Coronin-1 promotes directional cell rearrangement in Drosophila wing epithelium

Abstract

Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. In this study, we investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. We observed that Coronin-1 localizes at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1) and cofilin, actin disassemblers and known regulators of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1 and cofilin, highlighting the role of ABPs in the complex process of morphogenesis.

Key words: morphogenesis, cell rearrangement, actin binding proteins (ABPs)

Introduction

Cell rearrangement plays a critical role in morphogenesis (Takeichi, 2014). It involves shrinkage, reconnection, and elongation of the adherence junction (AJ) to alter the relative cell positions. The reorganization of the F-actin network by actin binding proteins (ABPs) is essential for remodeling the junction, as the AJ complex is tightly linked to the actomyosin network (Yap et al., 2015; Clarke and Martin, 2021). However, the precise molecular mechanisms governing F-actin dynamics during cell rearrangement remain unclear.

Drosophila pupal wing is an ideal model system to study the mechanisms of cell rearrangement (Matamoro-Vidal et al., 2015). In the wing tissue, the direction of cell rearrangement is precisely regulated. Approximately 21–22 hours after puparium formation (h APF), the anterior-posterior (AP) junction shrinks, while the newly generated junction elongates along the proximal-distal (PD) axis, resulting in contraction-elongation of the wing tissue (Aigouy et al., 2010; Sugimura and Ishihara, 2013). The myosin-II (myo-II) and its regulatory molecules trigger junction shrinkage and elongation in the wing (Bardet et al., 2013). In addition, actin interacting protein 1 (AIP1) and cofilin, which disassemble F-actin, guide cell rearrangement by promoting actin turnover along the shrinking AP junctions (Ikawa and Sugimura, 2018). Along the short junctions that undergo adhesion remodeling for reconnecting junctions, the myo-II cables detach from the AJ and form rectangle-shaped myo-II cables, called rsMCs (magenta square in Fig. 1A). This formation of rsMC is supported by AIP1 and cofilin and promotes junction reconnection (Ikawa and Sugimura, 2018; Ikawa et al., 2023). However, the role of other ABPs in cell rearrangement remains largely unexplored.

Fig. 1

Endogenous Coronin-1 localizes at reconnecting junctions

(A) A schematic of the Drosophila wing and cell rearrangement during wing development. The vertical and horizontal directions are aligned with the anterior-posterior (AP) and the proximal-distal (PD) axes, respectively. The axes are conserved throughout all figures. ptc-Gal4 is expressed in the C region, shaded grey. Approximately 21–22 h after puparium formation (APF) and afterwards, wing cells shorten AP junctions and intercalate along the PD axis (Aigouy et al., 2010; Sugimura and Ishihara, 2013). During cell rearrangement, myo-II cables (magenta) are detached from short reconnecting junctions (designated as rectangle-shaped myo-II cables (rsMC); Ikawa et al., 2023). (B) Generation of an EGFP knock-in allele at the coronin locus. See Methods for details. (C, D) Low magnification images of Coronin-1-EGFP of the WT and coronin RNAi wings at 24 h APF. (E) Images of Coronin-1-EGFP (gray in left panels and green in right panels) and myo-II-mKate2 (gray in middle panels and magenta in right panels). Arrowheads point to short, reconnecting junctions. (F) Quantifications of Coronin-1-EGFP and myo-II mKate2 signal intensities around the rsMC based on images in (E). (G) Time-lapse images of Coronin-1-EGFP (gray in upper panels, green in bottom panels) and myo-II-mKate2 (gray in bottom panels, magenta in bottom panels) during cell rearrangement. Blue and orange arrowheads indicate the AP and PD junctions, respectively. (H) Quantifications of Coronin-1-EGFP and myo-II mKate2 signal intensities around the rsMC based on timelapse images in (G). The number of regions of interest (ROIs) is indicated (F, H). The data are presented as mean ± SD (F, H). Scale bars: 100 μm (C, D) and 5 μm (E, G).

Cofilin relies on co-factors, including AIP1, for efficient actin filament severing (McCall et al., 2019; Nadkarni and Brieher, 2014). Coronin-1 (Drosophila gene name, coronin) is another cofactor of cofilin. It exerts bidirectional control over F-actin dynamics—it facilitates both cofilin-mediated F-actin severing and Arp2/3-mediated F-actin branching (Brieher et al., 2006; Gandhi et al., 2009). Notably, it has been reported that coronin mutants exhibit malformed wing shapes (Bharathi et al., 2004), suggesting cell rearrangement defects.

Here, we show that Coronin-1 promotes directional cell rearrangement in the Drosophila pupal wing. Using CRISPR-Cas9 techniques, we generated transgenic flies expressing Coronin-1-EGFP to observe its localization and dynamics. The observations indicated that Coronin-1 is localized at the reconnecting junction during cell rearrangement and that this localization is dependent on AIP1 and cofilin. Moreover, the loss of Coronin-1 functions resulted in directional cell rearrangement defects. These findings suggest that cofilin, AIP1, and Coronin-1 interplay promotes directional cell rearrangement.

Methods

Generation of transgenic flies

The Coronin-1-EGFP knock-in line was generated by targeted integration of an EGFP sequence into the 3' end of the coronin (Drosophila coronin-1 gene) gene using CRISPR/Cas9. An EGFP knock-in cassette vector, pPExRF3, was constructed by replacing the Venus sequence with the EGFP sequence in pPVxRF3 (Kondo et al., 2020). The left and right homology arms of approximately 2000-bp were PCR-amplified from the genomic DNA of the standard y¹; cn¹ bw¹ sp¹ strain. Their sequences were designed such that EGFP was translated as an in-frame C-terminal fusion with the target protein. The reporter cassette excised from pPExRF3 and the left and right homology arms were assembled and cloned into a linearized pUC19 in a single enzymatic reaction using the In-Fusion Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). A gRNA vector was constructed by cloning a 20-bp target sequence surrounding the stop codon of coronin into pU6 (FlyCRISPR). A mixture of the donor vector (100 ng/μL) and the gRNA vector (100 ng/μL) were injected into fertilized eggs of y¹, w¹¹¹⁸; attP40{nos-Cas9}/CyO, which maternally express Cas9 protein (Kondo and Ueda, 2013); 100–200 eggs were injected, either in-house or by BestGene Inc. The surviving larvae were reared to adulthood and crossed to y¹, w¹¹¹⁸. Transformants in the F1 progeny were selected by eye-specific RFP expression from a 3xP3-RFP marker gene in adults and a single transformant was used to establish a transgenic line. The 3xP3-RFP marker was subsequently removed by crossing them to CyO-Crew, a balancer chromosome carrying hs-Cre (Siegal and Hartl, 1996). The resulting males were then crossed to generic balancer lines, and their progeny were screened for loss of the eye-specific RFP expression. Leaky expression of Cre from the hs-Cre transgene in the absence of heat shock was sufficient to induce excision in almost all of the progeny.

Drosophila genetics

The fly strains and a list of genotypes are summarized in the Supplementary Methods.

Phalloidin staining

The phalloidin staining was performed as described previously (Ikawa and Sugimura, 2018). Details are described in the Supplementary Methods.

Image analysis

Subcellular distribution of proteins

The quantification of signal intensities at rsMC was performed as described previously (Ikawa et al., 2023). Briefly, Z-projection was performed to extract the fluorescent signals on the AJ planes. The background signal of myo-II-mKate2 or Coronin-1-EGFP was subtracted using the ‘subtract background’ command (r = 50), and the signal intensity in manually selected regions of interest (ROIs) was measured using the ROI manager in ImageJ. The myo-II-mKate2 or Coronin-1-EGFP signal intensities at the rsMC were calculated using the ‘Plot Profile’ in ImageJ with a line width of 10 pixels.

Direction of cell rearrangement

Cell rearrangement was manually detected from time-lapse movies captured at 1-min intervals starting from 24 h APF at 25°C (Ikawa and Sugimura, 2018).

Fraction of hexagonal cells

DE-cad-GFP images were skeletonized using ImageJ. The position and connectivity of vertices were extracted from the skeletonized images, and the polygonal distribution of cells was measured in OpenCV (Sugimura and Ishihara, 2013).

Statistical analysis

P-values were calculated based on the Fisher’s exact test and Student’s t-test using EZR, a graphical user interface for R (Saitama Medical Center, Jichi Medical University, Saitama, Japan, https://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed​EN.​html) (Kanda, 2012).

Results

Coronin-1 is localized at the reconnecting junction

To investigate the dynamics of Coronin-1 during cell rearrangement, we used CRISPR-Cas9 techniques to generate the transgenic fly expressing Coronin-1-EGFP (Fig. 1B). Live imaging confirmed the expression of the EGFP signal in the transgenic flies (Fig. 1C). Furthermore, the EGFP signal was specifically reduced in the C-region of the wing upon the expression of dsRNA against coronin using the ptc-Gal4 driver (Fig. 1A, D). These results demonstrate the successful generation of a Coronin-1-EGFP fly.

Using the Coronin-1-EGFP fly, we analyzed the subcellular localization of Coronin-1 during cell rearrangement. We observed a strong signal of Coronin-1-EGFP at reconnecting junctions (Fig. 1E). Previous studies have shown that during junction reconnection, the myo-II cables detach from the AJ and form rsMCs (Fig. 1A; Ikawa and Sugimura, 2018; Ikawa et al., 2023). Our image analysis revealed a single peak of Coronin-1-EGFP between two peaks of myo-II-mKate2, indicating Coronin-1 accumulation within the rsMC (Fig. 1E, F). Furthermore, time-lapse analysis showed that the level of Coronin-1 increased at reconnecting junctions (Fig. 1G, H).

coronin RNAi resulted in defects in directional cell rearrangement

We measured the angle of newly generated junctions following cell rearrangement to determine whether Coronin-1 is required for directional cell rearrangement in the wing (Fig. 2A). In WT wings, cell rearrangement was biased towards the PD axis, with ~70% of the cell rearrangement occurring along this axis (Fig. 2B). In contrast, the proportion of PD cell rearrangement was reduced to ~50% by RNAi of coronin (Fig. 2B). The PD cell rearrangement is known to increase the number of hexagonal cells and thereby contribute to the formation of hexagonal cell arrays in the wing (Aigouy et al., 2010; Sugimura and Ishihara, 2013). To assess the role of Coronin-1 in the formation of hexagonal cell arrays, we analyzed the fraction of hexagonal cells at 32 h APF, when the cell rearrangement process is complete and the fraction of hexagonal cells reaches a plateau. Our results demonstrate that the fraction of hexagonal cells decreased in coronin RNAi wings (75.6 ± 3.1% in WT and 57.8 ± 3.3% in coronin RNAi wings; Fig. 2C–E), consistent with the decrease in directional cell rearrangement. Collectively, these data indicate that Coronin-1 promotes directional cell rearrangement, thereby supporting hexagonal cell packing in the wing.

Fig. 2

coronin RNAi results in defects in directional cell rearrangement

(A) Schematic of cell rearrangement analysis. We tracked individual junctions that appeared in a movie and measured their angle relative to the PD axes (θ) of newly generated junctions following cell rearrangement. (B) Quantification of the direction of cell rearrangement for each genotype based on time-lapse data captured at 24–27 h after puparium formation (APF) at 25°C (WT, coronin RNAi). The classification of θ is illustrated with a semicircle (red: PD, gray: others). WT timelapse data acquired by Ikawa and Sugimura (2018). (C, D) Images of DE-cad-GFP with the indicated genotypes (C: WT at 32 h APF, D: coronin RNAi at 32 h APF). Cells are colored according to the number of junctions (red, square; green, pentagon; gray, hexagon; blue, heptagon; and magenta, octagon). (E) The percentage of hexagonal cells at 32 h APF in the WT and coronin RNAi wings. WT data acquired by Ikawa and Sugimura (2018). The number of cell rearrangements (B) and wings (E) is indicated. The data are presented as mean ± SD (B, E). Fisher’s exact test: WT vs. coronin dsRNA, ** P < 0.001 (B) and Student’s t-test: WT vs. coronin dsRNA, ** P < 0.01 (E). Scale bar: 20 μm (D).

AIP1 and cofilin are required for the junctional localization of Coronin-1

We examined the dependence of Coronin-1, AIP1 and cofilin on their subcellular localization. It has been shown that AIP1 localizes at reconnecting junctions and inside rsMCs (Fig. 3A; Ikawa and Sugimura, 2018; Ikawa et al., 2023). We found that AIP1 localization was largely unaffected in coronin RNAi wings. AIP1-GFP localized at the reconnecting junctions and inside rsMCs with a moderate increase in the GFP signal intensity (Fig. 3B). In contrast, RNAi of flare (flr; Drosophila aip1 gene) and twinstar (tsr; Drosophila cofilin gene) severely disrupted the junctional localization of Coronin-1 and induced cytoplasmic patch formation (Fig. 3C, D; Supplementary Fig. 1). These results suggest differential requirements for cofilin cofactors according to their subcellular localization: whereas AIP1 can localize along the remodeling AP junctions in the absence of coronin function, AIP1 and cofilin support the junctional localization of Coronin-1.

Fig. 3

Coronin-1 localization is dependent on AIP1 and cofilin

(A, B) Images of AIP1-GFP (gray in left panels and green in right panels) and myo-II-mKate2 (gray in middle panels and magenta in right panels) in control (A) and coronin RNAi (B) wings at 24 h APF. (C, D) Images of Coronin-1-EGFP (gray in left panels and green in right panels) and myo-II-mKate2 (gray in middle panels and magenta in right panels) in WT (C) and aip1/flr RNAi (D) wings at 58 h APF at 17°C, which corresponds to 24 h APF at 25°C. (E, F) Images of Coronin-1-EGFP (gray in left panels and green in right panels) and Phalloidin (gray in middle panels and magenta in right panels) in the B region of the wing at 21 h APF at 29°C, which corresponds to 24 h APF at 25°C. Cells in the B region of the wing were used as an internal control in the wing expressing tsr dsRNA using ptc-Gal4. The vertical section along the orange dashed line in (E) is shown in (F). Blue arrow heads indicate the reconnecting junction. (G, H) Images of Coronin-1-EGFP (gray in left panels and green in right panels) and Phalloidin (gray in middle panels and magenta in right panels) in the C region of the wing expressing cofilin/tsr dsRNA using ptc-Gal4 at 21 h APF at 29°C, which corresponds to 24 h APF at 25°C. The vertical section along the orange dashed line in (G) is shown in (H). Scale bars: 5 μm (B, D, E–H).

Finally, we investigated the relationship between the Coronin-1 localization and the F-actin distribution. In control cells, the Coronin-1 signal overlapped with F-actin, especially at the reconnecting junctions (arrowheads in Fig. 3E, F). In tsr RNAi cells, we observed a strong colocalization of Coronin-1 with F-actin accumulated in the apical cytoplasm (Fig. 3G; boxes in Fig. 3H). These observations indicate that the Coronin-1 localization follows the pattern of F-actin localization in both control and tsr RNAi cells, suggesting the possibility that AIP1 and cofilin regulate Coronin-1 localization through F-actin regulation.

Discussion

During cell rearrangement, F-actin, which is tightly linked to the junction structure, undergoes reorganization alongside junction remodeling (Yap et al., 2015; Clarke and Martin, 2021). In addition, F-actin remodeling has been implicated in the dissipation of excess stress along the shrinking junctions (Charras and Yap, 2018). ABPs play a critical role in controlling F-actin remodeling in eukaryotic cells (Munjal and Lecuit, 2014). However, the specific mechanisms and ABPs involved in regulating cell rearrangement remain unclear. In the present study, we demonstrated that Coronin-1, a cofilin cofactor, is localized at reconnecting junctions and promotes directional cell rearrangement in the Drosophila wing. Moreover, we found that AIP1 and cofilin are required for the junctional localization of Coronin-1. Exploring the interplay between cofilin, AIP1, and Coronin-1 is expected to provide insights into the mechanisms underlying F-actin remodeling during cell rearrangement.

Cofilin plays a critical role in cellular mechanosensing via actin filament structure (Ngo et al., 2015; Hayakawa et al., 2011). Single actin filaments adopt a twisted configuration when mechanically relaxed. Cofilin shows a higher affinity to twisted actin filaments and induces further twisting of actin filaments. This leads to positive feedback between cofilin binding and F-actin twisting. In the Drosophila pupal wing, the corporative binding of cofilin to twisted actin filaments localizes AIP1 to remodeling AP junctions that run perpendicular to the axis of tissue stretch, thereby supporting directional cell rearrangement (Ikawa and Sugimura, 2018). Exploring how Coronin-1 contributes to the mechanosensing mediated by actin filaments, AIP1 and cofilin will lead to new insights on actin regulation via ABPs for controlling cell rearrangement.

Several in vitro studies have demonstrated that Coronin-1 promotes F-actin branching by Arp2/3 at the barbed ends, while both Coronin-1 and AIP1 promote F-actin severing by cofilin at the pointed ends (Brieher et al., 2006; Gandhi et al., 2009; Jansen et al., 2015). These studies have indicated that Coronin-1 promotes the localization of AIP1 and cofilin at the pointed ends (Gandhi et al., 2009; Jansen et al., 2015), which is inconsistent with our observation that the localization of AIP1 in the wing tissue was unaffected by coronin RNAi. There are two possible explanations for this inconsistency. First, in vitro experiments have shown that AIP1 and cofilin can still localize at the pointed ends, albeit to a lesser extent, in the absence of Coronin-1 (Ngo et al., 2015; Nadkarni and Brieher, 2014), suggesting that coronin RNAi may not significantly affect the subcellular localizations of AIP1 in wing tissue. Second, the function of Coronin-1 in F-actin branching may outweigh its role in F-actin severing in the wing tissue. Considering the significance of F-actin disassemblers in wing cell rearrangement (Ikawa and Sugimura, 2018), the former explanation appears more plausible. To address the inconsistencies between in vitro and in vivo studies, it is crucial to uncover the molecular mechanisms through which cofilin, AIP1, and Coronin-1 regulate F-actin remodeling during cell rearrangement.

Given that AIP1, and Coronin-1 are involved in other cellular processes, such as cell division and extrusion (Chen et al., 2015; Michael et al., 2016), studying the function of ABPs in these processes offers promising avenues for future research.

Author Contributions

K.I. and K.S. designed the research. K.I. and S.H. performed the experiments. K.I., K.S and S.H. analyzed the data. S.K. generated transgenic flies. K.I. drafted the manuscript. K.I., K.S, S.O. and S.K revised the manuscript. All authors approved the final manuscript.

Declaration of Interests

The authors declare no competing interests.

Acknowledgments

The authors would like to thank Yohanns Bellaïche, Yang Hong, Roger Kares, and the Bloomington Stock Center for reagents; Miho Aruga, Kyoko Komano, and Risa Matsui for technical assistance; and the iCeMS Analysis Center for imaging equipment. This study was financially supported by a JSPS KAKENHI Grant (17K15125), the AMED PRIME program (20gm5810025h9904), the Sumitomo Foundation (200303), and the Takeda Science Foundation to K.S., a JSPS KAKENHI Grant (19K16139) and the Uehara Memorial Foundation (202110172) to K.I., a JSPS KAKENHI Grant (20H03246) to S.K., and a JSPS KAKENHI Grant (20H05945) to S.O.

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