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. 2021 Nov 15:10:e66574.
doi: 10.7554/eLife.66574.

Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle

Chet Huan Oon  1 , Kenneth E Prehoda  1
Affiliations

Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle

Chet Huan Oon et al. Elife. .

Abstract

The Par complex dynamically polarizes to the apical cortex of asymmetrically dividing Drosophila neuroblasts where it directs fate determinant segregation. Previously, we showed that apically directed cortical movements that polarize the Par complex require F-actin (Oon and Prehoda, 2019). Here, we report the discovery of cortical actomyosin dynamics that begin in interphase when the Par complex is cytoplasmic but ultimately become tightly coupled to cortical Par dynamics. Interphase cortical actomyosin dynamics are unoriented and pulsatile but rapidly become sustained and apically-directed in early mitosis when the Par protein aPKC accumulates on the cortex. Apical actomyosin flows drive the coalescence of aPKC into an apical cap that depolarizes in anaphase when the flow reverses direction. Together with the previously characterized role of anaphase flows in specifying daughter cell size asymmetry, our results indicate that multiple phases of cortical actomyosin dynamics regulate asymmetric cell division.

Keywords: D. melanogaster; actomyosin; cell biology; cell polarity; differentiation; regenerative medicine; stem cells.

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Conflict of interest statement

CO, KP No competing interests declared

Figures

Figure 1.
Figure 1.. Cortical F-actin dynamics in asymmetrically dividing Drosophila larval brain neuroblasts.
(A) Selected frames from Video 1 showing cortical actin pulses during interphase. mRuby-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. In this example, the pulse moves from the upper left of the cell to the lower right. Arrowheads mark several cortical actin patches. Time (mm:ss) is relative to nuclear envelope breakdown. (B) Selected frames from Video 1 as in panel A showing cortical actin moving apically. Arrowheads delineate apical and basal extent of dynamic actin. (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 nuclear envelope breakdown (NEB). A single 0.4 µM medial section of the cortical actin signal is relatively discontinuous before NEB, with areas of very low actin signal, but becomes more evenly distributed as the cell rounds in mitosis (3:00 time point). (E) Kymograph constructed from frames of Video 1 using sections along the apical-basal axis as indicated (NB, neuroblast). A legend with the features in the kymograph depicting the cortical dynamic phases and apical enrichment of actin is included below.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. 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 10–15 s and used to construct maximum intensity projections.
Figure 2.
Figure 2.. Coordinated actin and aPKC dynamics during the neuroblast polarity cycle.
Figure 3.
Figure 3.. Cortical F-actin is required for aPKC coalescence and polarity maintenance.
(A) Disruption of cortical F-actin dynamics using a low dosage (50 µM) of cytochalasin D (CytoD) causes immediate cessation of aPKC cortical movement. Selected frames from Figure 3—video 1 showing the inhibition of actin dynamics and accompanying loss of apically-directed aPKC movements. 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. (A’) Kymograph made from Figure 3—video 1 using a section of each frame along the apical-basal axis, as indicated. (B) Selected frames from Figure 3—video 3 showing significant entry of aPKC into the basal hemisphere (i.e. depolarization) following LatA treatment. Upper arrowhead highlights a localized enrichment of aPKC that is retained in the apical region. Lower arrowhead highlights increased aPKC in basal hemisphere. (B’) Kymograph made from Figure 3—video 3 using a section of each frame along the basal membrane near the cell’s equator as indicated. (C) Selected frames from Figure 3—video 4 showing the maintenance of aPKC polarization following CytoD treatment. (C’) Kymograph made from Figure 3—video 4 using a section of each frame along the basal membrane as indicated. (D) Gardner-Altman estimation plot of the fold increase in basal aPKC membrane signal following Latrunculin A (LatA) and CytoD treatments. The ratio of aPKC membrane signal on the basal membrane shortly after NEB to that at NEB is shown for individual LatA- and CytoD-treated neuroblasts (shown in Figure 3—video 5), along with the difference in means of the measurements. Statistics: bootstrap 95 % confidence interval (bar in ‘CytoD minus LatA’ column shown with bootstrap resampling distribution).
Figure 4.
Figure 4.. Dynamics of cortical actomyosin in asymmetrically dividing Drosophila larval brain neuroblasts.
(A) Selected frames from Video 3 showing cortical actomyosin dynamics. 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.
Figure 5.
Figure 5.. Model for role of actomyosin in neuroblast Par polarity.
During interphase when aPKC is cytoplasmic, actomyosin pulsatile contractions are predominantly equatorial. During apical polarity initiation in prophase and shortly before when discrete aPKC cortical patches begin to coalesce, actomyosin transitions to more continuous movements directed toward the apical cortex. At anaphase apical actomyosin is cleared as it flows toward the cleavage furrow while the aPKC cap is disassembled.
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