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βH‐spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination

2020; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês

10.15252/embr.201949858

1469-3178

Daniel Krueger, Cristina Pallares Cartes, Thijs Makaske, Stefano De Renzis,

Cellular Mechanics and Interactions

Report26 June 2020Open Access Transparent process βH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination Daniel Krueger orcid.org/0000-0003-1139-7755 Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Cristina Pallares Cartes Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Thijs Makaske Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Stefano De Renzis Corresponding Author [email protected] orcid.org/0000-0003-4764-2070 Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Daniel Krueger orcid.org/0000-0003-1139-7755 Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Cristina Pallares Cartes Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Thijs Makaske Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Stefano De Renzis Corresponding Author [email protected] orcid.org/0000-0003-4764-2070 Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Author Information Daniel Krueger1, Cristina Pallares Cartes1, Thijs Makaske1 and Stefano De Renzis *,1 1Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany *Corresponding author. Tel: +49 6221 387 8109; Fax: +49 6221 387 8166; E-mail: [email protected] *Corresponding author. Tel: +49 6221 387 8109; Fax: +49 6221 387 8166; E-mail: [email protected] EMBO Rep (2020)21:e49858https://doi.org/10.15252/embr.201949858 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Actomyosin-mediated apical constriction drives a wide range of morphogenetic processes. Activation of myosin-II initiates pulsatile cycles of apical constrictions followed by either relaxation or stabilization (ratcheting) of the apical surface. While relaxation leads to dissipation of contractile forces, ratcheting is critical for the generation of tissue-level tension and changes in tissue shape. How ratcheting is controlled at the molecular level is unknown. Here, we show that the actin crosslinker βH-spectrin is upregulated at the apical surface of invaginating mesodermal cells during Drosophila gastrulation. βH-spectrin forms a network of filaments which co-localize with medio-apical actomyosin fibers, in a process that depends on the mesoderm-transcription factor Twist and activation of Rho signaling. βH-spectrin knockdown results in non-ratcheted apical constrictions and inhibition of mesoderm invagination, recapitulating twist mutant embryos. βH-spectrin is thus a key regulator of apical ratcheting during tissue invagination, suggesting that actin cross-linking plays a critical role in this process. Synopsis This study shows that βH-spectrin is a key regulator of apical ratcheting during tissue invagination, suggesting that actin crosslinking plays a critical role in this process. The actin crosslinker βH-spectrin is upregulated at the apical surface of invaginating mesodermal cells during Drosophila gastrulation. βH-spectrin forms a network of filaments which co-localize with medioapical actomyosin fiber in a Twist and Rho signaling dependent manner. βH-spectrin knockdown results in non-ratcheted apical constrictions and inhibition of mesoderm invagination. Introduction Cell shape changes driven by contraction of cortical actomyosin filaments are of fundamental importance during animal development, underlying key morphogenetic processes such as cytokinesis (Pollard, 2010; Sedzinski et al, 2011), cell migration (Blanchoin et al, 2014), and localized remodeling of tissue shape (Bertet et al, 2004; Dawes-Hoang et al, 2005; Butler et al, 2009; Fernandez-Gonzalez et al, 2009; Heisenberg & Bellaiche, 2013; Izquierdo et al, 2018; Krueger et al, 2018). Fast in vivo imaging and in vitro studies demonstrate that contraction of cortical actomyosin networks is controlled by pulsatile flows of myosin-II molecules, which move centripetally as actin filaments contract and cell surface shrinks (Martin et al, 2009; Solon et al, 2009; Reymann et al, 2012; Banerjee et al, 2017). Myosin-II pulses are followed by either relaxation or stabilization of the cell surface, before a new contractile cycle starts. While it remains unclear whether pulsatile behavior plays an active role during morphogenesis, stabilization of the cell surface, usually referred to as ratcheting, has been suggested to be critical for generation of supracellular forces and large-scale tissue remodeling (Martin et al, 2009; Solon et al, 2009; Clement et al, 2017). According to this view, ratcheting might prevent dissipation of forces, which would otherwise occur during relaxation of the cell surface, thus facilitating force transmission through adherens junctions and building of tissue-level tension. Ratcheting might also serve as a mechanism to protect tissues from tearing apart as cells constrict (Ducuing & Vincent, 2016). As yet however, how ratcheting is controlled at the molecular level remains poorly understood (Munjal et al, 2015; Sumi et al, 2018; Miao et al, 2019). Here, we have identified the actin crosslinker βH-spectrin as a key regulator of apical ratcheting during Drosophila ventral furrow invagination. This morphogenetic process is driven by pulsatile apical constrictions and cell shape changes of a group of ~ 1,000 cells arranged in a rectangular pattern on the ventral surface of the embryo (Martin et al, 2009; Guglielmi et al, 2015). Apical constriction in ventral cells requires expression of the transcription factors Twist and Snail, which also confer mesodermal fate to the invaginating cells (Leptin, 1991). Twist and Snail control expression of several downstream targets (Rembold et al, 2014) converging on Rho signaling and myosin-II activation (Dawes-Hoang et al, 2005; Kolsch et al, 2007). However, while in snail mutant embryos cells do not apically constrict and change shape, in twist mutants cells undergo uncoordinated cycles of apical constriction and relaxation without maintaining the constricted state, suggesting that molecular mechanisms downstream of Twist control ratcheting of the apical surface (Martin et al, 2009). These contractile alterations result in impaired tissue invagination and are phenocopied by treating embryos with low doses of actin depolymerizing drugs. In both, twist mutants and cytochalasin D-treated embryos, the network of medio-apical actomyosin filaments that forms at the onset of ventral furrow formation (see cartoon in Fig 1A–C) either does not assemble or when present it loses its attachment to the junctions causing cells to expand abnormally when neighboring cells constrict (Mason et al, 2013). Figure 1. βH-spectrin co-localizes with myosin-II to medio-apical fibers in mesodermal cells during ventral furrow formation A, B. Schematic illustration of a constricting cell undergoing a cycle of ratcheted pulsatile constriction. Upregulation of myosin-II (blue) causes coalescence of actin fibers (red) in a radially polarized manner. Contraction of medio-apical actomyosin fibers, which are anchored to the plasma membrane (black) through adherens junctions (purple dots) and are mechanically coupled to a junctional actomyosin belt (red), causes constriction of the cell surface. Disassembly of medial actomyosin is accompanied by surface relaxation, which is contained by a ratcheting mechanism that stabilizes the constricted state. Repeated cycles of actomyosin-mediated ratcheted contraction result in an incremental shrinkage of the cell surface. (B) Schematic illustrating medio-apical (cyan) and junctional (purple) actomyosin fibers. C. Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm D. Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm. E. Z-Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry at 5 min (top), 7 min (middle) and 9 min (bottom) after initial medio-apical accumulation of βH-spectrin. Scale bar, 20 μm F, G. Immunostaining of βH-spectrin (green) visualized by STED nanoscopy revealed medio-apical βH-spectrin supracellular fibers in mesodermal cells (F). In ectodermal cells, βH-spectrin localizes to apical cell junctions (G). Note, in (F) the junctional βH-spectrin signal is shown in magenta as a proxy for cell membranes. Scale bars, 2.5 μm. H–J. Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (H), the myosin-II marker Sqh::mCherry (I) and a merge of the two (J) with βH-spectrin in green and Sqh::mCherry in magenta. Scale bars: 20 μm. K, L. Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin staining of sub-apical confocal sections. Scale bars: 5 μm. Download figure Download PowerPoint The results presented in this study show that the actin crosslinker βH-spectrin is upregulated at the apical surface of mesodermal cells during ventral furrow invagination in a process that requires the zygotic expression of twist and Rho signaling activation. βH-spectrin localizes to medio-apical actomyosin fibers, and its activity is required for ratcheting apical constrictions as demonstrated by nanobody-mediated protein knockdown. Similar to the twist mutant phenotype, reducing βH-spectrin protein levels does not inhibit apical constrictions. Rather it causes cells to pulse without stabilizing the apical surface resulting in defects in tissue invagination and integrity. Together these results support a model in which apical ratcheting during tissue invagination is controlled by βH-spectrin-dependent actin cross-linking and surface organization. Results and Discussion We have recently characterized a mechanism based on actin cross-linking that regulates the contraction of a basally localized actomyosin network during cellularization, a morphogenetic process that immediately precedes ventral furrow invagination (Krueger et al, 2019). In particular, we found that bottleneck, a protein expressed for only a short time during cellularization, bundles actin filaments and counteracts actomyosin contractility allowing formation of cells with the proper shape. These results prompted us to test whether actin crosslinkers would also be involved in modulating the dynamics of actomyosin contraction during ventral furrow formation, a paradigm of tissue invagination. By screening a collection of homozygous viable fly lines expressing endogenously tagged crosslinkers with fluorescent proteins (Lye et al, 2014), we identified βH-spectrin as being upregulated at the apical surface of ventral mesodermal cells during invagination (Fig 1D, Movie EV1). Live imaging of embryos co-expressing mVenus::βH-spectrin and the plasma membrane marker GAP43::mCherry demonstrated that as mesodermal cells started constricting, βH-spectrin formed a network of medio-apical fibers, whereas in the ectoderm it remained localized at the junctions demarcating cell boundaries (Fig 1E–G, Movie EV2). Imaging mVenus::βH-spectrin and the myosin-II probe Sqh::mCherry showed that in mesodermal cells βH-spectrin localized to the medio-apical actomyosin network (Fig 1H–J and cartoon in Fig 1A–C). Phalloidin staining confirmed that βH-spectrin and F-actin colocalized in medio-apical fibers (Figs 1K and L and EV1A–C). These results showing upregulation and medio-apical localization of βH-spectrin in mesodermal cells are suggestive of its potential involvement in tissue invagination. To test this hypothesis, we established a protein knockdown strategy based on a maternally expressed deGradFP-nanobody (Caussinus et al, 2011) to achieve efficient βH-spectrin depletion during early embryogenesis (Fig 2A). βH-spectrin depleted embryos underwent normal cellularization but displayed several defects during ventral furrow invagination as demonstrated by live imaging analysis of βH-spectrin depleted embryos expressing GAP43::mCherry. Whereas in 92% of wild-type embryos ventral furrow invagination occurred normally, only 24% of βH-spectrin depleted embryos completed invagination with no or minor abnormalities (Fig 2B–J). The remaining 76% displayed several defects including impaired apical constrictions (Fig 2K,L) and either irregular or complete failure of tissue internalization (Fig 2G–J, Movie EV3). In wild-type embryos, mesodermal cells constrict in a coordinated manner aligning their axis of contractility to the geometry of the ventral furrow primordium resulting in a shrinkage along the dorsoventral (D-V) axis and elongating along the anteroposterior (A-P) axis (Martin et al, 2010; Guglielmi et al, 2015; Chanet et al, 2017). In βH-spectrin knockdown embryos, cells constricted with a lower degree of A-P anisotropy compared with controls (Fig 2L) and displayed irregular shapes with some cells being more constricted than others. Together these alterations resembled the morphological abnormalities characteristic of the twist mutant phenotype as described in the introduction. Click here to expand this figure. Figure EV1. βH-spectrin co-localizes with F-actin at the apical surface during ventral furrow formation A–C. Surface projection of the apical cell surface of a Drosophila embryo during ventral furrow formation co-stained for F-actin using phalloidin (A) and mVenus::βH-spectrin using FluoTag®-X4 anti-GFP (B). Panel (C) shows a merge of the phalloidin (magenta) and βH-spectrin (green) staining (co-localization analysis: Pearson's R value > 0.7). White dashed lines indicate the cell boundaries based on the phalloidin staining of sub-apical confocal sections. Scale bars: 20 μm. Download figure Download PowerPoint Figure 2. Knockdown of βH-spectrin impairs apical constriction and tissue invagination A. Z-Projection of confocal image stacks spanning 5 μm of a control embryo (left) co-expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry (magenta) and of an embryo additionally expressing a maternally driven anti-GFP nanobody KD module to knockdown βH-spectrin (right). Co-expression of the anti-GFP nanobody KD system resulted in a drastic decrease of βH-spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit. B. Pie diagrams quantifying the percentage of control embryos (top, n = 50) and βH-spectrin knockdown embryos (bottom, n = 40) that formed ventral furrow either normally or with minor phenotypes (green) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination (red). The assessment of phenotypes was done in an unbiased randomized approach. C–J. Confocal images of a control embryo (C, D) and βH-spectrin knockdown embryos with a minor phenotype (E, F), with a severe phenotype that underwent tissue invagination (G, H) and with a severe phenotype that did not undergo tissue invagination (I, J). Surface view is shown in (C, E, G, I) and the cross section through the tissue at two different time points in (D, F, H, J). All embryos expressed the plasma membrane marker GAP43::mCherry. Scale bars, 20 μm. K, L. Quantification of apical surface area (K) and anisotropy (L) in control embryos (green, nControl: six embryos) and in βH-spectrin knockdown embryos (red, nβH-spectrin KD: seven embryos). Solid lines indicate the mean values and the semi-transparent regions the corresponding standard deviation. Download figure Download PowerPoint To test whether βH-spectrin localization in mesodermal cells is Twist-dependent (see schematics in Fig 3A and B), we generated homozygous twist mutant embryos in a homozygous mVenus::βH-spectrin background and followed mVenus::βH-spectrin localization using live imaging. twist mutant embryos formed an irregular ventral furrow, but βH-spectrin protein levels were not notably different than in control embryos (Fig 3D and E). In contrast, βH-spectrin localization changed and became predominately junctional, as in lateral ectodermal cells, instead of medio-apical (Fig 3F and G and Movie EV4). In agreement with a previous study showing that βH-spectrin transcription starts at later stages of embryogenesis (Thomas & Kiehart, 1994), these results argue that Twist does not control βH-spectrin expression directly, but rather affects its localization indirectly. Because Twist regulates the spatiotemporal organization of Rho signaling (Mason et al, 2013) (see Fig 3A and B for a schematic), we tested whether Rho activation controls βH-spectrin dynamics and localization. We employed optogenetics to stimulate RhoGEF2 and induce an acute burst of Rho signaling (Izquierdo et al, 2018) (see schematic in Fig 3C) on the dorsal surface of the embryo in ectodermal cells where Twist is not expressed and βH-spectrin is localized to the junctions (Fig 3H). Spatially confined optogenetic activation using two-photon illumination (Guglielmi & De Renzis, 2017) caused both βH-spectrin medio-apical localization and a ~ 1.7-fold increase in its overall levels at the apical surface (Fig 3H–R), thus demonstrating that Rho signaling is sufficient to induce βH-spectrin apical upregulation and medio-apical assembly. Injection of the Rock inhibitor Y-27632 before optogenetic activation did not result in βH-spectrin or myosin-II upregulation. Thus, medio-apical βH-spectrin upregulation is dependent on the Rock-myosin-II branch of the Rho signaling system (Figs 3S and T and EV2A–I), consistent with the role of Rock in organizing the medio-apical actomyosin network (Mason et al, 2013). Figure 3. Medio-apical βH-spectrin localization is controlled by the mesoderm-specific transcription factor Twist and Rho signaling activation A. Schematic showing the expression domain of the transcription factor Twist on the ventral side of the Drosophila embryo along the anterior-posterior axis (top: lateral view; bottom: cross section). B. Schematic showing the genetic network that induces actomyosin contractility at the apical surface of mesodermal cells. The transcription factors Twist and Snail regulate expression of the G-protein-coupled receptor (GPCR) Mist, its ligand Fog and the Gα Cta, as well as T48, which recruits the RhoA activating factor RhoGEF2 to the apical surface. Fog-mediated activation of Mist triggers RhoGEF2 activity through Cta and thus induction of actomyosin contractility. C. Schematic of the optogenetic system employed to activate Rho signaling. RhoGEF2-CRY2 is cytosolic in the dark and interacts upon photo-activation with blue light with its binding partner CIBN which is anchored at the plasma membrane. D, E. Still frames of a movie showing endogenously tagged mVenus::βH-spectrin in the cross section of a gastrulating wild-type (D) and twist mutant (E) embryo imaged using two-photon microscopy. The top frame corresponds to the time point where an initial bending of the apical surface was observed, the middle frame was taken 2 min and the lower frame 3 min afterward. Scale bars, 30 μm. F, G. Confocal images showing apical βH-spectrin (endogenously tagged mVenus::βH-spectrin) in a wild-type (F) and twist mutant (G) embryo. Scale bars, 20 μm. Medio-apical βH-spectrin fibers observed in wild-type embryos were absent in twist mutant embryos. H–Q. Confocal or two-photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co-expressing endogenously tagged mVenus::βH-spectrin (green, H, K, M, P), the myosin-II marker Sqh::mCherry (magenta, I, N), and the optogenetic modules as explained in (C). The apical surface is shown before photo-activation (H–L) and 1 min after photo-activation (M–Q). Panels (J, O) show a merge of βH-spectrin and myosin-II and the dashed rectangle indicates regions shown in (L and Q) at higher magnification. Panels (K, L) and (P, Q) are zoom-ins into the region indicated in (J) an (O), respectively. Scale bars, 20 μm (H–J, M–O), 2.5 μm (K, L, P, Q). R. Quantification of apical βH-spectrin upon optogenetic stimulation of Rho signaling. The graph shows the mean intensity of βH-spectrin (green) at the apical surface over time as the fold change relative to the initial time point (before photo-activation). The semi-transparent region indicates the standard deviation, and dots indicate individual data points (n = 5 embryos). S, T. Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. βH-spectrin upregulation depends on ROCK activity A–I. Confocal or two-photon microscopy images of Drosophila embryos co-expressing the optogenetic module to photo-activate Rho signaling (related to Fig 3C), the myosin-II probe Sqh::mCherry (magenta, A, C, E, G) and mVenus::βH-spectrin (green, B, D, F, H, I). A subset of cells (dashed boxes in A, B and E, F) of the dorsal tissue was photo-activated at the apical surface in embryos previously injected with water (A–D) or the ROCK inhibitor Y-27632 (E–I). The Sqh::mCherry and mVenus::βH-spectrin signal at the apical cell surface was recorded before photo-activation (A, B and E, F) and 1.5 min after photo-activation (C, D and G–I). Panel (I) shows an overview of the mVenus::βH-spectrin signal after photo-activation of the region indicated by a white dashed box. While in water-injected embryos, myosin-II and βH-spectrin levels increased and the cells constricted, in Y-27632-injected embryos their level did not change and the cells did not constrict. Scale bars: 20 μm. Download figure Download PowerPoint These results, showing Twist and Rho control over βH-spectrin medio-apical localization, prompted us to test whether the impaired contractile behavior caused by βH-spectrin knockdown results from impaired pulsatile behavior rather than lack of apical constriction per se. We followed myosin-II (Sqh::mCherry) dynamics in embryos expressing the junctional protein E-cad::mNeonGreen demarcating the cell boundaries using high-temporal resolution confocal imaging (Movie EV5). In βH-spectrin knockdown embryos, myosin-II displayed normal medio-apical accumulation (Movie EV5). However, over time the apical actomyosin network broke in cells that enlarged abnormally resulting in tissue tears (Fig 4A–C) and local disruption of adherens junctions (Fig EV3A–F). A similar phenotype was observed in βH-spectrin knockdown embryos expressing the F-actin reporter LifeAct::mNeonGreen (Movie EV6), suggesting that βH-spectrin is not required for initial assembly of the medio-apical actomyosin network but rather for maintaining its structural integrity as cells constrict. Quantitative analysis of mVenus::βH-spectrin signal over the course of apical constriction reveals that its levels progressively increased during the constriction phase and remained constant during relaxation (Fig EV4A–F). By inspecting the contractile behavior of βH-spectrin knockdown cells, we noticed that while some cells constricted in a ratcheted manner maintaining the constricted states also in the interval between subsequent myosin-II pulses, some others constricted in a non-ratcheted manner (Fig 4D and E). Quantification of the contractile behavior in more than 500 individual cells revealed that in control embryo cells constricted in a coordinated manner over time in a such a way that after 7 min from the onset of ventral furrow formation most of the cells (91%) shrank their apical area to < 50% of the initial value. In contrast, βH-spectrin knockdown caused loss of coordination with as many as 44% of the cells displaying impaired constriction (i.e., apical area > 50% and < 100% of initial values) and 6% of the cells expanding (Fig 4F–J). While in control embryos cells displayed positive constriction rate values throughout ventral furrow invagination, in βH-spectrin knockdowns cells with impaired contractions displayed both positive and negative rates indicating alternating cycles of surface contraction and expansion (Fig EV5A and B). Quantification of the constriction and expansion rates show that the constriction rate was reduced by ~ 24%, whereas the expansion rate increased by ~ 60% on average (Figs 4K and EV5C and D), demonstrating that the change in the expansion rate is 2.5 times higher than the change in the constriction rate. Taken together, these results are consistent with a role of βH-spectrin in apical ratcheting. Figure 4. βH-spectrin is required for ratcheting pulsatile apical constrictions A–C. Confocal images of the apical cell surface of a βH-spectrin knockdown embryo showing the myosin-II marker Sqh::mCherry (green) and E-cadherin::mNeonGreen (magenta) during ventral furrow formation. (A) myosin-II accumulates normally at the apical surface and cells undergo an initial phase of constriction. The dashed rectangle indicates the region shown in (B and C) at higher magnification. Scale bar, 30 μm. Panels (B, C) show a sub-region within the ventral tissue at different time points. Initially, a supracellular myosin-II network formed (0 s), before intercellular connections broke (13 s) resulting in membrane tears (dashed line) with tissue retractions (small arrow heads) along the anterior-posterior axis. The intercellular network re-established (39, 65 s) as new actomyosin fibers formed (big arrow heads) to reconnect neighboring cells. The intercellular network broke again causing tears (dashed lines) at different positions (104 s). Scale bars, 5 μm. D, E. Confocal images showing the myosin-II marker Sqh::mCherry (green), E-cadherin::mNeonGreen (magenta) and the segmented membrane (red) of a single cell at different time points in a control embryo undergoing ratcheted constriction (D) and in a βH-spectrin knockdown embryo undergoing non-ratcheted pulsations (E). Scale bars, 2.5 μm. F. Heatmaps showing the apical surface of individual cells over time of control embryos (left) and βH-spectrin knockdown embryos (right) normalized to the initial time point (nControl = 233; nβH-spectrinKD = 368; of five different embryos each). The color-coded scale bar is depicted on the left. Dark blue indicates a cell area bigger than the initial value (> 1), and yellow indicates an area constricted to less than 1/5 of the initial area (< 0.2). Three different cell behaviors in βH-spectrin knockdown embryos were identified: cells that constricted, cells that showed impaired constriction, and cells that expanded. G. Quantification of the percentage of cells that constricted (to an area < 50%, yellow), cells that showed impaired constriction (to an area > 50 and < 100%, green), and cells that expanded (to an area > 100%, blue) after 7 min in control embryos and in βH-spectrin knockdown embryos. H, I. Graph showing the average area over time of control cells (H, n = 235 cells) and cells in βH-spectrin knockdown embryos (I) that constricted (yellow, n = 274), of cells that showed impaired constriction (green, n = 232), and of cells that expanded (blue, n = 32). The lines indicate the mean value and the semi-transparent region the standard deviation. J. Boxplots showing the distribution of apical cell area values 7.5 min after the onset of ventral furrow formation of ventral cells in control embryos, of ventral cells in βH-spectrin knockdown embryos that constricted, and ventral cells in βH-spectrin knockdown embryos that displayed impaired constriction and expanded. Sample numbers are equal to I. The values are presented as percentage of initial cell area. ANOVA result (all data points): F(3,717) = 763.9, P = 9.6e-223; Cohen's d compared with the control: dconstricted = 0.1, dimpaired = 1.5, dexpanded = 3.7. Due to a big effect size (high sample number), statistical significance was assessed based on Cohen's d. A Cohen's d < 0.5 was considered not significant (n.s.). K. Quantification of the peak constriction rate and expansion rate relative to control cells. Control cells and cells in βH-spectrin knockdown embryos that showed impaired constriction or expanded were compared. The mean value and standard deviation