Moesin is involved in polarity maintenance and cortical remodeling during asymmetric cell division

Abstract

An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts (NBs). However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. We find that the actin-binding protein, Moesin, is essential for NB proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex, and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. Our findings reveal Moesin as a novel apical polarity protein that drives cortical remodeling of dividing NBs, which is essential for polarity maintenance and initial establishment of cell size asymmetry.

FIGURE 1:

Phosphorylated Moesin localizes asymmetrically in mitotic NBs. (A) w¹¹¹⁸ third instar larval central brain (CB) and optic lobe (OL) was fluorescently labeled with anti–p-Moesin (green) and anti-Prospero (Pros; magenta). P-Moesin localizes to the cortex of NBs with an asymmetric p-Moesin enrichment indicated by yellow arrows. (B, C) P-Moesin and the basal polarity protein (Numb) are enriched at opposite cortical poles during metaphase. (C) The relative mean FI of p-Moesin along the lateral cortex (indicated by the blue line in the schematic diagram) shows that p-Moesin is enriched at the apical cortex (compartment I) during metaphase (n = 5). (D, E) P-Moesin is reduced at the apical cortex during anaphase, with the relative mean FI of p-Moesin along the lateral cortex shown (n = 5). (F–H) P-Moesin is enriched at the basal cortex of the dividing NB and accumulates at the cleavage furrow site during telophase. (H) The relative mean FI of p-Moesin along the lateral cortex shows that p-Moesin is enriched at the basal NB cortex where the cleavage furrow forms (compartment IV; n = 5). (B, D, F, G) Merged panels are single focal plane images and show DAPI (blue), p-Moesin (green), Numb (red), and β-tubulin (cyan). Grayscale images are maximum intensity projections. Error bars represent SD. Scale bars represent (A) 50 μm and (B, D, F, G) 5 μm.

FIGURE 2:

Moesin is essential for NB proliferation and mitotic progression. UAS-Dicer;;UAS-Moe^dsRNA was crossed to w¹¹¹⁸ (Ctrl) and Insc-GAL4 (Moe^dsRNA). UAS-Dicer alone was crossed to Insc-GAL4 (Dicer). (A–C) The larval CNS of Control, Dicer, and Moe^dsRNA labeled with anti-Deadpan (Dpn; green) and anti–phospho-histone H3 (PH3; magenta) at 96 h after larval hatching (ALH) are shown. (D) The mean number of Dpn-positive cells and (E) mean proportion of PH3-positive, Dpn-positive cells per central brain lobes of Control, Dicer, and Moe^dsRNA at ∼24, 48, 72, and 96 h ALH (n = minimum of 28 brain lobes; see Materials and Methods for exact sample sizes). (F) The diameter of Control and Moe^dsRNA metaphase NBs at 48 h (n = 35 and 44 NBs, respectively) and 96 h (n = 23 NBs each) ALH. The red line marks the mean diameter. (G) The area of Control and Moe^dsRNA interphase NBs at 48 h ALH (n = 30 and 39 NBs, respectively). The red line marks the mean area. (H) The mean proportion of PH3-positive, Dpn-positive cells undergoing the specific stages of mitosis per central brain lobe of Control (n = 28) and Moe^dsRNA (n = 55) at 96 h ALH. In control brain lobes, the mean proportions (± SE) of mitotic NBs in the specific stages are 29.6 ± 2.0% (prophase), 43.4 ± 2.0% (metaphase), 7.2 ± 1.6% (anaphase), and 19.7 ± 1.6% (telophase). In Moe^dsRNA brain lobes, the mean proportions (± SE) of mitotic NBs are 60 ± 4.6% (mitotic defective), 13.9 ± 2.8% (prophase), 16.7 ± 2.6% (metaphase), 0.7 ± 0.3% (anaphase), and 4.6 ± 2.1% (telophase). Scale bars represent (A–C) 100 μm. Error bars represent SE. *p < 0.05, **p < 0.001, ***p < 0.0001, and ns = not significant using an unpaired t test.

FIGURE 3:

Slik is essential for NB proliferation and Moesin phosphorylation. (A) w¹¹¹⁸ larval NBs labeled with DAPI (blue), anti-Moesin (green), anti-Slik (red), and anti-Prospero (cyan). Slik partially colocalizes with Moesin at the NB cortex and localizes to the GMC cortex. (B) w¹¹¹⁸ larval NB undergoing metaphase labeled with DAPI (blue), anti–p-Moesin (cyan), anti-Slik (green), and anti–α-tubulin (red) as shown in merged panel. (C) The apical/basal FI ratios of p-Moesin and Slik in metaphase NBs (n = 23) show that p-Moesin is apically enriched (1.99 ± 0.12; mean ± SE) and Slik is uniformly distributed at the apical and basal cortices (0.93 ± 0.04, p < 0.0001). The red line marks the mean ratio. (D, E) w¹¹¹⁸ and slik¹ larval CNS labeled with anti-Deadpan (Dpn; green) and anti–phospho-histone H3 (PH3; magenta) at 96 h ALH. (F) The mean number of Dpn-positive cells and (G) the mean proportion of PH3-positive, Dpn-positive cells per central brain lobes of w¹¹¹⁸ (n = 35) and slik¹ (n = 26) at 96 h ALH. (H) The diameter of metaphase NBs in w¹¹¹⁸ (n = 25) and slik¹ (n = 29) ∼5–6 d AEL. The red line marks the mean diameter. (I, J) w¹¹¹⁸ and slik¹ larval metaphase NBs labeled with DAPI (blue), anti–p-Moesin (cyan), anti-Numb (green), and anti–β-tubulin (red) at ∼5–6 d AEL. (J) The p-Moesin signal is reduced in slik¹ mutant NBs during metaphase, compared with w¹¹¹⁸ controls. Scale bars represent (A) 10 μm, (B, I, J) 5 μm, and (D, E) 100 μm. Error bars represent SE. ***p < 0.0001 using an unpaired t test.

FIGURE 4:

Moesin is involved in apical polarity maintenance in NBs undergoing prophase and metaphase. UAS-Dicer;;UAS-Moesin^dsRNA was crossed to w¹¹¹⁸ (Ctrl) and Insc-GAL4 (Moe^dsRNA) and larval NBs undergoing (A–D) prophase and (E–J) metaphase were analyzed ∼96 h ALH. (A) Bazooka (Baz) and (C) aPKC crescents form in control NBs during prophase. (B) Baz and (D) aPKC crescents are not observed in a proportion of Moe^dsRNA NBs during prophase. (C, D) Filamentous actin (Phalloidin; red) appears discontinuous in Moesin knockdown NBs undergoing prophase (yellow arrows) compared with controls. (E) Par-6 crescents form in control NBs during metaphase. (F) A weak polar Par-6 signal (yellow asterisk) and (G) an absent polar Par-6 signal is observed in Moe^dsRNA NBs during metaphase. (H) aPKC crescents form in control NBs during metaphase. (I) A weak polar aPKC signal (yellow asterisk) and polar Miranda is observed in Moe^dsRNA NBs during metaphase. (J) An absent polar aPKC and polar Miranda is observed in Moe^dsRNA NBs. Refer to Table 1 for summary of phenotypic proportions observed. Merged panels are single focal plane images and show DAPI (blue), Miranda (cyan), α-tubulin or aPKC/PH3 (green), and specified apical polarity protein or Phalloidin (red). Grayscale images are maximum intensity projections, with the exception of Phalloidin panels, which are single focal plane images. Scale bars represent 5 μm.

FIGURE 5:

Slik is important for aPKC polarity maintenance in NBs. w¹¹¹⁸ and slik¹ larval NBs labeled with DAPI (blue), anti–α-tubulin (green), anti-aPKC/anti–phospho-histone H3 (aPKC/PH3; red), and anti-Miranda (Mira; cyan), ∼5–6 d AEL. (A) An aPKC crescent forms in w¹¹¹⁸ NBs undergoing prophase. (B) An aPKC crescent is not observed in 87% of slik¹ mutant NBs undergoing prophase (n = 23). (C) The apical/basal FI ratios of aPKC in w¹¹¹⁸ (n = 20) and slik¹ (n = 23) prophase NBs are shown. The red line marks the mean ratio. (D) An aPKC crescent is observed in w¹¹¹⁸ metaphase NBs. (E) A weak polar aPKC signal is observed in 65% of slik¹ NBs, indicated by the yellow asterisk, and (F) a polar aPKC signal is absent in 27% slik¹ NBs during metaphase (n = 26). (G) The apical/basal FI ratios of aPKC in w¹¹¹⁸ (n = 26) and slik¹ (n = 26) metaphase NBs are shown. The red line marks the mean ratio. Merged panels are single focal plane images, and grayscale images are maximum intensity projections. Scale bars represent 5 μm. ***p < 0.0001 using an unpaired t test.

FIGURE 6:

Apical polarity proteins are important for the asymmetric distribution of p-Moesin during metaphase. Third instar larval NBs of (A) w¹¹¹⁸, (B) UAS-Cdc42^dsRNA, (C) UAS-Par6^dsRNA, (D) UAS-aPKC^dsRNA, and (E) UAS-Lgl^dsRNA crossed to Insc-GAL4. The p-Moesin immunofluorescence signal appears reduced at the apical cortex of metaphase NBs in the respective knockdowns, when compared with controls. (F) The apical/basal FI ratios of p-Moesin in metaphase NBs of controls (n = 20), Cdc42^dsRNA (n = 10), Par-6^dsRNA (n = 30), aPKC^dsRNA (n = 33), and Lgl^dsRNA (n = 13). The red line marks the mean ratio. (G–L) w¹¹¹⁸ and pins¹⁹³ larval NBs undergoing metaphase ∼5–6 AEL. (H–L) Multiple phenotypes are observed in pins¹⁹³ mutant NBs during metaphase (n = 138), compared with (G) w¹¹¹⁸ metaphase NBs, which display polar p-Moesin and polar Numb at opposite poles (100%, n = 57). Polar p-Moesin and Numb are enriched at (H) opposite poles (17%, n = 138) and (I) the same pole (19%, n = 138) in pins¹⁹³ mutant NBs. (J) Polar p-Moesin and uniform Numb (17%, n = 138), (K) uniform/discontinuous p-Moesin and polar Numb (16%, n = 138), and (L) uniform/discontinuous p-Moesin and uniform Numb (20%, n = 138) distributions are observed in pins¹⁹³ mutant NBs during metaphase. Polar enrichment is indicated by yellow asterisk. Merged panels are single focal plane images and show DAPI (blue), p-Moesin (cyan), Numb (red), and β-tubulin (green). Grayscale images in A–E are maximum intensity projections and in G–L are single focal plane images. Scale bars represent 5 μm. *p < 0.05 and ***p < 0.0001 using an unpaired t test.

FIGURE 7:

Moesin is important for apical integrity and cortical asymmetric divisions. NBs from w¹¹¹⁸ and Moe^G0323 third instar larvae were labeled with DAPI (blue), anti–α-tubulin (green), anti-aPKC/anti–phospho-histone H3 (aPKC/PH3; red) and anti-Miranda (Mira; cyan). (A) A polar aPKC crescent forms in w¹¹¹⁸ metaphase NBs. (B) A polar aPKC crescent, similar to controls, forms in 65% of Moe^G0323 metaphase NBs (n = 116); however, aPKC localizes to ectopic cortical sites in 35% of these Moe^G0323 NBs (yellow arrow; n = 75). Cortical blebbing is present in Moe^G0323 NBs (yellow asterisk). (C) The polar aPKC domain appears disorganized in 21% of Moe^G0323 NBs during metaphase (n = 116). (B, C) Miranda localizes to the opposite cortical pole of aPKC in 74% of Moe^G0323 NBs (n = 116), although polar Miranda crescents appear reduced in size or diffuse compared with control NBs. (D) An asymmetric basal furrow is observed in w¹¹¹⁸ NBs undergoing anaphase (yellow arrowheads). (E) Initial constriction at the presumptive cleavage furrow site (yellow arrowheads) appears symmetric in Moe^G0323 NBs undergoing anaphase. (F) The quantification of A/B ratios in w¹¹¹⁸ and Moe^G0323 NBs undergoing anaphase (n = 8 and 9, respectively) and telophase (n = 5). The red line marks the mean ratio. Merged panels are single focal plane images, and grayscale images are maximum intensity projections. Scale bars represent 5 μm. ns = not significant using an unpaired t test.

FIGURE 8:

Moesin is essential for maintaining a stable actomyosin cortex during metaphase. (A, B) Live imaging of Insc-GAL4; UAS-ChRFP-Tub, UAS-Lifeact-GFP crossed to w¹¹¹⁸ and Moe^G0323 to visualize filamentous actin (green) and the mitotic spindle (magenta) in NBs. Single focal plane images were selected from Supplemental Videos 1 and 2 to further demonstrate cortical dynamics during metaphase. The time value in the top left corner of the merged panels is shown in minutes:seconds. NEB occurs at 0:00. (A) A round and stable metaphase cortex is observed in w¹¹¹⁸ NBs. (B) The NB cortex appears unstable and nonuniform cortical actin dynamics (yellow arrows) are observed in Moe^G0323 mutant NBs during metaphase. (C–F) Metaphase NBs observed in larval brains isolated from Ubi-Rok-GFP crossed to (C, D) w¹¹¹⁸ and (E, F) Moe^G0323. (C, D) Rok-GFP and p-Myosin appear cortical with a slight apical enrichment, as shown in D where the relative FI was measured around the entire metaphase cortex (white dashed line in merged panel). (E, F) Rok-GFP and p-Myosin displayed a nonuniform distribution at the Moe^G0323 metaphase cortex and was enriched at the lateral cortex (yellow arrows), as shown in F, where the relative FI was measured around the entire cortex (white dashed line in merge). Merged panels show DAPI (blue), anti–p-Myosin (red), and anti-GFP (green). Scale bars represent 5 μm.

FIGURE 9:

Moesin regulates Myosin-induced furrow positioning during early anaphase. (A, B) Live imaging of Insc-GAL4; UAS-ChRFP-Tub, UAS-Lifeact-GFP crossed to w¹¹¹⁸ and Moe^G0323 to visualize filamentous actin (green) and the mitotic spindle (magenta) in the NBs. The time value in the top left corner of the merged panels is shown in minutes:seconds. NEB occurs at 0:00. (A) Furrow induction (yellow arrowheads) shows restricted cortical actin at this site in the w¹¹¹⁸ control NB. (B) Cortical actin does not appear to be as restricted to the furrow site (yellow arrowheads) in the Moe^G0323, as in the w¹¹¹⁸ NB. Cortical blebbing (yellow arrows) is also observed throughout ACD of Moe^G0323 NB. (C–F) w¹¹¹⁸ and Moe^G0323 larval NBs undergoing early anaphase were labeled with DAPI (blue), anti–α-tubulin (green), anti–p-Myosin (red), and anti-Miranda (Mira; cyan). (C, D) P-Myosin localizes to the basal furrow in 80% of w¹¹¹⁸ NBs undergoing early anaphase (n = 5). The white dashed line from the apical to basal pole in the merged panel indicates the region of the cortex that was measured in D. (D) The relative p-Myosin FI along the w¹¹¹⁸ lateral cortex from the apical to basal cortical pole shows that p-Myosin is enriched at the basal cortex. (E, F) P-Myosin localizes to the cell equator in the Moe^G0323 NB shown and the p-Myosin basal furrow was not observed during early anaphase (100%; n = 5). The white dashed line from the apical to basal pole in the merge panel indicates the region of the cortex that was measured in F. (F) The relative p-Myosin FI along the Moe^G0323 lateral cortex from the apical to basal cortical pole shows that p-Myosin peaks near the cell equator. All panels shown are a single focal plane images. Scale bars represent 5 μm.