Abstract
The Par complex is polarized to the apical cortex of asymmetrically dividing Drosophila neuroblasts. Previously we showed that Par proteins are polarized by apically directed cortical movements that require F-actin (Oon and Prehoda, 2019). Here we report the discovery of cortical actin pulses that begin before the Par complex is recruited to the cell cortex and ultimately become tightly coupled to Par protein dynamics. Pulses are initially unoriented in interphase but are rapidly directed towards the apical pole in early mitosis, shortly before the Par protein aPKC accumulates on the cortex. The movements of cortical aPKC that lead to its polarization are precisely correlated with cortical actin pulses and F-actin disruption coincides with immediate loss of movement followed by depolarization. We find that myosin II is a component of the cortical pulses, suggesting that actomyosin pulsatile contractions initiate and maintain apical Par polarity during the neuroblast polarity cycle.
Introduction
The Par complex polarizes animal cells by excluding specific cortical factors from the Par cortical domain (Lang and Munro, 2017; Venkei and Yamashita, 2018). In Drosophila neuroblasts, for example, the Par domain forms at the apical cortex during mitosis where it prevents the spread of cortical neuronal fate determinants, effectively restricting them to the basal cortex. The resulting cortical domains are bisected by the cleavage furrow segregating the neuronal fate determinants into the basal daughter cell where they promote differentiation (Homem and Knoblich, 2012). It was recently discovered that apical Par polarization in the neuroblast is a multistep process in which the complex is initially targeted to the apical hemisphere early in mitosis where it forms a discontinuous meshwork (Kono et al., 2019; Oon and Prehoda, 2019). Cortical Par proteins then move along the cortex towards the apical pole, ultimately leading to formation of an apical cap that is maintained until shortly after anaphase onset (Oon and Prehoda, 2019). Here we examine how the cortical movements that initiate and potentially maintain neuroblast Par polarity are generated.
An intact actin cytoskeleton is known to be required for the movements that polarize Par proteins to the neuroblast apical cortex, but its role in the process has been unclear. Depolymerization of F-actin causes apical aPKC to spread to the basal cortex (Hannaford et al., 2018; Oon and Prehoda, 2019), prevents aPKC coalescence, and induces disassembly of the apical aPKC cap (Oon and Prehoda, 2019), suggesting that actin filaments are important for both apical polarity initiation and its maintenance. How the actin cytoskeleton participates in polarizing the Par complex in neuroblasts has been unclear, but actomyosin plays a central role in generating the anterior Par cortical domain in the C. elegans zygote. Pulsatile contractions oriented towards the anterior pole transport the Par complex from an evenly distributed state (Illukkumbura et al., 2019; Lang and Munro, 2017). Bulk transport is mediated by advective flows generated by highly dynamic, transient actomyosin accumulations on the cell cortex (Goehring et al., 2011). While pulsatile movements of actomyosin drive formation of the Par domain in the worm zygote, and F-actin is required for apical Par polarity in the neuroblast, no pulsatile contractions of actomyosin have been observed during the neuroblast polarization process, despite extensive examination (Barros et al., 2003; Cabernard et al., 2010; Connell et al., 2011; Koe et al., 2018; Roth et al., 2015; Roubinet et al., 2017; Tsankova et al., 2017). Instead, both F-actin and myosin II have been reported to be cytoplasmic or uniformly cortical in interphase, and apically enriched at metaphase (Barros et al., 2003; Koe et al., 2018; Tsankova et al., 2017), before undergoing cortical flows towards the cleavage furrow that are important for cell size asymmetry (Cabernard et al., 2010; Roubinet et al., 2017).
The current model for neuroblast actomyosin dynamics is primarily based on the analysis of fixed cells or by imaging a small number of central optical sections in live imaging experiments and we have recently found that rapid imaging of the full neuroblast volume can reveal dynamic phases of protein movements (Oon and Prehoda, 2019). Here we use rapid full volume neuroblast imaging to investigate whether pulsatile movements of cortical actomyosin occur during early mitosis when the Par complex becomes polarized to the apical cortex.
Results and Discussion
Pulsatile dynamics of cortical actin during neuroblast asymmetric divisions
To gain insight into how actin participates in the neuroblast polarity cycle, we imaged larval brain neuroblasts expressing an mRuby fusion of the actin sensor LifeAct (mRuby-LA) using spinning disk confocal microscopy. The localization of this sensor in neuroblasts has been reported (Abeysundara et al., 2018; Roubinet et al., 2017), but only during late mitosis. To follow cortical actin dynamics across full asymmetric division cycles, we collected optical sections through the entire neuroblast volume (~40 0.5 μm sections) at 10 second intervals beginning in interphase and through at least one mitosis (Figure 1-figure supplement 1). Acquiring full cell volume optical sections at this frequency required careful optimization to prevent photobleaching while maintaining sufficient signal levels. Maximum intensity projections constructed from these data revealed localized actin enrichments on the cortex, some of which were highly dynamic (Figure 1 and Video 1). We observed four discrete phases of cortical actin dynamics during neuroblast asymmetric divisions that we describe in detail below.
Imaging and analysis scheme for rapid, full volume imaging of Drosophila neuroblasts from larval brain explants. Larval brains from 3rd instar larvae were mounted and imaged along the neuroblast polarity axis (“apical” and “basal”). Optical sections across the full cell volume were acquired every 7-10 seconds and used to construct maximum intensity projections.
Cortical F-actin dynamics in asymmetrically dividing Drosophila larval brain neuroblasts. (A) Selected frames from Video 1 showing cortical actin pulses during interphase. mRu-by-LifeAct expressed via insc-GAL4/UAS (“actin”) is shown via a maximum intensity projection (MIP) constructed from optical sections through the front hemisphere of the cell.The outline of the neuroblast is shown by a dashed yellow circle. Arrowhead marks an example cortical actin patch. Time (mm:ss) is relative to nuclear envelope breakdown. (B) Selected frames from Video 1 as in panel A showing cortical actin moving apically. (C) Selected frames from Video 1 as in panel A showing cortical actin enriched on the apical cortex. (D) Selected frames from Video 1 showing how actin becomes cortically enriched near NEB. Medial cross sections show the cortical actin signal is relatively discontinuous until NEB approaches. (E) Kymograph constructed from frames of Video 1 using sections along the apical-basal axis as indicated. A legend for the features in the kymograph is included below.
The interphase neuroblast cortex was a mixture of patches of concentrated actin, highly dynamic pulsatile waves that traveled across the entire width of the cell, and areas with little to no detectable actin (Figure 1 and Video 1). Pulsatile movements consisted of the appearance of actin on a localized area and moved rapidly across the cortex for approximately one minute before disappearing (Figure 1A,E). Concentrated actin patches were relatively static, but sometimes changed size over the course of several minutes but were mostly unaffected by the pulsatile waves that occasionally passed over them. Pulses were sporadic in early interphase but became more regular near mitosis, with a new pulse appearing immediately following the completion of the prior one (Figure 1E and Video 1). The direction of the pulses during interphase was highly variable, but often along the cell’s equator (i.e. orthogonal to the polarity/division axis). In general, actin in the interphase cortex was highly discontinuous and included large areas with little to no detectable actin in addition to the patches and dynamic pulses described above. Interphase pulses were correlated with cellular scale morphological deformations in which these areas of low actin signal became distorted away from the cell center while the cortex containing the actin pulse was compressed towards the center of the cell (Figure 1D and Video 1).
A clear transition in the neuroblast cortex occurred several minutes before nuclear envelope breakdown in which pulsatile movements shifted towards the apical pole and actin accumulated at cortical regions with minimal actin in interphase (Figure 1B-E and Video 1). The seemingly randomly oriented interphase pulses gave way to a highly regular progression of apically-directed ones that traveled from the basal pole towards the apical pole (i.e. along the polarity division axis). Pulses remained apically-directed until ceasing near the onset of anaphase, leading to the apical actin enrichment that has been described previously (Barros et al., 2003; Tsankova et al., 2017). Additionally, while the interphase cortex had areas with very little actin, actin was more evenly-distributed following the transition (Figure 1D and Video 1). Another rapid transition occurred shortly after anaphase onset, in which the apically-directed cortical actin movements reversed direction such that the F-actin that had accumulated in the apical hemisphere began to move basally towards the emerging cleavage furrow (Roubinet et al., 2017).
Apically directed actin pulses polarize aPKC
Previously we showed that Par polarity proteins undergo complex cortical dynamics during neuroblast asymmetric cell division, and that Par cortical movements require an intact actin cytoskeleton (Oon and Prehoda, 2019). Examination of the cortical actin cytoskeleton revealed that it also highly dynamic (Figure 1 and Video 1), with key transitions in cortical movements at points in the cell cycle that are similar to those that occur in the protein polarity cycle. We determined the extent to which cortical actin and aPKC dynamics are correlated by simultaneously imaging GFP-aPKC expressed from its endogenous promoter with mRuby-Lifeact (Figure 2 and Video 2). We observed aPKC targeting to the apical membrane beginning approximately ten minutes before NEB, when small foci start to appear. The cortical pulses of actin that passed over these aPKC enrichments had no noticeable effect on them, suggesting that interphase cortical actin dynamics are not coupled to aPKC movement. Near NEB (e.g. 0:30 in Video 2), the continued accumulation of aPKC lead to a diffusely scattered distribution over the apical hemisphere when the transition in actin cortical dynamics began. Continued accumulation led to a discontinuous distribution of aPKC in the apical hemisphere near NEB (e.g. −2:30 in Video 2). At this point, cortical actin pulses transitioned to the apically-directed phase. While interphase pulses had no apparent effect on cortical aPKC, aPKC began moving towards the apical pole when the apically-directed pulses began, and these movements continued until it became fully polarized (3:50 in Video 2). Cortical actin pulses continued with no apparent change in the polarized aPKC apical cap for several minutes (until approximately 7:20 in Video 2) when cortical actin and aPKC began simultaneously moving basally, toward the emerging cleavage furrow. Thus, cortical aPKC does not appear to be coupled to interphase actin pulses, but its movements are highly correlated with the apically-directed pulses that begin in early mitosis. Cortical actin pulses continue even after aPKC is fully polarized and both actin and aPKC simultaneously begin moving basally towards the cleavage furrow in anaphase.
Coordinated actin and aPKC dynamics during the neuroblast polarity cycle. (A) Selected frames from Video 2 showing the correlated dynamics of aPKC and actin during polarization and depolarization. aPKC-GFP expressed from its endogenous promoter (“aPKC”) and mRu-by-LifeAct expressed via insc-GAL4/UAS (“actin”) are shown via a maximum intensity projection (MIP) constructed from optical sections through the front hemisphere of the cell. (B) Kymograph made from a segment along the apical-basal axis of the neuroblast in Video 2 showing the correlated dynamics of aPKC and actin.
Simultaneous imaging of aPKC and actin allowed us to examine precisely when disruption of the actin cytoskeleton influences aPKC dynamics (Figure 3 and Figure 3-videos 1-3). We introduced the actin depolymerizing drug Latrunculin A (LatA) at different phases of the cell cycle and examined how the movement of cortical aPKC was influenced as the cortical actin signal dissipated. We previously found that the actin cytoskeleton is required for the apically-directed polarizing movements of aPKC. Here we find that when cortical actin signal dissipates immediately before the targeting phase (Figure 3A,A’ and Figure 3-video 1), aPKC is recruited to the apical cortex but rapidly depolarizes. When actin dissipates after aPKC is targeted to the apical cortex and has begun coalescing at the apical pole (Figure 3B,B’ and Figure 3-video 2), aPKC movement ceases immediately following the loss of cortical actin and the remaining aPKC diffuses into the basal cortex and becomes depolarized as observed previously. Finally, when cortical actin dissipates after the aPKC apical cap is formed, we see aPKC begins diffusing away from the apical immediately following the disappearance of the cortical actin signal (Figure 3C,C’ and Figure 3-video 3). Thus, cortical actin and aPKC dynamics are highly correlated, and cortical aPKC movement is dependent on cortical actin, ceasing immediately following the loss of cortical actin.
Effect of LatA on actin and aPKC dynamics. (A-C) Selected frames from Figure 3-videos 1-3 showing how loss of cortical actin before mitosis (A), at NEB (B), and during metaphase (C) influences aPKC dynamics. aPKC-GFP expressed from its endogenous promoter (“aPKC”) and mRuby-LifeAct expressed via insc-GAL4/UAS (“actin”) are shown via a maximum intensity projection (MIP) constructed from optical sections through the front hemisphere of the cell. The scale bar applies to all panels in A-C. (A’-C’) Kymographs for A’ and C’ are made from Figure 3-videos 1 and 3 using a section of each frame along the apical-basal axis. The kymograph in B’ is made from a different cell than shown in (B) and FIgure 3-video 2.
Myosin II is a component of neuroblast cortical pulses
We observed morphological changes in interphase cells (Figure 1D and Video 1), and cortical aPKC movements that were correlated with cortical actin dynamics in early mitosis (Figure 2 and Video 2). These phenomena are consistent with force generation by the cortical actin pulses. While actin can generate force through polymerization, contractile forces are generated when it is paired with myosin II, and cortical pulsatile contractions of actomyosin have been observed in other systems (Michaux et al., 2018; Munro et al., 2004). Although there are numerous reports of myosin II dynamics in neuroblasts (Barros et al., 2003; Koe et al., 2018; Tsankova et al., 2017), no cortical pulses have been described and its localization has been described as uniformly cortical or cytoplasmic in interphase and before metaphase in mitosis. We used rapid imaging of the full cell volume, simultaneously following a GFP fusion of the myosin II regulatory light chain Spaghetti squash (GFP-Sqh) with mRuby-Lifeact, to determine if myosin II is part of the cortical actin pulses we observed. We found that myosin II is a component of every phase of the actin pulses (Figure 4 and Video 3), including the apically-directed pulses that polarize aPKC. Interestingly, however, while myosin II localized with actin and had very similar dynamics, the localization between the two was not absolute and there were often large cortical regions where the two did not colocalize in addition to the region where they overlapped (Figure 4 and Video 3). This is similar to the localization of the two proteins in the polarizing worm zygote (Michaux et al., 2018; Reymann et al., 2016). We also noticed that myosin II pulses were less persistent than their actin counterparts during the apically-directed phase of dynamics (Figure 4C). Thus, while there are some differences in the dynamics of the two proteins, myosin II is a component of interphase and early mitotic neuroblast cortical pulses.
Dynamics of cortical actomyosin in asymmetrically dividing Drosophila larval brain neuroblasts. (A) Selected frames from Video 3 showing cortical actomyosin traveling across the equatorial face of the cell. GFP-Sqh expressed from its endogenous promoter (“Myosin II”) and mRuby-LifeAct expressed via worniu-GAL4/UAS (“actin”) are shown via a maximum intensity projection (MIP) constructed from optical sections through the front hemisphere of the cell.The outline of the neuroblast is shown by a dashed yellow line and arrowheads indicate the starting position of the cortical patches. Time is relative to nuclear envelope breakdown. (B) Kymograph constructed from frames of Video 3 during interphase using sections through the equatorial region of the cell as indicated. (C) Kymograph constructed from frames of Video 3 during mitosis using sections along the polarity axis of the cell as indicated.
Model for role of actomyosin in neuroblast Par polarity. During interphase when aPKC is cytoplasmic, myosin II pulsatile contractions are predominantly equatorial. During apical polarity initiation in prophase and shortly before when discrete aPKC cortical patches begin to undergo coordinated movements towards the apical pole, myosin II pulsatile contractions reorient towards the apical cortex. Contractions are initially over a large surface area but become concentrated to the apical cortex as aPKC apical cap assembly is completed and the maintenance phase begins. At anaphase apical myosin II is cleared as it flows towards the cleavage furrow while the aPKC cap is disassembled.
A role for actomyosin pulsatile contractions in the initiation and maintenance of apical Par polarity in neuroblasts
Our results reveal previously unrecognized phases of pulsatile contractions during cycles of neuroblast asymmetric divisions. During interphase, transient cortical patches of actomyosin undergo highly dynamic movements in which they rapidly traverse the cell cortex, predominantly along the cell’s equator, before dissipating and a new cycle begins (Figure 1A). Shortly after mitotic entry the pulsatile movements reorient to align with the polarity axis. Importantly, the transition between these phases occurs shortly before the establishment of apical Par polarity, when discrete cortical patches of aPKC undergo coordinated movements towards the apical pole to form an apical cap. Pulsatile movements continue past metaphase when apical cap assembly is completed, suggesting that they may also be involved in cap maintenance.
The actomyosin dynamics we have uncovered provide a framework for understanding how actomyosin participates in neuroblast apical polarity. First, apically directed pulsatile movements of actomyosin are consistent with the requirement for F-actin in the cortical flows that lead to coalescence of discrete aPKC patches (Figure 3) (Oon and Prehoda, 2019). Furthermore, the continuation of myosin II pulsatile movements after cap assembly is completed implies that they are also important for polarity maintenance (Figure 3). A role for actomyosin in cap maintenance would explain why the cap becomes dissociated when F-actin is depolymerized shortly after cap assembly (Oon and Prehoda, 2019). How might myosin II pulsatile contractions lead to the cortical flows we have observed during the polarization of the neuroblast apical cortex? Studies of worm zygote Par polarity provide a possible explanation. In this system, pulsatile contractions generate bulk cortical flows (i.e. advection) that lead to non-specific transport of cortically localized components (Goehring et al., 2011; Illukkumbura et al., 2019). Whether the cortical flows that occur during apical polarization of the neuroblast are also driven by advection will require further study.
Materials and Methods
Key Resources Table
Fly strains and genetics
UAS-LifeAct-Ruby (Bloomington stock 35545), BAC-encoded aPKC-GFP (Besson et al., 2015) and Sqh-GFP (Royou et al., 2002) transgenes were used to assess F-actin, aPKC and myosin II dynamics, respectively. Expression of LifeAct was specifically driven in nerve cells upon crossing UAS-LifeAct-Ruby to insc-Gal4 (1407-Gal4, Bloomington stock 8751) or to wor-Gal4 (Bloomington stock 56553). The following genotypes were examined through dual channel live imaging: BAC-aPKC-GFP/Y; insc-Gal4, UAS-LifeAct-Ruby/+ and; worGal4, Sqh-GFP, UAS-LifeAct-Ruby/+;.
Live imaging
Third instar larvae were incubated in 30°C overnight (~12 hours) prior to imaging and were dissected to isolate the brain lobes and ventral nerve cord, which were placed in Schneider’s Insect media (SIM). Larval brain explants were placed in lysine-coated 35 mm cover slip dishes (WPI) containing modified minimal hemolymph-like solution (HL3.1). Explants were imaged on a Nikon Ti2 microscope equipped with a Yokogawa CSU-W1 spinning disk that was configured to two identical Photometrics Prime BSI Scientific CMOS cameras for simultaneous dual channel live imaging. Using the 1.2 NA Plan Apo VC water immersion objective, explants were magnified at 60x for visualization. Explants expressing LifeAct-Ruby, aPKC-GFP and Sqh-GFP were illuminated with 488 nm and 561 nm laser light throughout approximately 41 optical sections with step size of 0.5 μm and time interval of 10 seconds.
Image processing, analysis and visualization
Movies were analyzed in ImageJ (using the FIJI package) and in Imaris (Bitplane). Neuroblasts whose apical-basal polarity axis is positioned parallel to the imaging plane were cropped out to generate representative images and movies. Cortical edge and central maximum intensity projections (MIP) were derived from optical slices capturing the surface and center of the cell, respectively. Cortical MIPs were also used to perform kymograph analysis, where the change in localization profile of fluorescently-tagged fusion proteins within a 3 to 5 pixels wide region was examined across time. To track cortical movements over the length of the apical-basal axis, a vertical region parallel to the polarity axis was specified for the kymograph analysis. Similarly, a horizontal line orthogonal to the polarity axis that is superimposing on the presumptive equator was specified for examining equatorial motions. Optical sections capturing the whole of the cell were assembled for 3D rendering and visualization in FIJI or Imaris. These volumetric reconstructions were then used to determine the timing of cortical motions characterized in this paper.
Video Legends
Video 1 Actin dynamics in a larval brain neuroblast.
The mRuby-Lifeact sensor expressed from the UAS promoter and worniu-GAL4 (drives expressing in neuroblasts and progeny) is shown with a maximum intensity projection of the front hemisphere of the cell.
Video 2 Correlated dynamics of the Par protein aPKC and Actin in a larval brain neuroblast. GFP-aPKC expressed from its endogenous promoter and the mRuby-Lifeact sensor expressed from the UAS promoter and worniu-GAL4 (drives expressing in neuroblasts and progeny) are shown from simultaneously acquired optical sections with a maximum intensity projection of the front hemisphere of the cell.
Figure 3-video 1 Correlated dynamics of the Par protein aPKC and Actin in a larval brain neuroblast treated with Latrunculin A before mitosis.
GFP-aPKC expressed from its endogenous promoter and the mRuby-Lifeact sensor expressed from the UAS promoter and worniu-GAL4 (drives expressing in neuroblasts and progeny) are shown from simultaneously acquired optical sections with a maximum intensity projection of the front hemisphere of the cell. Lat A was added to the media surrounding the larval brain explant at the indicated time.
Figure 3-video 2 Correlated dynamics of the Par protein aPKC and Actin in a larval brain neuroblast treated with Latrunculin A during prophase.
GFP-aPKC expressed from its endogenous promoter and the mRuby-Lifeact sensor expressed from the UAS promoter and worniu-GAL4 (drives expressing in neuroblasts and progeny) are shown from simultaneously acquired optical sections with a maximum intensity projection of the front hemisphere of the cell. Lat A was added to the media surrounding the larval brain explant at the indicated time.
Figure 3-video 3 Correlated dynamics of the Par protein aPKC and Actin in a larval brain neuroblast treated with Latrunculin A during metaphase.
GFP-aPKC expressed from its endogenous promoter and the mRuby-Lifeact sensor expressed from the UAS promoter and worniu-GAL4 (drives expressing in neuroblasts and progeny) are shown from simultaneously acquired optical sections with a maximum intensity projection of the front hemisphere of the cell. Lat A was added to the media surrounding the larval brain explant at the indicated time.
Video 3 Correlated dynamics of myosin II and Actin in a larval brain neuroblast.
GFP-Sqh (the myosin II regulatory light chain, Spaghetti Squash) expressed from its endogenous promoter and the mRuby-Lifeact sensor expressed from the UAS promoter and worniu-GAL4 (drives expressing in neuroblasts and progeny) are shown from simultaneously acquired optical sections with a maximum intensity projection of the front hemisphere of the cell and the medial optical section. The neuroblast is highlighted by a dashed circle.
References
