Cellular contractility coordinates cytoskeletal dynamics and cell behaviour during Drosophila abdominal morphogenesis

During morphogenesis, cells undergo various behaviours, such as migration and constriction, which need to be coordinated. How this is achieved remains elusive. During morphogenesis of the Drosophila adult abdominal epidermis, larval epithelial cells (LECs) migrate directedly before constricting apically and undergoing apoptosis. Here, we study the mechanisms underlying the transition from migration to constriction. We show that LECs possess a pulsatile apical actomyosin network and that a change in network polarity underlies behavioural change. Exploring the properties of the contractile network, we find that the level of cell contractility impacts on the network’s behaviour, as well as on overall cytoskeletal architecture and cell behaviour. We also find that pulsed contractions occur only in cells with intermediate levels of contractility. Furthermore, increasing levels of the small Rho GTPase Rho1 disrupts pulsed contractions, and instead leading to cells that cycle between two states, characterised by a junctional cortical and an apicomedial actin network. Our results highlight that behavioural change relies on tightly controlled cellular contractility. Moreover, we show that constriction can occur without pulsed contractions, raising questions about their contribution to constriction.


Introduction 69
During development, morphogenetic processes ultimately shape the organism (Lecuit 70 and Le Goff, 2007). Such processes are driven by various cell behaviours, e.g.  The transition between phases 2 and 3 was characterised by lamellipodium 156 disappearance and loss of two actin foci, with one focus appearing in the cell centre 157 1D; S1C). 165 Cytoskeletal activity was not only correlated with cell behaviour, but also with 166 cell shape. During migration, cells tended to be elongated along the dorso-ventral (d-167 v) axis, with actin foci in the back, whereas during constriction cells were rounder, with 168 a central actin focus (Fig. 1G). To become rounder, cells mainly changed shape along 169 the d-v axis (Fig. S1D). 170 Overall, pulsed contractions of the LEC actin cytoskeleton adopt a highly 171 coordinated pattern, which correlates with cell behaviour. we would expect the rhythmic activity of actin foci to be correlated with rhythmic 178 changes in apical cell area. We therefore quantified the apical area change in the 179 different phases of LEC behaviour ( Fig. 2A). can be divided into an 'early' phase, in which the apical area was not reduced but 185 changed its shape, with cells becoming rounder (Figs. 1G; 2A; S1D), and a 'late' 186 phase, in which apical area decreased ( Fig. 2A). This 'late' constriction was 187 characterised by a further significant increase in apical area fluctuation (Fig. 2C) as 188 well as ratcheted constrictions, which ultimately led to delamination. Thus, the 'early' 189 constriction phase can be considered a transition phase in which LECs alter their 190 apical shape and undergo pulsed contractions but do not considerably reduce apical 191 area (as they do later on). We next asked whether pulsed contractions correlate with cell area fluctuations (Fig.  200 3A). We found that, in 51% of fluctuations in migrating LECs and in 74% of fluctuations 201 in constricting LECs, one actin focus occurred during one fluctuation (Fig. 3B). We 202 also found that foci appeared around 30±5.3s (n=118 foci) before LEC area was 203 smallest (Fig. 3C). This suggests that area fluctuations correlate with actin foci and 204 that the contractile event drives cell area reduction. 205 Besides area fluctuations that correlated with foci, we also observed 206 fluctuations without foci (Fig. 3B). We hypothesised that these fluctuations might be 207 due to external forces exerted by contracting neighbouring cells, which could interfere 208 with regular fluctuations. Such events might have a shorter duration and lead to 209 smaller area reduction than pulsed contractions (Fig. 3D). Categorising individual 210 fluctuations by duration, we found that the majority of fluctuations not coinciding with 211 an focus were 'short' (≤90s) (Fig. 3B). These 'short' fluctuations did not reduce the cell 212 area much -the reduction, both during migration (-1.19±0.2%; n=7) and constriction 213 (-1.41±0.4%; n=7) ( Fig. 3E), was comparable to that in LECs that migrated without 214 visible pulsatile activity (-1.36±0.1%; p=0.64). However, for 'longer' fluctuations 215 (>90s), there was a significant difference in area reduction per fluctuation between 216 migration and constriction (Fig. 3E). Overall, this suggests that the majority of area 217 fluctuations that occur without an accompanying actin focus are 'short' non-contractile 218 fluctuations that might be due to external forces by neighbouring LECs. 219 Furthermore, in migrating LECs, the correlation between area fluctuations and 220 actin foci was less strong than in constricting LECs -around 25% of the fluctuations 221 in migrating LECs showed two foci, and overall the number of 'short' fluctuations 222 involving foci was higher than in constricting LECs (Fig. 3B). The weaker correlation 223 could be due to the two alternating contractile events in different cell regions affecting 224 cell shape change unevenly (Fig. 3F).   Rok-RNAi did not interfere with LEC migration (Fig. S3A,B), but LECs showed 268 a phenotype indicative of reduced contractility. Cells had an increased apical area 269 compared to controls (Fig. 5A  In addition, Rok-RNAi affected pulsed contractions. 75% of analysed LECs 284 showed some actin foci, whereas in 25%, foci were absent (n=8; Fig. 5E). Where foci 285 could be observed, their pulsation period was comparable to wild-type (180s±1.7s; 286 n=5; p=0.11), but foci were more diffuse (Fig. 5E). In strong phenotypes, GMA-GFP 287 labelled a less dynamic apicomedial network, which did not generate any foci and 288 To ask whether defects in LEC contractility impact on morphogenesis, we 294 assessed the time of abdominal closure in control, Rok-RNAi and MbsN300 pupae. 295 We found that in both experiments, closure was delayed (Fig. 5F'). In some cases, 296 this led to closure defects, where LECs did not complete morphogenesis successfully 297 (2.6% of Rok-RNAi pupae (n=39); 60.8% of MbsN300 pupae (n=51); Fig. 5F"). This 298 suggests that impaired contractility can lead to morphogenesis defects, but that in 299 many cases, LECs still delaminate. 7A,B; Movies S11,S12), which is reflected in the lack of cell area fluctuation (Fig. 7C). 336 However, unlike Rok-CAT cells, Rho-CA LECs did not create excessive cortical actin 337 bundles (Fig. 7A,B). With respect to cell migration, Rho1-CA pupae showed two 338 phenotypes: 'migrating' phenotypes (29%, n=7; Fig. 7A; Movie S11), and 'non- Our results suggest that adding additional Rho1, which can be switched on and 363 off by the cells' endogenous machinery, causes LECs to cycle between two states 364 characterised by distinct cytoskeletal networks. Thus, rhythmical activity appears not 365 to be limited to pulsed contractions of an actomyosin network, but also extend to more 366 general rhythmical remodelling of cytoskeletal architecture (Fig. 8E). 367 368

Discussion 369
We show that the apicomedial actin cytoskeleton of LECs undergoes pulsed 370 contractions, while cells transit from migration to constriction (Fig. 1A,B). During this 371 transition, cytoskeletal activity is highly coordinated and correlates with cell behaviour 372 and shape (Fig. 1B-G). Pulsed contractions are correlated with cell area fluctuations 373 (Figs. 2; 3) and LECs go through distinct phases of contractile activity ( Fig. 1C; 2A). 374 We find that a cell's level of contractile activity determines the behaviour of its 375 actin cytoskeleton, as well as cytoskeletal architecture and cell behaviour (Fig. 8E). 376 Not only are pulsed contractions dependent on intermediate levels of contractility, but 377 moderately increasing contractility causes LECs to cycle between two states, 378 characterised by a junctional cortical and an apicomedial actin network, respectively 379 ( Fig. 8). Moreover, our data suggest that constriction can occur without pulsed 380 contractions, raising questions about their contribution to constriction. They also show radial actin bundles that appear to connect the apical network to cell-392 cell junctions, resembling a spider's web (Fig. 4D). In addition, Sqh::GFP is distributed 393 evenly at all interfaces ( Fig. S2A''). Thus, a crucial step during LEC behavioural 394 change is a change in cell polarity from PCP to RCP that underlies a change in 395 cytoskeletal activity as well as an overall reorganisation of cytoskeletal architecture 396 (Fig. S4). Pulsed contractions are not involved in this change, as Rho-CA cells, which 397 do not pulse, also transit from migration to constriction (Movie S11). 398 What regulates this polarity change requires further investigation. We have 399 previously shown that LEC polarity depends on PCP signalling (Bischoff, 2012). contractile event might not suffice to constrict the whole apicomedial network (Fig. 3F). 420 Constricting cells, however, are rounder (Figs. 1G; S1D), which might allow radial actin 421 recruitment over the whole apical area (Fig. 4D). Alternatively, the two foci could be a 422 pulsatile activity (Fig. 6A,B). Interestingly, LECs that create actin foci migrate faster 428 than LECs of an earlier stage, which do not undergo pulsed contractions (Fig. S5A).  Increasing wild-type Rho1 levels proved particularly informative (Fig. 8A-D). Firstly, 481 UAS.rho1 cells show a phenotype consistent with an increase in contractility, i.e. loss 482 of pulsed contractions (Fig. 8A'') and blebbing (Fig. 8A',B). This suggests excess 483 endogenous Rho1-activating machinery, which constitutively activates some of the 484 additional Rho1. Secondly, when adding additional Rho1, its rhythmical activation and 485 de-activation via the endogenous machinery still appears to occur, leading to the 486 cycling between a state of higher contractility, where actin accumulates at the 487 junctional cortex and cells show blebbing (Fig. 8A',B), and a state of lower contractility, 488 where the apicomedial network is present but non-pulsatile (Fig. 8A,B). There are two  Compared to pulsed contractions, the duration of the two states in Rho1 504 overexpressing LECs is far longer and more irregular (Fig. 8C). This could be due to 505 the endogenous machinery having to deal with large amounts of Rho1 that need to be 506 switched on/off, which unbalances the system. LECs begin pulsed contractions while still migrating (Fig. 1C), at a time when LEC 526 shape changes, and thus tissue remodelling, intensify. Also, for most of 527 morphogenesis, LECs undergo pulsed contractions without constricting, merely 528 changing shape; only when ratcheted constrictions begin, cell area reduces notably 529 ( Fig. 2A). This suggests that pulsed contractions do not drive apical constriction per 530 se. Instead, they might have other roles, such as helping to maintain cell shape in an 531 environment where the activity of neighbouring cells creates pushing and pulling 532 forces. Ultimately, this will help to maintain tissue integrity during morphogenesis 533 (Coravos et al., 2017). Alternatively, pulsed contractions could cooperate with 534 junctional cortical contractility to create sufficient forces to drive apical constriction 535 more effectively. LEC constriction that is accompanied by pulsed contractions is faster 536 than constriction of boundary LECs without actin foci (Fig. 4G). 537 538

Control of cellular contractility is crucial for enabling behavioural change 539
Overall, our results highlight the importance of contractility levels mediated by the 540 amount of activated myosin, not only for the contractile network, but also for cellular 541 architecture and cell behaviour (Fig. 8E). Contractility needs to be tightly controlled, 542 otherwise LECs will change their behaviour. For wild-type LEC migration and the 543 subsequent behavioural transition to constriction, a polarised cytoskeletal network and 544 intermediate contractility levels seem to be crucial. However, rhythmical cytoskeletal 545 activity appears not to be limited to rhythmical contractions of an actomyosin network, 546 but also to extend to more general dynamic and rhythmical remodeling of the 547 cytoskeletal architecture. repression of Gal4 by Gal80ts is leaky, 100% of pupae showed a weak phenotype 575 (n=5). All UAS.rho1 pupae were grown at 25ºC. 576

4D microscopy 577
For microscopy, pupae were staged according to (Bainbridge and Bownes, 1981). 578 Pupae were dissected and filmed as described in (Seijo-Barandiarán et al., 2015). In 579 all images and movies, anterior is to the left. All flies developed into pharate adults 580 and many hatched. Z-stacks with a step size of 0.5 to 2.5 µm were recorded every 2 581 to 150s, depending on the experiment. Imaging was done with a Leica SP8 confocal 582 To obtain a single value that describes the shape of the cell, the cell shape coefficient 617 was calculated using the most anterior, posterior, dorsal and ventral coordinates 618 Cell area was tracked manually using ImageJ. The polygon selection tool was used to 627 draw the cell area, using as many vertices as needed to have an accurate outline. 628 From one frame to the next, this selection was adjusted if cell area changed over time. 629 To estimate the error associated with this technique, we tracked a cell for three frames 630 and repeated the tracking 22 times. We found that the average error between 631 repetitions was 0.37±0.07%. For seven wild-type LECs, cell area was measured every 632 frame (30s) for the entire length of the recording, covering all four behavioural phases 633 Using ImageJ, a region of 20 µm 2 was drawn in the cell centre. Relative fluorescence 665 intensities in this region were calculated for each channel using the plot profile function 666 (Fig. 4A,B). This function creates a y-projection of the selected region before 667 measuring intensities along the resulting line. 668 669 i) Cell size determination 670 As cells change shape dynamically and no two cells are in exactly the same stage of 671 development at the same time, we needed to make sure that cell sizes could still be 672 compared. For this, we measured the largest P compartment cell during migration 673 and/or constriction using the polygon tool of ImageJ (Fig. 5B). 674

j) Kymographs 675
Kymographs were used to present cytoskeletal dynamics in a single image (Figs. 5E; 676 6B,E; 7A,B; 8A",B). Kymographs were created from a rectangular region of interest 677 (ROI), using the ImageJ tool 'Reslice', followed by a maximum z-projection of the 678 obtained stack. This operation leads to vertical pixel rows, each of which depicts a y-679 projection of the ROI for each frame of the analysed movie (scheme in Fig. 5E). These  Table S2. For normally distributed data, Two-sample Student's t-tests were 718 used to compare the means of two groups. For more than two groups, one-way 719 analyses of variance tests (ANOVA) were conducted. For non-parametric data of 720 more than two groups, Kruskal-Wallis H tests were used, followed by a pairwise 721 Wilcoxon-Mann-Whitney test to compare individual pairs. Medians were used for the 722 non-parametric data, as they are better measurements of the central tendency of the 723 data for skewed distributions. In order to calculate the standard errors and confidence 724 intervals for the medians, a bootstrap method was applied (Efron, 1979), using a plug-