© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
RESEARCH ARTICLE
A rapid, membrane-dependent pathway directs furrow formation
through RalA in the early Drosophila embryo
ABSTRACT
Plasma membrane furrow formation is crucial in cell division and
cytokinesis. Furrow formation in early syncytial Drosophila embryos is
exceptionally rapid, with furrows forming in as little as 3.75 min. Here,
we use 4D imaging to identify furrow formation, stabilization, and
regression periods, and identify a rapid, membrane-dependent
pathway that is essential for plasma membrane furrow formation
in vivo. Myosin II function is thought to provide the ingression force for
cytokinetic furrows, but the role of membrane trafficking pathways
in guiding furrow formation is less clear. We demonstrate that a
membrane trafficking pathway centered on Ras-like protein A (RalA)
is required for fast furrow ingression in the early fly embryo. RalA
function is absolutely required for furrow formation and initiation. In
the absence of RalA and furrow function, chromosomal segregation is
aberrant and polyploid nuclei are observed. RalA localizes to
syncytial furrows, and mediates the movement of exocytic vesicles
to the plasma membrane. Sec5, which is an exocyst complex subunit
and localizes to ingressing furrows in wild-type embryos, becomes
punctate and loses its cortical association in the absence of RalA
function. Rab8 also fails to traffic to the plasma membrane and
accumulates aberrantly in the cytoplasm in RalA disrupted embryos.
RalA localization precedes F-actin recruitment to the furrow tip,
suggesting that membrane trafficking might function upstream of
cytoskeletal remodeling. These studies identify a pathway, which
stretches from Rab8 to RalA and the exocyst complex, that mediates
rapid furrow formation in early Drosophila embryos.
KEY WORDS: Syncytial divisions, RalA, Rab8, Exocytosis
INTRODUCTION
The ability to form a plasma membrane furrow is essential to
most cellular and tissue-level developmental processes. Plasma
membrane furrow formation is required for cytokinesis and the
generation of multicellular tissues. In the Drosophila embryo, the
first nine rounds of nuclear mitoses occur deep within the syncytial
yolk. However, at cycle 10, nuclei migrate out to the embryonic
periphery and sequential transient rounds of plasma membrane
furrow formation occur in rapid succession during mitotic
cycles 10-13. These furrow processes then culminate in a final,
permanent furrowing event that encapsulates individual nuclei in a
contiguous plasma membrane forming the embryonic epithelium at
cycle 14 (reviewed by Schejter and Wieschaus, 1993; Sullivan and
Theurkauf, 1995). The early syncytial fly embryo is therefore a
furrow-making machine, rapidly making and disassembling
thousands of interconnected furrows in the time-scale of a few
Department of Biological Sciences, University of Denver, Denver, CO 80208, USA.
*Author for correspondence ([email protected])
Received 11 December 2014; Accepted 13 May 2015
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minutes. Understanding how cells are able to coordinate changes in
cytoskeletal and membrane trafficking networks to produce these
dynamic ingressions of the plasma membrane should inform our
understanding of the general processes that are available to cells to
drive furrow formation in animal cells.
Ras-like protein A (RalA) is a small GTPase that was originally
identified as a key downstream target of the Ras oncoprotein (Hofer
et al., 1994; Kikuchi et al., 1994; Spaargaren and Bischoff, 1994;
White et al., 1996). Subsequent studies have demonstrated that RalA
can function through the exocyst complex to control directed
membrane addition (Moskalenko et al., 2002). The exocyst
complex is an octameric protein complex that directs the targeting
and tethering of vesicles to the plasma membrane. The Sec5 and
Exo84 exocyst subunits directly bind to active GTP-bound RalA, and
this interaction drives exocyst complex assembly and function
(Moskalenko et al., 2002, 2003; Fukai et al., 2003). In multicellular
organisms, exocyst components are required for many cellular
processes involving directed membrane trafficking, including
epithelial polarity (Grindstaff et al., 1998; Yeaman et al., 2001;
Langevin et al., 2005; Blankenship et al., 2007), photoreceptor
morphogenesis (Beronja et al., 2005), synapse formation (Mehta
et al., 2005) and cell abscission (Fielding et al., 2005; Gromley et al.,
2005). The exocyst complex has additionally been found to bind to a
different class of small GTPases, the Rab proteins, which are key
mediators of membrane trafficking pathways. During lumen
formation and ciliogenesis in mammalian cells, Rab8 binds to the
Sec15 subunit of the exocyst complex (Bryant et al., 2010; Knödler
et al., 2010; Feng et al., 2012), and the Rab11 recycling endosome
protein can directly associate with the exocyst subunits Sec5 and
Sec15 (Zhang et al., 2004; Beronja et al., 2005; Jafar-Nejad et al.,
2005; Langevin et al., 2005; Wu et al., 2005).
The formation of a plasma membrane cleavage furrow is an
obligate step in successful cytokinesis. It is well established that
both an actomyosin contractile ring as well as membrane trafficking
are required for animal cell cytokinesis (reviewed by Pollard, 2010;
Neto et al., 2011; Schiel and Prekeris, 2013). However, the relative
contributions of these two pathways in directing the progression of
the cytokinetic furrow are unclear. One advantage of studying the
syncytial furrows in early Drosophila embryos is that furrow
formation occurs in vivo, at a predictable time, and their occurrence
at the surface of the embryo facilitates imaging with modern
confocal microscopy approaches. Additionally, working in
Drosophila permits the use of defined genetic alleles, whereas
studies on tissue culture cells rely on partial disruption through
RNAi. Finally, syncytial cell cycles and furrow formation are
exceptionally rapid, enabling the visualization of several rounds of
furrow formation in a short time period (Zalokar and Erk, 1976; Foe
and Alberts, 1983; Foe et al., 2000).
Here, we use 4D time-lapse analysis to characterize some of the
very first morphological events that occur at the embryonic plasma
membrane. Our results define the temporal and spatial dynamics of
DEVELOPMENT
Ryan M. Holly, Lauren M. Mavor, Zhongyuan Zuo and J. Todd Blankenship*
furrow formation during mitotic cycles 10-13, and show that
furrows possess three distinct phases encompassing ingression,
stabilization, and disassembly. We additionally demonstrate a
requirement for RalA protein function in the formation of plasma
membrane furrows. In the absence of RalA function, furrow
formation does not initiate, Rab8 fails to traffic to the cell surface,
and the exocyst complex subunit Sec5 loses its cortical localization.
These results are consistent with a fundamental requirement for
directed membrane addition in the initiation and ingression of
plasma membrane furrows.
RESULTS
Rapid furrow formation and regression in the early syncytial
embryo
The early Drosophila embryo undergoes 13 rounds of division in
the absence of cytokinesis before the process of cellularization
packages individual nuclei into an epithelial array. These rounds of
division are rapid, with the 13 cycles occurring in a 2-h time span at
25°C. The last four divisions occur after nuclei have migrated out to
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
the embryonic periphery. With the nuclei arranged into a common
cortical plane, cytokinetic-like plasma membrane furrows rapidly
ingress to separate the nuclei, prevent chromosomal missegregation,
and provide attachment points for spindle assembly and positioning
(Foe and Alberts, 1983; Sullivan et al., 1993). We have used highresolution temporal and spatial imaging to examine the timing and
dynamics of furrow formation and regression during these divisions
(Fig. 1; supplementary material Movie 1).
We first characterized the furrows that form during cycles 10-13
by analyzing embryos expressing a plasma membrane marker,
Gap43:mCherry (Gap43:mCh). We observed that furrow dynamics
can be separated into three distinct phases: (1) furrow formation; (2)
furrow stabilization; and (3) furrow regression. The events of furrow
formation, stabilization and regression occur continuously over the
total cycle time. As successive cycles proceed, the nuclei crowd
closer together, and furrows reach deeper into the embryo (Fig. 1I).
Furrow ingression occurs on average in 3.8, 5.7, 6.1 and 11.3 min
during cycles 10, 11, 12 and 13, respectively (Table 1). Furrows are
more irregular in the earliest cortical cycle (cycle 10) but become
Fig. 1. Spatial and temporal dynamics of syncytial furrow formation. (A-D) Still-frame views at a single z-plane of a wild-type (WT) Drosophila embryo
expressing the membrane marker Gap43:mCh at t=0, 3, 7.5 and 15 min during cycle 12. (A) At the onset of cycle 12, Gap43 remnant furrows from cycle 11 persist
near the apical surface. (B,C) As the cycle proceeds, the broader furrow tip ingresses into the viewing plane (B), and then moves deeper into the embryo as
thinner, more medial portions of the furrow come into the viewing plane (C). (D) The furrow retracts apically and is lost from the viewing plane. (E-H) Schematic
depicting the corresponding apical-basal views for orientation. (I) Quantification of mean furrow lengths for syncytial cycles 10-13 (n=10). Mean furrow lengths
are 2.3 µm, 4.2 µm, 6.1 µm and 8.2 µm at cycle 10, 11, 12 and 13, respectively. (J) Quantification of calculated mean ingression speeds and retraction
speeds for cycles 10-13 (n=10) (see Table 1). Error bars indicate s.e. Scale bar: 10 µm.
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RESEARCH ARTICLE
successively more regular and hexagonally packed with each
proceeding cycle. After furrow ingression has occurred, furrows are
relatively stable in depth for a period of 2-3 min (Table 1). After this
short stable period, the syncytial furrows begin to deform as mitosis
proceeds, and furrow regression rapidly occurs with different
temporal dynamics than furrow formation. Furrow regression occurs
on average in 2.5, 3.3, 3.9 and 4.8 min during cycles 10, 11, 12 and
13, respectively (Table 1). Furrow regression is often not complete,
with small regions of remnant furrows (0.5-1.5 µm in depth)
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
apparent as a new cycle of furrow formation initiates (Fig. 1A).
Interestingly, ingression speeds for each cycle remain fairly
consistent (Fig. 1J), at ∼0.7 µm/min. However, furrow retraction
rates progressively increase with development (Fig. 1J), suggesting
that ingression and regression are controlled by different
mechanisms.
As furrow formation is one of the first morphological events that
occurs at the plasma membrane in the Drosophila embryo, and as
the embryo makes hundreds of furrows in a matter of a few minutes,
Fig. 2. RalA localizes to the cortex and furrows during syncytial cell cycles. (A-D) Still frames from live imaging of mCh:RalA at t=0, 2.5, 6.5 and 11 min
during cycle 11. RalA is localized to the plasma membrane during furrow formation. (E-H) mCh:RalA localization at t=0, 3, 12.5 and 22.5 min during cycle 13.
(I-I‴) Planar views of fixed WT embryos stained with anti-RalA (red), and for F-Actin (green, phalloidin) and DNA (blue, Hoechst) during cycle 12. Consistent
with results obtained from mCh:RalA live imaging, immunostaining indicates RalA localization to the plasma membrane during furrow formation. (J-J‴) Apicalbasal views of fixed WT embryos stained as in I during cycle 12. RalA is present at furrows and the plasma membrane during furrow ingression.
(K-O) Co-expression of mCh:RalA and Histone:GFP at t=0, 3.5, 5.5, 8.5 and 12 min during cycle 12. (K) During interphase, furrows begin to ingress but are
not present at the level of the nuclei. (L,M) As the nuclei begin to condense (L) and align across the metaphase plate (M), plasma membrane furrows are fully
extended basally (M) and are present at the level of the nuclei. (N) As anaphase proceeds, furrows deform and lose their tight hexagonal spacing and regression
begins. (O) Furrows regress below the level of the nuclei during telophase. Scale bars: 10 µm.
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we chose to focus further on the mechanisms that drive rapid furrow
formation in the early embryo.
RalA localizes to the plasma membrane and furrows during
syncytial cell cycles
The RalA small GTPase is an essential regulator of targeted
membrane addition and directs vesicular tethering activity at the
plasma membrane (Moskalenko et al., 2002, 2003; Fukai et al.,
2003). RalA is therefore a good candidate protein to probe the role
of directed membrane addition during furrow formation in the early
embryo. To examine whether RalA is involved in furrow formation,
we first imaged RalA localization in vivo. During cycles 1-9 of the
syncytial nuclear divisions, UAS-mCherry:RalA (mCh:RalA) is
localized in puncta basal to the plasma membrane (supplementary
material Fig. S1A-C). At the onset of cycle 10, these puncta
disappear, and RalA localizes to the plasma membrane
(supplementary material Fig. S1B,C). Throughout cycles 10-13,
RalA localizes to the plasma membrane as furrows ingress around
the nuclei (Fig. 2A-H). To ensure that the localization of the mCh:
RalA construct represented that of endogenous RalA populations,
we fixed wild-type (WT) embryos and stained with a RalA antibody
(Teodoro et al., 2013). Planar and apical-basal images of fixed
embryos show that endogenous RalA is present during syncytial
stages and localizes in a manner similar to mCh:RalA (Fig. 2I-J‴).
We also examined mCh:RalA; Histone:GFP-expressing embryos
in order to study the relationship between RalA localization and
the cell cycle as indicated by chromosomal morphologies (Fig. 2K-O;
supplementary material Movie 2). RalA-marked furrows are
apparent from the earliest stages of furrow formation (Fig. 2B), but
are not present at the level of the nuclei until prophase (Fig. 2K,L).
At the end of prophase, furrow ingression halts and the furrows enter
a stabilization phase that persists through prometaphase and
metaphase (Fig. 2M). During the initiation of anaphase, the
extended furrow begins to regress, and regression continues
through the end of telophase (Fig. 2N,O). Furrow behaviors appear
to occur continuously at these stages, as new furrows form
immediately after regression of the furrows of the previous cycle
(supplementary material Movies 1 and 2).
Defective early development and furrow formation in RalA
mutant embryos
Given the localization of RalA to furrows and the plasma
membrane, we suspected that it might be required for furrow
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
formation in the early Drosophila embryo. To address this, we
examined the effect of disrupting RalA (Rala – FlyBase) function
during early development. Embryos maternally deficient for RalA
function (RalAPL56 germline clone embryos; Ghiglione et al., 2008)
were scored under oil for gross tissue morphologies to determine if
RalA is required for early morphogenesis. RalAPL56 embryos were
severely disrupted in development: 86.5% of embryos showed
defects during the syncytial nuclear divisions, 7.7% first displayed
defects during cellularization, and 5.8% became defective during
germband extension (Fig. 3A). These results suggest an essential
maternal requirement for RalA function in the early embryo.
We further performed RNAi analysis of RalA function in the
embryo. OreR (WT) embryos injected with RalA-specific dsRNA
also demonstrated early morphological defects. In the early embryo,
dsRNA injection often results in a partial knockdown of RalA,
destabilizing maternally and zygotically expressed mRNAs while
maternal protein stores may remain unaffected. RalA dsRNAinjected embryos were unable to complete germband extension,
with 11.5% of embryos showing defects during germband
extension, 50% first demonstrating defects during cellularization,
and 38.5% becoming defective during the syncytical nuclear
divisions (Fig. 3B). These results confirm the RalAPL56 mutant
analysis, and suggest the possibility that all furrow-forming events
(i.e. those that generate the transient syncytial and permanent
cellularization furrows) require RalA function.
To examine the effects of disrupting RalA function on furrow
formation, we examined GFP:MoeABD (an F-actin marker) in WT
and RalA mutant backgrounds. In WT embryos, GFP:MoeABD
localizes to furrows (Fig. 4A-D), illustrating the canonical model
that the F-actin cytoskeleton supports furrow formation (Foe et al.,
2000; Albertson et al., 2008; Cao et al., 2008). We then examined
GFP:MoeABD in RalAPL56 germline clone embryos to address
whether furrows and their associated F-actin networks could form in
the absence of RalA function. RalAPL56 embryos were unable to
produce regular F-actin-coated furrow canals, with only the borders
of the apically associated F-actin nuclear caps remaining (Fig. 4E-H).
We then examined F-actin behaviors in fixed embryos in which the
mitotic stage could be determined. In WT embryos, F-actin-marked
furrows are present in embryos undergoing DNA condensation and
chromosome alignment (Fig. 4I,K). In RalAPL56 embryos, F-actin
is present in puncta basal to the plasma membrane during
prophase (Fig. 4J,L), but is never found on furrows. This suggests
either that RalA may direct F-actin formation at the furrow, or that
Fig. 3. Early developmental defects in RalA mutant embryos. (A) Quantification of the onset of developmental defects in WT and maternal RalAPL56 embryos
as a percentage of total embryos scored. (B) Percentage of WT embryos injected with RalA-specific dsRNA or water that first display defective phenotypes during
each stage of early development. GBE, germband extension.
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Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
the F-actin cytoskeleton at furrows is dependent on a membranedriven ingression pathway.
Disruption of syncytial nuclear divisions in RalA mutant
embryos
If furrow formation is defective during the syncytial divisions,
then RalA mutant embryos would be expected to demonstrate
defects in the ability to properly segregate chromosomes (Zalokar
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and Erk, 1976; Foe and Alberts, 1983; Sullivan et al., 1993). To
better understand the mechanisms by which furrows support
chromosomal segregation, we imaged the chromosomal marker
Histone:RFP in RalAPL56 embryos. We observed that widespread
nuclear fusions occur during cycles 10-13, affecting nearly 90%
of nuclei. Early syncytial cycles, in which nuclei are spaced
further apart, are not as dependent on RalA function, with ∼12%
of mitotic divisions failing and producing polyploid nuclei
(Table 2). However, by cycle 13, nearly all nuclei have suffered
a chromosomal missegregation event (Table 2). We further
observed that there are two major types of nuclear fusion event
in RalAPL56 embryos. The first type we classified as ‘mitotic
collapse’ nuclear fusion events, which are a result of the failure of
sister chromosomes to remain separated at the end of anaphase
(Fig. 5E-E‴; supplementary material Movie 3). The second type
of nuclear fusion event observed was a fusion of chromosomes
originating from adjacent dividing nuclei, which we classified as
‘adjacent nuclear fusion’ (Fig. 5F-F‴; supplementary material
Movie 4). Both types of fusion resulted in 4n nuclei; however,
adjacent nuclear fusions can drive larger, highly polyploid nuclei
if more than two nuclei fuse.
Different syncytial cycles in RalA mutant embryos were
characterized by different ratios of mitotic collapse nuclear
fusions and adjacent nuclear fusions. In WT embryos, nuclear
fusion events are rare throughout the syncytial divisions (1.2% of
nuclear divisions, supplementary material Table S1). During cycle
10, RalAPL56; Histone:RFP germline clone embryos display mitotic
collapse-driven fusions in 11.7% of mitoses (Table 2). No adjacent
nuclear fusions are observed during cycle 10 (Table 2). Cycle 11
also shows predominately mitotic collapse nuclear fusions in RalA
mutant embryos (Fig. 5B, Table 2), whereas cycle 12 demonstrates a
mix of mitotic collapse and adjacent nuclear fusions (Table 2), and
cycle 13 has mostly adjacent nuclear fusions (Fig. 5D, Table 2).
Since RalAPL56 embryos display increasingly defective
phenotypes as nuclei become more numerous and more crowded,
we hypothesized that RalA might function by regulating the
formation of furrows, which in turn provide potential anchor points
for the spindle machinery and maintain the physical separation of
chromosomes during mitosis.
RalA directs membrane addition required during furrow
formation and ingression
As our data suggest that RalA function may drive a furrow
ingression pathway, and published data demonstrate that RalA can
physically bind to members of the exocyst complex and direct
exocyst assembly (Moskalenko et al., 2002, 2003; Fukai et al.,
DEVELOPMENT
Fig. 4. Defective furrow and F-actin formation in RalA mutant embryos.
(A-D″) Live imaging planar views of embryos expressing mCh:RalA and
GFP:MoeABD at t=0, 3.5, 12.5 and 20.5 min during cycle 13. RalA and F-actin
colocalize to the forming furrow in an optical plane 2 µm basal to the apical
surface. (E-H) Live imaging planar views of a RalAPL56 embryo expressing
GFP:MoeABD at t=0, 5, 11 and 21 min during cycle 13. F-actin-marked plasma
membrane furrows fail to form, while borders of the apical F-actin caps are
partly apparent in an optical plane 2 µm basal to the apical surface. (I-I″) Fixed
WT embryo stained for F-actin ( phalloidin) and DNA (Hoechst) during cycle 11.
F-actin is localized to the furrow during ingression, consistent with the live
imaging results. (J-J″) Fixed RalAPL56 embryo stained for F-actin and DNA
during cycle 11. F-actin is present as cytoplasmic aggregations, but no
F-actin-based furrows are observed. (K-K″) Apical-basal views of a fixed
WT embryo during cycle 11. F-actin coats ingressing syncytial furrows.
(L-L″) Apical-basal views of a fixed RalAPL56 embryo during cycle 11. F-actin is
localized in large aggregates basal to the apical surface, but no F-actin-based
furrows form. Scale bars: 10 µm.
RESEARCH ARTICLE
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
Table 1. Quantification of syncytial furrow periods and speeds
Cycle
Ingression time
Stabilization time
Retraction time
Ingression speed
Retraction speed
10
11
12
13
3.75±0.31
5.67±0.89
6.05±0.67
11.25±4.91
2.06±0.09
2.58±0.18
2.90±0.29
3.05±0.62
2.53±0.15
3.25±0.41
3.93±1.53
4.80±1.06
0.66±0.02
0.70±0.02
0.82±0.01
0.70±0.01
0.68±0.05
0.74±0.04
0.96±0.10
1.14±0.06
Mean (±s.e.) furrow ingression, stabilization and retraction times (min) and ingression and retraction speeds (μm/min) during cycles 10-13 of syncytial furrow
behaviors, as determined from Gap43:mCherry-expressing embryos (n=10).
2003), we hypothesized that RalA might guide a membrane delivery
mechanism required to form furrows. To test this, we examined the
in vivo delivery of a cell surface-associated protein (Gap43:mCh)
that is trafficked to the plasma membrane (Martin et al., 2010). In
WT embryos, Gap43:mCh is present in punctate, cytoplasmic
populations prior to cycle 10. During cycles 10 and 11 these large,
punctate pools of Gap43:mCh disappear as the protein begins to
localize to the plasma membrane (supplementary material Fig. S1DF). In a RalAPL56 mutant background, Gap43:mCh is present in
vesicular pools prior to cycle 10, similar to the Gap43:mCh
population in WT embryos (data not shown). However, with each
syncytial cycle these Gap43:mCh pools remain in their punctate
localization and delivery to the plasma membrane is severely
disrupted (Fig. 6D-F). Additionally, no evidence of furrow
formation, as marked by Gap43:mCh, is observed (Fig. 6D-F).
This strongly suggests that RalA is required for exocytic trafficking
to the plasma membrane during these early cycles, and that RalAdependent trafficking is necessary for the earliest events of furrow
formation.
To examine a potential molecular mechanism of RalA function,
we determined the localization of a component of the exocyst
complex, Sec5, which has been shown to physically interact with
RalA in MDCK cells (Moskalenko et al., 2002, 2003). Anti-Sec5
staining in WT embryos revealed that the localization of Sec5 was
strikingly similar to that of RalA during cycles 10-13. In RalA
mutant embryos, Sec5 localization to the cortex is lost, and it
accumulates within the cytoplasm (Fig. 6H′,J′). Prior to cycle
10 and the onset of furrow formation, Sec5 does not localize to
RalA puncta in WT embryos, suggesting that these puncta might
represent inactive RalA protein populations (supplementary material
Fig. S1G). These results are consistent with a model in which active
RalA directs the localization of exocytic machinery to the incipient
furrows for the delivery of cytoplasmic membrane stores.
accumulate at the furrow in a cortical array (Fig. 7C). At these
stages, mCh:RalA is also present in a cortical localization at the
furrow and demonstrates colocalization with YFP:Rab8 (Fig. 7C).
As RalA and Rab8 colocalize at the furrow cortex, and both RalA
and Rab8 have been shown to be essential for furrow formation
(Fig. 4I-L; L.M.M., Z.Z., J.T.B., unpublished), we tested their
functional interdependence. To determine if Rab8 localization is
dependent on RalA function, RalA dsRNA was injected into a YFP:
Rab8 background. Indeed, RalA knockdown resulted in the
mislocalization of YFP:Rab8 rather than cycling from a punctate to
cortical location, Rab8 remained in cytoplasmic puncta (Fig. 7F-F‴)
and these puncta also appeared larger in size. These results are
suggestive of a failure of Rab8 trafficking to the cell surface and might
explain the underlying failure of furrow formation in RalA mutant
embryos. YFP:Rab8 does not localize to the apical surface in waterinjected or RalA dsRNA-injected embryos (Fig. 7G,H).
Next, we performed the converse experiment of injecting Rab8
dsRNA into a mCh:RalA background. RalA localization at the
apical surface of the plasma membrane was unaffected (Fig. 7K-K‴).
However, furrows were unable to extend in embryos with
compromised Rab8 function (Fig. 7L′-L‴). RalA puncta, which
normally clear from the cytoplasm by the end of cycle 10, were still
present at cycle 12 (Fig. 7L-L‴). As Rab8 localization is severely
affected by disrupting RalA function, and RalA still localizes
appropriately to the cell cortex, it is likely that RalA functions
upstream of Rab8 in a membrane delivery pathway. RalA, in turn,
might require a Rab8-dependent exocytic trafficking pathway to
reinforce cortical RalA populations.
Finally, we examined the effect of disrupting Sec5 function on
RalA localization (Fig. 7M,N). Similar to Rab8 RNAi, apical
RalA localization is unaffected in Sec5 dsRNA-injected embryos,
while furrow formation is disrupted, suggesting that RalA might
function upstream of exocyst complex assembly and Sec5
function.
RalA directs membrane addition through the Rab8 GTPase
RalA is enriched at the furrow tip relative to F-actin during
furrow formation
As furrows fail to form from their earliest stages in RalA mutant
embryos, we further examined the relationship between the F-actin
cytoskeleton and the RalA/Sec5/Rab8 membrane trafficking
pathway. Embryos co-expressing mCh:RalA and GFP:MoeABD
were imaged at a single focal plane basal to a forming furrow. We
then asked whether either protein appears first in the focal plane on
Table 2. Chromosomal segregation defects in RalA mutant embryos
Cycle
WT divisions completed
Mitotic collapse nuclear fusions
Adjacent nuclear fusions
WT divisions started
10
11
12
13
91 (88.3%)
135 (88.8%)
114 (64.4%)
18 (12.7%)
12 (11.7%)
15 (9.8%)
36 (20.3%)
33 (23.4%)
0 (0%)
1 (0.7%)
14 (7.9%)
65 (46.1%)
103
152
177
141
Quantification (number and percentage) of mitotic nuclear fusions and adjacent nuclear fusions observed in RalAPL56 embryos. As mitotic cycles progress, the
number of adjacent nuclear fusions increases exponentially (n=9).
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Recent work in our laboratory suggests that Rab8 is a key mediator
of membrane addition during furrow formation in the early
Drosophila embryo (L.M.M., Z.Z., J.T.B., unpublished). To
determine Rab8 localization relative to RalA, embryos coexpressing mCh:RalA and YFP:Rab8 were examined. Rab8
compartments are present in the cytoplasm as furrow formation
initiates (Fig. 7B). As furrow ingression proceeds, cytoplasmic
Rab8 compartments become depleted and low levels of Rab8
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Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
Fig. 5. Mitotic collapse and adjacent nuclear fusions in RalA mutant embryos. (A-A‴′) Still-frame images of embryo expressing Histone:GFP at (A)
t=0 min – interphase, (A′) 1.8 min – prophase, (A″) 3.4 min – metaphase, (A‴) 5.1 min – anaphase and (A‴′) 6.9 min – telophase, during cycle 11. (B-D‴′) The
corresponding cell cycle stages are shown for: (B-B‴′) RalAPL56 embryo expressing Histone:RFP at t=0, 1.5, 2.5, 4.5 and 9.5 min of cycle 11; (C-C‴′) Histone:
GFP-expressing embryo at t=0, 8.6, 10.1, 13.0 and 16.1 min during cycle 13; and (D-D‴′) RalAPL56 embryo expressing Histone:RFP at t=0, 8.3, 11.7, 12.5
and 16.3 min during cycle 13. (E-E‴) Example of the mitotic nuclear fusion phenotype. Yellow and red arrows track separate mitoses in which chromosomes fail
to fully separate leading to anaphase collapse and nuclear fusion. (F-F‴) Example of the adjacent nuclear fusion phenotype. Yellow and red arrows track separate
mitotic divisions that fail to maintain separation between adjacent mitoses and then fuse into a common nucleus. Scale bars: 10 µm.
2322
occurs during anaphase, and further deformation of the regularity of
the furrow correlates with this period (Fig. 8E).
The above results are consistent with a model whereby RalAdependent membrane trafficking is the core machinery responsible
for furrow formation, with F-actin coating of the furrow acting as a
structural reinforcement.
DISCUSSION
We have demonstrated the furrow dynamics of one of the earliest
membrane-shaping morphological events in the Drosophila
embryo. We identified formation, stabilization and retraction
phases in the plasma membrane furrows of the early syncytial
embryo, and measured furrow lengths and ingression and retraction
rates. We show that furrow formation can be exceptionally rapid,
occurring in as little as 3.75 min. Interestingly, the rate of furrow
formation appears relatively consistent regardless of the cycle or the
number of furrows that are forming in the embryo, in contrast to
DEVELOPMENT
the ingressing furrow. We observed that RalA is strongly detected
at the furrow from the earliest moments that the ingressing furrow
can be imaged (Fig. 8B). RalA is present in the broader furrow tip at
levels comparable to the rest of the furrow (Fig. 8B,C). F-actin,
however, appears to have an asymmetric enrichment on syncytial
furrows: it is present at low levels at the furrow tip, but then is
more strongly enriched on the more apical portions of the furrow
(Fig. 8B,C). These observations, in combination with the finding
that F-actin is localized in puncta in the absence of RalA function
(Fig. 4J,L and Fig. 6H,J), suggest that membrane trafficking, as
indicated by RalA, might precede F-actin cytoskeletal remodeling
during furrow formation.
We also examined furrow retraction, and asked whether RalA or
F-actin disassembly occurs first. Here, again, we observed a
differential behavior of RalA and F-actin. During retraction, F-actin
depolymerization occurs first and the furrow loses the F-actin
cortical assembly (Fig. 8E). Interestingly, this F-actin disassembly
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Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
syncytial furrow retraction rates. The rate of furrow formation is
similar to those reported for furrow ingression during the fast, but
not slow, phase of cellularization (0.8 µm/min; Lecuit et al., 2002).
The fast phase of cellularization is a process that, like syncytial
furrow formation, is heavily dependent on targeted membrane
addition (Lecuit and Wieschaus, 2000; Pelissier et al., 2003; Murthy
et al., 2010; L.M.M., Z.Z., J.T.B., unpublished). Furrow retraction
rates, by contrast, are not consistent between different mitotic
cycles, and retraction speeds increase with syncytial mitotic cycles.
This suggests that a simple reversal of the trafficking pathways
might not drive furrow retractions. It is also worth noting that small
regions of the furrows often never fully retract at the end of a cycle,
and short ridges (0.5-1.5 µm) are present as a new cycle of furrow
formation begins. This demonstrates that furrow formation
and regression are continuously occurring throughout mitotic
cycles 10-13, which would be consistent with a model in which
cell cycle length drives furrow depth.
We have also shown that RalA function is essential from the
earliest stages of furrow initiation. In the absence of RalA function,
furrow failures lead to two distinct classes of failed nuclear
divisions. These classes suggest that furrows are required for both
spindle anchoring (the failure of which leads to the mitotic collapse
phenotype) and for the separation of adjacent genomes (the failure
of which leads to the fusion of adjacent nuclei). In the earliest
2323
DEVELOPMENT
Fig. 6. RalA is required for membrane addition and
syncytial furrow formation. (A-C) Control embryo
expressing the plasma membrane marker Gap43:mCh at
t=0, 5 and 13 min of cycle 12. (D-F) RalAPL56 embryo
expressing Gap43:mCh at t=0, 4 and 10.5 min during
cycle 12. Cytoplasmic membrane pools are unable to
traffic to the plasma membrane to allow for furrow
ingression. (G-G‴) Fixed WT embryo stained with antiSec5 (green) and for F-actin (red, phalloidin) and DNA
(blue, Hoechst). Sec5 localizes to furrows during cycle 12
furrow ingression. (H-H‴) Fixed RalAPL56 embryos during
mitotic cycle 12. Sec5 becomes delocalized and is
distributed broadly within the cytoplasm. (I-I‴) Apicalbasal views of WT fixed embryos stained for Sec5,
F-actin and DNA. Sec5 localizes to the cortex and at
furrows. (J-J‴) A RalAPL56 embryo demonstrates loss of
Sec5 localization to the cortex and an absence of
syncytial furrows. Scale bars: 10 µm.
RESEARCH ARTICLE
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
cycles of furrow formation (cycles 10, 11), furrow function is
most important for proper spindle function and the successful
completion of individual mitoses. However, as the cycles progress
and nuclei become more numerous (cycles 12, 13), furrows become
increasingly important to each division through their maintenance
of a physical barrier between chromosomes. In the absence of these
furrows, adjacent mitotic figures are not properly fenced in, and
mitotic fusions occur.
We further demonstrate the existence of a membrane trafficking
pathway that directs rapid furrow formation. This pathway involves
the combined function of RalA, Sec5 and Rab8. Sec5 and Rab8
become aberrantly localized in RalA mutant embryos, suggesting
that RalA functions through the exocyst complex and Rab8 to direct
2324
furrow formation. Indeed, active GTP-bound RalA has been shown
to directly bind to the Sec5 and Exo84 exocyst subunits, and this
interaction drives exocyst complex assembly and function
(Moskalenko et al., 2002, 2003; Fukai et al., 2003). Interestingly,
Sec5 does not colocalize with RalA prior to cycle 10, suggesting
that RalA is inactive prior to furrow formation. Activation of RalA
might therefore initiate furrow formation through the recruitment of
a vesicular targeting and tethering complex, i.e. the exocyst, to the
plasma membrane (Fig. 9A). The exocyst, in turn, may bind to and
recruit exocytic vesicles through direct subunit interactions with
Rab8 (Fig. 9A) (Bryant et al., 2010; Knödler et al., 2010; Feng et al.,
2012). It is interesting to note that both Rab8 and RalA are present as
cytoplasmic puncta prior to cycle 10, and that RalA partially
DEVELOPMENT
Fig. 7. RalA directs membrane growth through Rab8. (A-D″) Planar views of embryo expressing mCh:RalA and YFP:Rab8 at t=0, 3, 11 and 18.5 min
during cycle 13. Rab8 is present in cytoplasmic compartments, but also colocalizes cortically with RalA on the ingressing furrow (C). (E-E‴) Transverse section
through furrows of water-injected YFP:Rab8-expressing embryo at t=0, 3, 6 and 15 min during cycle 13. Rab8 displays the normal transition from compartmental
to cortical localization at the furrow. (F-F‴) RalA dsRNA-injected embryos expressing YFP:Rab8 at t=0, 3, 6 and 15 min during cycle 13; view is 1.5 µm below
the apical surface. Rab8 fails to transition into a cortical array, and cytoplasmic puncta increase and grow with time. (G-H‴) YFP:Rab8 is not present at the
apical surface of the embryo in water-injected (G-G‴) nor RalA dsRNA-injected (H-H‴) embryos. (I-J‴) Water-injected mCh:RalA-expressing embryos at t=0, 2.5,
7.5 and 11.5 min during cycle 11, shown at the apical surface (I-I‴) and 1 µm basal to the apical surface (J-J‴). (K-L‴) Rab8 dsRNA-injected embryos expressing
mCh:RalA at t=0, 2.5, 7.5 and 11.5 min during cycle 11, shown at the apical surface (K-K‴) and 1 µm basal to the apical surface (L-L‴). RalA localization is not
disrupted at the apical surface; however, furrows are unable to ingress and RalA puncta remain basal to the plasma membrane. (M-N‴) Sec5 dsRNA-injected
embryos at t=0, 2.5, 7.5 and 11.5 min during cycle 11. Sec5 compromised embryos localize RalA at the apical surface (M-M‴), but lack furrow-associated RalA
and furrow formation fails as observed in a basal plane 1 µm down (N-N‴). Scale bars: 10 µm.
Fig. 8. Differential RalA and F-actin behaviors during furrow formation
and retraction. (A-C″) Still-frame planar images of embryos co-expressing
mCh:RalA and GFP:MoeABD at t=0, 9.5 and 12.5 min during cycle 13 furrow
ingression. (B) The first frame in which furrow-associated fluorescence was
observable in the plane of view. RalA is strongly detected at the furrow tip, while
F-actin localizes at the furrow tip at low levels. (C) F-actin and RalA are present
on apicolateral portions of the syncytial furrows as ingression proceeds.
(D-F″) mCh:RalA and GFP:MoeABD localization at t=15.5, 17.5 and 20.5 min
during cycle 13 furrow retraction. Images are of same embryo as in A-C.
F-actin disassembly at the furrow (E) occurs prior to the loss of RalA (F). Scale
bar: 10 µm.
remains in a punctate distribution when Rab8 function is knocked
down. Previous work has illustrated a reinforcing loop, in which
exocyst complex subunits traffic to the cell surface on vesicular
intermediates (Yeaman et al., 2001, 2004; Langevin et al., 2005). It
might be that RalA localization to the cortex depends on a similar
reinforcing loop. The subsequent fusion of exocyst-tethered Rab8
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
compartments would then potentiate furrow formation (Fig. 9B).
These incipient furrows would serve as a scaffold for F-actin
assembly, which may impart a structural rigidity to the furrow
(Fig. 9C) (Foe et al., 2000; Cao et al., 2008). Indeed, recent evidence
suggests a close relationship between membrane behaviors at the
syncytial and cellularization furrows and the recruitment and
remodeling of the F-actin cytoskeleton (Sokac and Wieschaus,
2008a,b; Yan et al., 2013; Reversi et al., 2014). Furrow retraction
may occur through the disassembly of the supporting F-actin
cytoskeleton (Fig. 8E), and furrows may collapse back into the
apical plasma membrane. This membrane could be stored in villous
projections for the next round of furrow formation, as has recently
been observed during cellularization (Figard et al., 2013).
Alternatively, membrane might be recycled back to cytoplasmic
Rab8 stores through endocytic pathways, a mechanism that has also
been shown to operate during early furrow formation through
Dynamin-dependent endocytosis (Lecuit and Wieschaus, 2000;
Pelissier et al., 2003; Rikhy et al., 2015).
It is interesting to contrast these results with the mechanisms
known to regulate cleavage furrow formation during animal cell
cytokinesis. The major force driving cleavage furrow ingression is
believed to originate through the action of a contractile actomyosin
ring (reviewed by Fededa and Gerlich, 2012). However, it is clear
that membrane trafficking pathways are required for cytokinesis as
well [reviewed by Albertson et al. (2005); Prekeris and Gould
(2008)]. Teasing apart the relative contributions of these pathways to
cytokinesis has been difficult. Syncytial furrow formation in the
early Drosophila embryo is believed to be a myosin-independent
process (Royou et al., 2004), and the geometric constraints of the
embryo make assembling and orienting myosin ring contraction into
the interior of the embryo challenging. Our results demonstrate that
exocytic membrane addition is required for syncytial furrow
formation and ingression, and are consistent with a model in
which targeted membrane addition directs furrow formation through
the accretion of membrane material at the incipient furrow. As our
antibody staining and fluorescent protein fusions reveal total
RalA protein dynamics, we could not distinguish between inactive
GDP-bound and active GTP-bound RalA. It therefore remains to be
seen whether RalA is activated locally in cortical subdomains, or if
it is generally activated at the plasma membrane and then further
processes, such as regulated vesicular fusion or cytoskeletal
function, guide the localization of furrow formation. However,
Rab8 localization to the cortex occurs specifically in an apicolateral
region of the furrow (Fig. 7E,G; L.M.M., Z.Z., J.T.B., unpublished),
suggesting that RalA/exocyst function might be locally active at the
furrow. Rgl has been identified as a potential RalA guanine
nucleotide exchange factor (GEF) in the adult fly and neuroblasts
(Mirey et al., 2003; Carmena et al., 2011), and the characterization of
Rgl in the early embryo might reveal the sites of active RalA protein.
Alternatively, polarized membrane addition may occur through the
functioning of the interphase F-actin caps, which could act to inhibit
vesicular trafficking to the apical region directly above the nucleus,
and thus bias trafficking to incipient furrows. In this context, it is
interesting to note that membrane trafficking is required for the local
enrichment of F-actin at the cell surface in the early embryo
(Albertson et al., 2008; Cao et al., 2008).
The ease of imaging early embryos and the availability of the rich
genetic and molecular tools that Drosophila can offer suggest that
the transient syncytial furrows should provide a potent system for
studying the interplay between membrane trafficking pathways and
cytoskeletal networks in driving the formation of plasma membrane
furrows.
2325
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2015) 142, 2316-2328 doi:10.1242/dev.120998
Fig. 9. Model of RalA/Sec5/Rab8-mediated
furrow formation. (A) Furrow formation initiates with
the activation of RalA at the plasma membrane and
the subsequent recruitment of exocyst complex
assembly (Sec5) by RalA. Exocyst complex
assembly functions to target and tether Rab8
vesicular compartments. (B) The fusion of Rab8
vesicles potentiates the ingression of incipient
furrows, and Rab8 begins to accumulate in a cortical
array. Cortical exocyst complex function may be
reinforced by trafficking of exocyst subunits on Rab8
vesicles. (C) Furrow ingression provides a scaffold
for F-actin assembly, which supports furrow
structural integrity. Cytoplasmic Rab8
compartments become depleted as trafficking to the
cell surface progresses. (D) The timing of furrow
formation, stabilization and retraction events relative
to cell cycle stage.
Fly stocks and genetics
Fly stocks were maintained at 25°C by standard procedures. All UAS
transgenic flies were crossed with matαTub-Gal4VP16 67C;15 (D. St
Johnston, Gurdon Institute, Cambridge, UK) maternal driver females, and
second generation embryos were analyzed. Fly stocks used: UAST-mCh:
RalA, RalAPL56, FRT19A/FM7 (Bourbon et al., 2002; Ghiglione et al.,
2008), Histone:GFP (Bloomington Stock Center), Histone:RFP
(Bloomington Stock Center), Sqh-GFP:MoesinABD (Kiehart et al., 2000)
and Sqh-Gap43:mCherry (Martin et al., 2010).
Confocal microscopy and time-lapse imaging
Confocal images were acquired with an Olympus Fluoview FV1000 confocal
laser scanning microscope with a 40×/1.35NA objective for fixed specimens.
Time-lapse imaging was performed on a spinning-disk confocal microscope
from Zeiss or Solamere Technologies Group with 40×/1.3NA or 63×/1.4NA
objectives. Live imaging was performed with embryos that were dechorionated
and placed on a gas-permeable membrane in Halocarbon 27 oil (Sigma). A
coverslip was placed on embryos, which were then imaged over time. Fixed
specimen confocal imaging was performed using 8 ms/pixel exposure settings,
and live imaging was performed using 150-300 ms exposure times. Live
imaging was acquired at fast (<1 image/s) or slow (1 image/30 s) rates. Furrow
lengths and depths were determined by fine z-scale movie acquisitions (0.5 µm
intervals). All movies were acquired at 25±1°C.
Embryo fixation and immunostaining
Embryos were dechorionated in 50% bleach solution and fixed for 1 h
15 min at the interface of heptane and 3.7% formaldehyde in 0.1 M sodium
phosphate buffer ( pH 7.4) before being manually devitellinized and stained
with Alexa 546-phalloidin (1:200; Molecular Probes), guinea pig anti-RalA
(1:500; Teodoro et al., 2013), Hoechst (1:500; Sigma), mouse anti-Lamin
(1:100; DSHB) or mouse anti-Sec5 (1:35; Murthy et al., 2010). Secondary
antibodies conjugated with Alexa 488 or Alexa 568 (Molecular Probes)
2326
were used at 1:500. Embryos were mounted in Prolong Gold (Molecular
Probes).
Fixed and time-lapse embryo analysis
Confocal and spinning disk images were edited using Adobe Photoshop.
Channels for fixed and time-lapse images were leveled in Photoshop to
show optimal protein populations.
Statistical analysis
Germline clone and siRNA scoring data were tested for statistical
significance using a two-dimensional contingency table with a χ2 test
with α=0.005.
siRNA preparation
Primers for siRNA treatments were selected to represent independent
regions of RalA through the use of the SNAPDRAGON RNAi design
program, which bioinformatically selects against off-target effects (DSRC,
Harvard). dsRNA was made using the Megascript T7 Transcription Kit
(Ambion) and purified using Qiagen RNeasy columns. Final concentration
was determined with a NanoDrop ND1000 spectrophotometer (1550 ng/µl).
Scoring embryonic development
OreR and RalAPL56 embryos were dechorionated in 50% bleach, and then
placed onto a transparent apple juice plate and observed over the course of
4 hours. RalA siRNA- and water-injected embryos were dechorionated in
50% bleach, then glued onto a coverslip. The embryos were dehydrated for
11 min, covered in Halocarbon 700 oil (Sigma), and then injected with RalA
siRNA or water. The embryos were observed over the course of 4 hours.
Scoring nuclear fusion events
RalAPL56 embryos were live imaged, and individual nuclei were tracked
throughout cycles 10-13. For each cycle, during prophase, all WT nuclei
were counted. During anaphase, each nucleus was tracked over time to
DEVELOPMENT
MATERIALS AND METHODS
identify if the nucleus underwent a WT division, a mitotic nuclear collapse,
or adjacent nuclear fusion(s). Since two nuclei are involved in a single
adjacent nuclear fusion, the total number of WT divisions started does not
equal the total number of WT divisions completed when an adjacent nuclear
division occurs. The fusion of one WT nucleus with an already polyploid
nucleus was still counted as an adjacent nuclear fusion.
siRNA injection and live imaging
Embryos were prepared in the same manner as scored siRNA-injected
embryos. After injection, embryos on a coverslip were immersed in
Halocarbon 27 oil and placed onto a gas-permeable slide and imaged on the
spinning disk confocal microscope.
Germline clone preparation
Heterozygous RalAPL56 mutant females were crossed with hemizygous
ovoD, FRT19A males. Larvae were heat shocked twice for 2 hours over the
course of 3 days to generate recombination events.
Acknowledgements
We are grateful to the generous colleagues who supplied antibodies and fly lines:
Hugo Bellen, Tom Schwarz and Tom Millard. Thanks are due to Dinah Loerke and
the J.T.B. lab for suggestions and critical reading of the manuscript. Stocks obtained
from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in
this study.
Competing interests
The authors declare no competing or financial interests.
Author contributions
R.M.H. performed the live imaging, immunostaining and knockdown experiments.
L.M.M. performed dsRNA injections into Rab8 and RalA backgrounds. Z.Z.
performed initial characterization of RalA nuclear phenotypes. R.M.H. and J.T.B.
designed research, analyzed data and wrote the article.
Funding
This work was supported by a National Institutes of Health grant [R01 GM090065] to
J.T.B. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.120998/-/DC1
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