Membrane-actin interactions in morphogenesis: Lessons learned from Drosophila cellularization.

During morphogenesis, changes in the shapes of individual cells are harnessed to mold an entire tissue. These changes in cell shapes require the coupled remodeling of the plasma membrane and underlying actin cytoskeleton. In this review, we highlight cellularization of the Drosophila embryo as a model system to uncover principles of how membrane and actin dynamics are co-regulated in space and time to drive morphogenesis.

Combining protein and mRNA quantification to decipher transcriptional regulation.

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.

Stochastic Kinetics of Nascent RNA.

The stochastic kinetics of transcription is typically inferred from the distribution of RNA numbers in individual cells. However, cellular RNA reflects additional processes downstream of transcription, hampering this analysis. In contrast, nascent (actively transcribed) RNA closely reflects the kinetics of transcription. We present a theoretical model for the stochastic kinetics of nascent RNA, which we solve to obtain the probability distribution of nascent RNA per gene. The model allows us to evaluate the kinetic parameters of transcription from single-cell measurements of nascent RNA. The model also predicts surprising discontinuities in the distribution of nascent RNA, a feature which we verify experimentally.

The maternal-to-zygotic transition targets actin to promote robustness during morphogenesis.

Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/α-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-α (Sry-α) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-α are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-α collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-α and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.

Glyoxal-based fixation of Drosophila embryos for immunofluorescence staining and RNA in situ hybridization.

The dialdehyde glyoxal is an alternative chemical fixative that cross-links tissues faster than formaldehyde, retains higher antigenicity, and is less hazardous than either formaldehyde or glutaraldehyde. Here we present a glyoxal-based fixation protocol for use with Drosophila embryos. We describe steps to prepare acid-free glyoxal, fix embryos, and then stain with antibodies for immunofluorescence (IF). We also describe methods for RNA fluorescence in situ hybridization (FISH) and FISH plus IF (FISH-IF) using glyoxal-fixed embryos. This protocol was adapted for Drosophila embryos from the methods of Bussolati et al.1 and Richter et al.2.

Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs.

The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos.

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Mechanical Networks Have Robustness Built into Their Topology, Too.

Network topology ensures robust outcomes for information-transferring networks in biology (e.g., signaling and gene regulatory networks). In this issue of Developmental Cell, Yevick et al. (2019) demonstrate that a similar principle holds for the mechanical networks that drive morphogenesis.

Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed Embryos.

Environmental stress threatens the fidelity of embryonic morphogenesis. Heat, for example, is a teratogen. Yet how heat affects morphogenesis is poorly understood. Here, we identify a heat-inducible actin stress response (ASR) in Drosophila embryos that is mediated by the activation of the actin regulator Cofilin. Similar to ASR in adult mammalian cells, heat stress in fly embryos triggers the assembly of intra-nuclear actin rods. Rods measure up to a few microns in length, and their assembly depends on elevated free nuclear actin concentration and Cofilin. Outside the nucleus, heat stress causes Cofilin-dependent destabilization of filamentous actin (F-actin) in actomyosin networks required for morphogenesis. F-actin destabilization increases the chance of morphogenesis mistakes. Blocking the ASR by reducing Cofilin dosage improves the viability of heat-stressed embryos. However, improved viability correlates with restoring F-actin stability, not rescuing morphogenesis. Thus, ASR endangers embryos, perhaps by shifting actin from cytoplasmic filaments to an elevated nuclear pool.

Seeing a Coastline Paradox in Membrane Reservoirs.

In this issue of Developmental Cell, Goudarzi et al. (2017) examine the membrane source that allows bleb-based cell migration in vivo. Their work reminds us of the fractal nature of cell surfaces and highlights how the unfolding of these convoluted surfaces contributes to physiologically relevant cell shape change in intact organisms.

-Back-to-back mechanisms drive actomyosin ring closure during Drosophila embryo cleavage.

Contraction of actomyosin rings during cytokinesis is typically attributed to actin filaments sliding toward each other via Myosin-2 motor activity. However, rings constrict in some cells in the absence of Myosin-2 activity. Thus, ring closure uses Myosin-2-dependent and -independent mechanisms. But what the Myosin-2-independent mechanisms are, and to what extent they are sufficient to drive closure, remains unclear. During cleavage in Drosophila melanogaster embryos, actomyosin rings constrict in two sequential and mechanistically distinct phases. We show that these phases differ in constriction speed and are genetically and pharmacologically separable. Further, Myosin-2 activity is required for slow constriction in "phase 1" but is largely dispensable for fast constriction in "phase 2," and F-actin disassembly is only required for fast constriction in phase 2. Switching from phase 1 to phase 2 seemingly relies on the spatial organization of F-actin as controlled by Cofilin, Anillin, and Septin. Our work shows that fly embryos present a singular opportunity to compare separable ring constriction mechanisms, with varying Myosin-2 dependencies, in one cell type and in vivo.

Membrane Supply and Demand Regulates F-Actin in a Cell Surface Reservoir.

Cells store membrane in surface reservoirs of pits and protrusions. These membrane reservoirs facilitate cell shape change and buffer mechanical stress, but we do not know how reservoir dynamics are regulated. During cellularization, the first cytokinesis in Drosophila embryos, a reservoir of microvilli unfolds to fuel cleavage furrow ingression. We find that regulated exocytosis adds membrane to the reservoir before and during unfolding. Dynamic F-actin deforms exocytosed membrane into microvilli. Single microvilli extend and retract in ∼20 s, while the overall reservoir is depleted in sync with furrow ingression over 60-70 min. Using pharmacological and genetic perturbations, we show that exocytosis promotes microvillar F-actin assembly, while furrow ingression controls microvillar F-actin disassembly. Thus, reservoir F-actin and, consequently, reservoir dynamics are regulated by membrane supply from exocytosis and membrane demand from furrow ingression.

A membrane reservoir at the cell surface: unfolding the plasma membrane to fuel cell shape change.

Cell surface expansion is a necessary part of cell shape change. One long-standing hypothesis proposes that membrane for this expansion comes from the flattening out of cell surface projections such as microvilli and membrane folds. Correlative EM data of cells undergoing phagocytosis, cytokinesis, and morphogenesis has hinted at the existence of such an unfolding mechanism for decades; but unfolding has only recently been confirmed using live-cell imaging and biophysical approaches. Considering the wide range of cells in which plasma membrane unfolding has now been reported, it likely represents a fundamental mechanism of cell shape change.

The plasma membrane flattens out to fuel cell-surface growth during Drosophila cellularization.

Cell-shape change demands cell-surface growth, but how growth is fueled and choreographed is still debated. Here we use cellularization, the first complete cytokinetic event in Drosophila embryos, to show that cleavage furrow ingression is kinetically coupled to the loss of surface microvilli. We modulate furrow kinetics with RNAi against the Rho1-GTPase regulator slam and show that furrow ingression controls the rate of microvillar depletion. Finally, we directly track the microvillar membrane and see it move along the cell surface and into ingressing furrows, independent of endocytosis. Together, our results demonstrate that the kinetics of the ingressing furrow regulate the utilization of a microvillar membrane reservoir. Because membranes of the furrow and microvilli are contiguous, we suggest that ingression drives unfolding of the microvilli and incorporation of microvillar membrane into the furrow. We conclude that plasma membrane folding/unfolding can contribute to the cell-shape changes that promote embryonic morphogenesis.

Imaging cell shape change in living Drosophila embryos.

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)(1-5). On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility(6), but is instead fueled largely by exocytosis of membrane from internal stores(7). Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)(8-19). In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques(9-10). Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization.

Cortical compartments partition proteins and membrane at the cell surface to define regions of specialized function. Here we ask how cortical compartments arise along the plasma membrane furrows that cellularize the early Drosophila embryo, and investigate the influence that this compartmentalization has on furrow ingression. We find that the zygotic gene product Nullo aids the establishment of discrete cortical compartments, called furrow canals, which form at the tip of incipient furrows. Upon nullo loss-of-function, proteins that are normally restricted to adjacent lateral regions of the furrow, such as Neurotactin and Discs large, spread into the furrow canals. At the same time, cortical components that should concentrate in furrow canals, such as Myosin 2 (Zipper) and Anillin (Scraps), are missing from some furrows. Depletion of these cortical components from the furrow canal compartments precipitates furrow regression. Contrary to previous models, we find that furrow compartmentalization does not require cell-cell junctions that border the furrow canals. Instead, compartmentalization is disrupted by treatments that reduce levels of cortical F-actin. Because the earliest uniform phenotype detected in nullo mutants is reduced levels of F-actin at furrow canals, we propose that Nullo compartmentalizes furrows via its regulation of F-actin, thus stabilizing furrows and insuring their ingression to complete cellularization.

Local actin-dependent endocytosis is zygotically controlled to initiate Drosophila cellularization.

In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. Here, we find that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. We propose that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis.