(D), some spindle fibres have penetrated the nucleus (E), and anillin
is dispersed in the cytoplasm (F). Bar, 5 µm.
Fig. 4. Fusome disintegration in a cyst containing mature primary
spermatocytes (S5 stage). (A) Hoechst 33258 staining (blue). (B) Factin staining (red). Note in B the regression and fragmentation of
the fusome. Bar, 5 µm.
The role of anillin in cytokinesis 2329
Fig. 5. Anillin localization during
meiotic divisions. Testis
preparations were fixed according
to protocol 1 (see Materials and
Methods), and sequentially stained
with anti-anillin and anti-α tubulin
antibodies, and with Hoechst
33258. (A-F) Anillin localization
during the first meiotic division.
(A-C) Anillin localization (red) in
early anaphase (A, stage M4a),
mid-anaphase (B, stage M4b) and
telophase (C, stage M5). (D-F) The
same anillin signals of A-C merged
with tubulin (green) and Hoechst
33258 (blue) stainings. In early
anaphase (A,D) anillin is dispersed
in the cytoplasm but concentrates in
the cleavage furrow from mid
anaphase (B,E) through telophase
(C,F). (G-J) Merged images of the
second meiotic division; colors as
above. In prometaphase (G, stage
M8) and early anaphase (H, stage
M10a) anillin is diffuse in the
cytoplasm; note that anillin is
excluded from prometaphse nuclei.
As in the first meiotic division, anillin concentrates in the cleavage furrow from mid anaphase (I, stage M10b) through telophase (J, stage M11).
(A,D,G,H) Examples of RCs that persist through meiotic divisions. Bar, 5 µm.
which would be subject to rupturing during the fixation
procedure.
During the second meiotic division of KLP3A and chic
mutants anillin exhibits the same aberrant behavior displayed
in the first meiotic division (not shown). As a consequence of
the aberrant meiotic divisions, KLP3A and chic testes exhibit
RCs of variable sizes. In both mutants the RCs present in
spermatogonia and primary spermatocytes are usually normal.
These apparently normal RCs persist through meiosis and are
also found in spermatids. However, spermatids also exhibit
larger, often severely misshaped RC-like structures (Fig. 8D).
In chic and KLP3A testes the abnormally large RCs are usually
associated with spermatids composed by a large nebenkern and
4 normally-sized nuclei, and represent 15% and 12% of the
total RCs, respectively. This suggests that these large RCs are
Fig. 6. Anillin and actin localization during meiotic anaphase and
telophase I. Testis preparations were fixed according to protocol 2
(see Materials and Methods) and sequentially stained with antianillin antibody (green), rhodamine-labelled phalloidin (red) and
Hoechst 33258 (blue). Note that Hoechst 33258 stains the
mitochondria associated with the central spindle, outlining this
structure. During mid- anaphase (A, stage M4b) anillin is already
concentrated in the equatorial cortex, while the F-actin ring is not yet
visible. From late anaphase (B, stage M4c) through telophase
(C, stage M5) the F-actin and anillin signals precisely overlap. Bar,
5 µm.
2330 M. G. Giansanti, S. Bonaccorsi and M. Gatti
derivatives of the abnormal anillin-enriched cytokinetic
structures present in these mutants.
In KLP3A mutants both the structure and the dynamic
behavior of the fusome do not differ from wild type. We have
previously described that in gonial and primary spermatocyte
cysts of chic mutants the fusome develops normally. However,
it fails to disassemble properly during the S4 stage. As a
consequence, cysts of mature chic spermatocytes (stage S5)
contain long-lived fusome remnants which disintegrate during
the first meiotic division (Giansanti et al., 1998). Although
spermatid cysts of chic mutants contain fewer RCs than wildtype cysts, they develop an apparently normal fusome that
connects all the extant RCs.
In tsr mutant testes anillin cycling from the nucleus to the
cytoplasm is completely normal, and anillin meiotic behavior
does not substantially differ from that seen in wild type. During
mid-anaphase of both meiotic divisions (stages M4b and
M10b) of tsr mutants anillin forms a regular equatorial band.
In late anaphase and telophase this band colocalizes with the
F-actin ring and contracts along with it (Fig. 9). In late
telophase I and II, when the actin rings of tsr mutants fail to
disassemble, overgrow and become misshaped, anillin no
longer colocalizes with F-actin but forms regularly-shaped
rings which are often smaller than their wild-type counterparts
(Figs 8E and 9B,C). The small size of these anillin rings is
likely to be a consequence of the abnormal persistence of the
contractile ring in tsr mutants, which may result in a higher
contraction of the anillin equatorial band.
An examination of tsr spermatid cysts revealed that they
contain two types of RCs: normally-sized RCs, and RCs
smaller than their wild-type counterparts (23% of total RCs).
Most of the RCs of normal size are probably formed during
the gonial divisions. This is suggested by the observation that
the RCs associated with tsr primary spermatocytes have
usually a normal size. The small RCs are likely to be
derivatives of meiotic cleavage furrows which have attained a
high degree of contraction due to the persistence of the
contractile apparatus.
We have already shown that in spermatogonial and primary
spermatocyte cysts of tsr mutants both the structure and the
behavior of the fusome are normal (Gunsalus et al., 1995).
During the meiotic divisions of these mutants there is little or
no fusome material, as occurs in wild type. In tsr spermatid
cysts the fusome reforms but it is often interrupted and fails to
connect all the extant RCs (not shown). It is possible that these
defects in fusome development are due to an insufficient
intracellular concentration of actin. In tsr spermatid cysts a
substantial fraction of actin is trapped in the actin masses
generated by the failure of disassembly of the meiosis II
contractile rings (Gunsalus et al., 1995).
DISCUSSION
Anillin localization in interphase cells
Field and Alberts (1995) have shown that anillin is present only
in dividing cells, with the exception of ovarian nurse cells
where anillin is presumably stored for later use during
embryogenesis. Accordingly, we have found that anillin is
enriched in spermatogonia and spermatocytes but not in cells
that have ceased dividing such as spermatids and sperm. Thus,
our findings reinforce the conclusion that the presence of
anillin in a cell correlates with its potential to divide (Field and
Alberts, 1995).
In most types of somatic cells examined by Field and
Alberts anillin concentrates in the nuclei in a cell cycledependent fashion; in recently divided cells anillin is
localized in the cytoplasm but it is imported into the nucleus
as cells proceed through the cell cycle. Anillin alternates
between a nuclear and cytoplasmic localization also in
spermatogonia and primary spermatocytes. In recently
divided spermatogonia and very young primary
spermatocytes anillin is cytoplasmic but it becomes restricted
to the nucleus as these cells progress through the cell cycle
or the growth phase. The mechanisms by which anillin is
imported into the nucleus are currently unknown, although
they are likely to exploit the three potential nuclear
localization sequences present in this protein (Field and
Alberts, 1995). Also unknown is the biological meaning of
the nuclear confinement of anillin. Another protein which
exhibits an intranuclear localization in spermatogonia and
primary spermatocytes is KLP3A, a kinesin-like protein
required for meiotic cytokinesis which localizes to the central
spindle midzone during anaphase and telophase (Williams et
al., 1995). Similarly, the Pavarotti protein, a kinesin-like
protein required for mitotic cytokinesis, concentrates in the
interphase nuclei of cellularized embryos (Adams et al.,
1998). The results on anillin, KLP3A and PAV-KLP suggest
that a nuclear localization may be a common feature of many
proteins involved in cytokinesis. The confinement of these
proteins to the nucleus during interphase may serve to prevent
untimely interactions with the cortical cytoplasm and
microtubules (Field and Alberts, 1995).
We have shown that in S5 spermatocytes anillin concentrates
in the nucleolus, to be released again in the nucleoplasm when
this structure breaks down during the S6 stage. An
accumulation of anillin into the nucleolus prior to cell division
is not evident in spermatogonia and has not been previously
described in somatic cells and female germ cells (Field and
Alberts, 1995). Moreover, although KLP3A is localized in
the primary spermatocyte nucleus like anillin, it does not
concentrate into the nucleolus. The reasons why anillin is
specifically imported into the nucleolus of S5 spermatocytes
are currently unknown. This further compartmentalization of
the protein may reflect the necessity of sequestering anillin to
avoid interactions with the complex intranuclear structures of
S5 primary spermatocytes (Bonaccorsi et al., 1988; Cenci et
al., 1994). Alternatively, anillin may play an active, as yet
unknown biological role, within the nucleolus.
There are two types of dividing cells that do not concentrate
anillin in the nucleus: the preblastoderm embryonic cells (Field
and Alberts, 1995) and the secondary spermatocytes (this
report). Interestingly, these two cell types also fail to
accumulate KLP3A (Williams et al., 1995). The reasons for the
exclusion of anillin from syncytial embryonic nuclei and
secondary spermatocyte nuclei are unclear. Field and Alberts
suggested that anillin uptake in precellularization embryonic
nuclei may be inactivated to allow anillin to interact with the
cortical structures of the embryo. Clearly, this explanation does
not apply to secondary spermatocytes which do not possess
anillin-enriched cortical structures comparable to those present
in syncitial embryos. A common feature of embryonic cells
The role of anillin in cytokinesis 2331
and secondary spermatocytes is the duration of interphase,
which is much shorter than that of spermatogonia, primary
spermatocytes or postcellularization somatic cells (Karr and
Alberts, 1986; Cenci et al., 1994). It is thus possible that the
relative brevity of embryonic and secondary spermatocyte
interphases does not allow sufficient time for anillin to be
imported into the nucleus.
Fusome morphogenesis
The analysis of testis preparations simultaneously stained
for anillin and actin provided information on fusome
morphogenesis in Drosophila males. The fusome was first
described by Giardina (1901) and subsequently found in
several orders of insects (reviewed by Telfer, 1975; de Cuevas
et al., 1997). In D. melanogaster females the fusome develops
during cystoblast divisions and eventually forms a branched
structure that traverses each ring canal within stage 1 cysts
(Storto and King, 1989; Lin and Spradling, 1995). Indirect
immunofluorescence studies have shown that the female
fusomes are highly enriched in α- and β-spectrin, in the
adducin-like Hu-li tai shao (Hts) protein, in the Bag-of-marble
(Bam) protein and in the cell cycle regulator cyclin A (Lin et
al., 1994; Lin and Spradling, 1995; Mc Kearin and Ohlstein,
1995; de Cuevas et al., 1996; de Cuevas and Spradling, 1998).
Phalloidin staining revealed that female fusomes do not contain
detectable amounts of F-actin (Warn et al., 1985; Spradling,
1993).
Male fusomes, like their female counterparts, contain αspectrin, the Hts and Bam proteins and cyclin A (Mc Kearin
and Ohlstein, 1995; Eberhart et al., 1996; Giansanti et al.,
1998). However, in contrast with oocyte fusomes, male
fusomes are highly enriched in F-actin (Gunsalus et al., 1995;
Hime et al., 1996). In the present study, we have examined
fusome morphogenesis during spermatogonial divisions and in
both spermatocyte and spermatid cysts. We have shown that
the male fusome grows by a cyclic process of formation and
fusion of fusome pieces. Nascent ring canals are progressively
filled up by fusome material approaching them from both sides.
As cell cycle proceeds, this fusome plugs gradually fuse with
the preexisting central fusome, giving rise to a continuous,
branched structure. This pattern of fusome growth is
essentially identical to that recently described for the ovarian
fusome (de Cuevas and Spradling, 1998). Thus, altough males
and females differ in both ring canal (Hime et al., 1996) and
fusome composition, they maintain the same mechanism of
fusome development.
Our observations on fusome formation in Drosophila
males do not help to define the functional role of this
organelle. Of the possible functions that have been proposed
for the fusome, two apply to the male germline (see Lin et
al., 1994). First, the fusome may be required to control the
pattern of germ cell interconnections by providing a
framework for spindle orientation during gonial division.
This is a likely hypothesis but certainly not exhaustive,
because spermatid cysts which contain non-dividing cells,
develop a highly branched fusome. Second, the fusome may
provide a physical support for intercellular signaling. We
favour this possibility, which we believe is in agreement with
all the extant observations on both male and female fusomes.
However, we would like to point out that there are no data
which specifically support this conclusion. Nor there are
clues on the possible molecular basis for fusome-mediated
intercellular signaling.
The role of anillin during meiotic cytokinesis
It has been previously reported that during anaphase and
telophase anillin becomes highly enriched in the cleavage
furrow in a variety of Drosophila mitotic cells and in male
meiotic cells (Field and Alberts, 1995; Hime et al., 1996; de
Cuevas and Spradling, 1998). In the present study we have
examined the relationships between anillin and the contractile
ring both in wild-type male meiosis and in meiotic divisions
of mutants defective in either contractile ring formation or
disassembly.
In wild type, anillin concentrates in a circumferential band
around the equator of cells in meiotic anaphase, before the
assembly of the F-actin-based contractile ring. A comparison
of anillin and tubulin immunostaining with the underlying
phase-contrast images, revealed that this anillin band is not
associated with the central spindle midzone but outlines the
cell equator from mid-anaphase through telophase. This
staining pattern, which is particularly evident in cells fixed with
formaldehyde prior to squashing, strongly suggests that anillin
is bound to the equatorial cortex. Additional evidence that
anillin concentration in the cleavage furrow area does not
depend upon either the central spindle or the F-actin contractile
ring is provided by the cytological analysis of meiotic divisions
in chic and KLP3A mutants. Mutations in these loci suppress
both central spindle and contractile ring assembly, but do not
affect anillin concentration at the equator of the cell at midanaphase.
Although the initial formation of the anillin cortical band
does not require the presence of an F-actin ring, these
structures, once formed, precisely colocalize throughout
anaphase and telophase. In the absence of a contractile ring, as
it happens in chic and KLP3A mutants, the anillin cortical band
does not shrink as in wild type; it maintains its initial size and,
in most cells, degenerates at the end of telophase. These
findings strongly suggest that the constriction of the anillin
band is driven by the acto-myosin contractile ring.
Taken together, our results suggest a model for the role of
anillin during cytokinesis (Fig. 10). Anillin would first
concentrate in the cleavage furrow area through an as yet
unknown mechanism, where it would bind the equatorial
cortex. This binding may be mediated by its carboxy-terminal
pleckstrin homology (PH) domain (Straight et al., 1998); PH
domains are found in numerous membrane-associated proteins
and have been implicated in protein-protein and proteinphospholipid interactions (reviewed by Shaw, 1996). Once
bound to the equatorial cortex anillin would interact with the
actin filaments of the contractile ring through its actin-binding
domain, anchoring this structure to the plasma membrane
throughout cytokinesis. With respect to the interaction
between anillin and F-actin-based contractile ring, we can
envisage two possibilities. The assembly of the F-actin ring
may be independent of the anillin equatorial band, and the
actin filaments would interact with anillin because of the close
proximity of these structures. Alternatively, it is conceivable
that the anillin band directs the formation of the F-actin ring,
perhaps providing a framework for the correct assembly of
this structure. At present we cannot discriminate between
these two alternatives. An understanding of the exact
2332 M. G. Giansanti, S. Bonaccorsi and M. Gatti
Fig. 7. Anillin behavior during
meiosis of chic and KLP3A
mutant males. Testis
preparations were fixed
according to protocol 3 (see
Materials and Methods) and
sequentially stained with antianillin (red) and anti-α tubulin
antibodies, and with Hoechst
33258 (blue). (A,C,E,G) Only
tubulin staining is shown;
(B,D,F,H) Hoechst and anillin
stainings are merged. (A,B) A
wild-type late anaphase;
(C,D) a chic late anaphase;
(E,F) a wild-type telophase;
(G,H) a KLP3A telophase.
Note the absence of central
spindle in both chic and
KLP3A ana-telophases, which
exhibit an unconstricted anillin
equatorial band. Bar, 5 µm.
Fig. 8. Late telophases and spermatids in chic and tsr
mutants. Testes, fixed according to protocol 1 (see
Materials and Methods), were sequentially stained with
anti-anillin antibody (red) and Hoechst 33258 (blue).
(A,C,E) Late telophases I in wild-type (A), chic (C) and
tsr (E) males. In the chic telophase (C) the anillin band
failed to contract and is dislocated from the cell equator,
whereas in the tsr telophase (E) the anillin ring is
overcontracted with respect to wild type.
(B,D,F) Spermatids in wild-type (B), chic (D) and tsr
(F) males. In wild-type spermatids each nucleus is
associated with a single nebenkern (NK). (D) Two
abnormal chic spermatids resulting from failures in
cytokinesis; one of them has four nuclei associated with
a large nebenkern (NK4), and the other is composed by
two nuclei and one large nebenkern (NK2). The two tsr
spermatids shown in F are also abnormal; they both
contain two nuclei associated with a large nebenkern
(NK2). The RCs are indicated by arrows; note that the
RC in chic spermatids is larger, and those in tsr
spermatids are smaller than wild-type RCs. Bar, 5 µm.
The role of anillin in cytokinesis 2333
Fig. 10. A model for the role of anillin during meiotic cytokinesis.
The plasma membrane is depicted in black, anillin in green, actin in
red, and chromatin in blue. (A) A mid-anaphase (stage M4b); (B)
late anaphase (stage M4c); (C) telophase (stage M5). In wild-type
mid-anaphase, anillin concentrates in a narrow equatorial band and
binds the plasma membrane prior to the assembly of the contractile
ring. In late anaphase the anillin band interacts with the actomyosin
ring, anchoring it to the cell cortex. Anillin remains intimately
associated with the cytokinetic ring throughout telophase and
contracts along with it, thus mediating membrane invagination. In
chic mutants, anillin concentration in the cleavage furrow occurs
normally, but the anillin band fails to contract and eventually
degenerates, due to the absence of the actomyosin ring. In tsr
mutants anillin behavior is regular till late telophase, when the
cytokinetic ring fails to disassemble and overgrows. This
phenomenon leads to an overcontraction of the anillin band and may
result in its detachment from the plasma membrane, with a
consequent impairment of cytokinesis. See text for a more detailed
explanation.
Fig. 9. Anillin and F-actin behavior during telophase of tsr mutant
males. Testes, fixed according to protocol 2 (see Materials and
Methods), were sequentially stained for anillin (green), actin (red)
and DNA (blue). (A) an early tsr telophase I in which the anillin and
F-actin ring precisely overlap, as occurs in wild type. (B,C) a late
telophase I (B) and a late telophase II (C) in which the anillin rings
only partially overlap with the abnormally prominent and misshaped
F-actin contractile structures. The anillin rings exhibit a higher
degree of contraction than in wild type. Bar, 5 µm.
structure. It is conceivable that the F-actin overgrowth can
result in a detachment of the plasma membrane from the anillin
ring, thus impairing the cytokinetic process (Figs 9 and 10).
We thank Chris Field for the anillin antibodies, and Anna Bodini
for the artwork. This work was supported in part by a TMR grant of
the EU.
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