Single-cell transcriptomics identifies new blood cell populations in Drosophila released at the onset of metamorphosis.

Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, we identified a highly migratory and immune-responsive pupal cell population expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, we present the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.

Restriction of subapical proteins during cellularization depends on the onset of zygotic transcription and the formin Dia.

Cortical domains are characterized by spatially restricted polarity proteins. The pattern of cortical domains is dynamic and changes during cell differentiation and development. Although there is a good understanding for how the cortical pattern is maintained, e. g. by mutual antagonism, less is known about how the initial pattern is established, and its dynamics coordinated with developmental progression. Here we investigate the initial restriction of subapical marker proteins during the syncytial-cellular transition in Drosophila embryos. The subapical markers Canoe/Afadin, the complex ELMO-Sponge, Baz and Arm become initially restricted between apical and lateral domains during cellularization. We define the role of zygotic genome activation as a timer for subapical domain formation. Subapical markers remained widely spread in embryos treated with α-amanitin and became precociously restricted in mutant embryos with premature zygotic transcription. In contrast, remodeling of the nuclear division cycle without cytokinesis to a full cell cycle is not a prerequisite for subapical domain formation, since we observed timely subapical restriction in embryos undergoing an extra nuclear cycle. We provide evidence that earliest subapical markers ELMO-Sponge and Canoe are required for subapical accumulation of Baz. Supporting an important role of cortical F-actin in subapical restriction, we found that the formin Dia was required for Baz restriction, and its distribution depended on the onset of zygotic gene expression. In summary, we define zygotic transcription as a timer, to which subapical markers respond in a dia-dependent mechanism.

Dia- and Rok-dependent enrichment of capping proteins in a cortical region.

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II (MyoII, encoded by zip in flies) control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos, Rho1 signaling is high between actin caps, i.e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. In the present study, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok-MyoII control Capping protein enrichment and support a model that Dia and Rok-MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling. This article has an associated First Person interview with the first authors of the paper.

Cytoskeletal mechanics and dynamics in the Drosophila syncytial embryo.

Cell and tissue functions rely on the genetic programmes and cascades of biochemical signals. It has become evident during the past decade that the physical properties of soft material that govern the mechanics of cells and tissues play an important role in cellular function and morphology. The biophysical properties of cells and tissues are determined by the cytoskeleton, consisting of dynamic networks of F-actin and microtubules, molecular motors, crosslinkers and other associated proteins, among other factors such as cell-cell interactions. The Drosophila syncytial embryo represents a simple pseudo-tissue, with its nuclei orderly embedded in a structured cytoskeletal matrix at the embryonic cortex with no physical separation by cellular membranes. Here, we review the stereotypic dynamics and regulation of the cytoskeleton in Drosophila syncytial embryos and how cytoskeletal dynamics underlies biophysical properties and the emergence of collective features. We highlight the specific features and processes of syncytial embryos and discuss the applicability of biophysical approaches.

Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos.

The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.

ELMO and Sponge specify subapical restriction of Canoe and formation of the subapical domain in early Drosophila embryos.

Canoe/Afadin and the GTPase Rap1 specify the subapical domain during cellularization in Drosophila embryos. The timing of domain formation is unclear. The subapical domain might gradually mature or emerge synchronously with the basal and lateral domains. The potential mechanism for activation of Rap1 by guanyl nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs) is unknown. Here, we retraced the emergence of the subapical domain at the onset of cellularization by in vivo imaging with CanoeYFP in comparison to the lateral and basal markers ScribbledGFP and CherrySlam. CanoeYFP accumulates at a subapical position at about the same time as the lateral marker ScribbledGFP but a few minutes prior to basal CherrySlam. Furthermore, we show that the unconventional GEF complex ELMO-Sponge is subapically enriched and is required for subapical restriction of Canoe. The localization dynamics of ELMO-Sponge suggests a patterning mechanism for positioning the subapical region adjacent to the apical region. While marking the disc-like apical regions before cellularization, ELMO-Sponge redistributes to a ring-like pattern surrounding the apical region at the onset of cellularization.

In vivo optochemical control of cell contractility at single-cell resolution.

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing, and cancer invasion. Here, we report a simple optochemical method to induce cell contractions in vivo during Drosophila morphogenesis at single-cell resolution. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis in the epithelial tissue. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, causing an approximately 50% drop in the cross-sectional area. Increased Ca2+ levels are reversible, and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (ROCK) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be, in principle, adapted to many experimental systems and species, as no specific genetic elements are required.

Dynamics of cortical domains in early Drosophila development.

Underlying the plasma membrane of eukaryotic cells is an actin cortex that includes actin filaments and associated proteins. A special feature of all polarized and epithelial cells are cortical domains, each of which is characterized by specific sets of proteins. Typically, an epithelial cell contains apical, subapical, lateral and basal domains. The domain-specific protein sets contain evolutionarily conserved proteins, as well as cell-type-specific factors. Among the conserved proteins are, the Par proteins, Crumbs complex and the lateral proteins Scribbled and Discs large 1. Organization of the plasma membrane into cortical domains is dynamic and depends on cell type, differentiation and developmental stage. The dynamics of cortical organization is strikingly visible in early Drosophila embryos, which increase the number of distinct cortical domains from one, during the pre-blastoderm stage, to two in syncytial blastoderm embryos, before finally acquiring the four domains that are typical for epithelial cells during cellularization. In this Review, we will describe the dynamics of cortical organization in early Drosophila embryos and discuss the processes and mechanisms underlying cortical remodeling.

A 'molecular guillotine' reveals the interphase function of Kinesin-5.

Motor proteins are important for transport and force generation in a variety of cellular processes and in morphogenesis. Here, we describe a general strategy for conditional motor mutants by inserting a protease cleavage site into the 'neck' between the head domain and the stalk of the motor protein, making the protein susceptible to proteolytic cleavage at the neck by the corresponding protease. To demonstrate the feasibility of this approach, we inserted the cleavage site of the tobacco etch virus (TEV) protease into the neck of the tetrameric motor Kinesin-5. Application of TEV protease led to a specific depletion and functional loss of Kinesin-5 in Drosophila embryos. With our approach, we revealed that Kinesin-5 stabilizes the microtubule network during interphase in syncytial embryos. The 'molecular guillotine' can potentially be applied to many motor proteins because Kinesins and myosins have conserved structures with accessible neck regions.This article has an associated First Person interview with the first author of the paper.

Rapid nonlinear image scanning microscopy.

Image scanning microscopy (ISM) doubles the resolution of a conventional confocal microscope for super-resolution imaging. Here, we describe an all-optical ISM design based on rescanning microscopy for two-photon-excited fluorescence and second-harmonic generation that allows straightforward implementation into existing microscopes. The design offers improved sensitivity and high frame rates relative to those of existing systems. We demonstrate its utility using fixed and living specimens as well as collagen hydrogels.

Slam protein dictates subcellular localization and translation of its own mRNA.

Many mRNAs specifically localize within the cytoplasm and are present in RNA-protein complexes. It is generally assumed that localization and complex formation of these RNAs are controlled by trans-acting proteins encoded by genes different than the RNAs themselves. Here, we analyze slow as molasses (slam) mRNA that prominently colocalizes with its encoded protein at the basal cortical compartment during cellularization. The functional implications of this striking colocalization have been unknown. Here, we show that slam mRNA translation is spatiotemporally controlled. We found that translation was largely restricted to the onset of cellularization when Slam protein levels at the basal domain sharply increase. slam mRNA was translated locally, at least partially, as not yet translated mRNA transiently accumulated at the basal region. Slam RNA accumulated at the basal domain only if Slam protein was present. Furthermore, a slam RNA with impaired localization but full coding capacity was only weakly translated. We detected a biochemical interaction of slam mRNA and protein as demonstrated by specific co-immunoprecipitation from embryonic lysate. The intimate relationship of slam mRNA and protein may constitute a positive feedback loop that facilitates and controls timely and rapid accumulation of Slam protein at the prospective basal region.

Nanobody-Conjugated Nanotubes for Targeted Near-Infrared In Vivo Imaging and Sensing.

Fluorescent nanomaterials such as single-walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP-binding nanobodies to DNA-wrapped SWCNTs. This approach combines the targeting capabilities of GFP-binding nanobodies and the nonbleaching near-infrared fluorescence (850-1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin-5-GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.

Mechanical Model of Nuclei Ordering in Drosophila Embryos Reveals Dilution of Stochastic Forces.

During the initial development of syncytial embryos, nuclei go through cycles of nuclear division and spatial rearrangement. The arising spatial pattern of nuclei is important for subsequent cellularization and morphing of the embryo. Although nuclei are contained within a common cytoplasm, cytoskeletal proteins are nonuniformly packaged into regions around every nucleus. In fact, cytoskeletal elements like microtubules and their associated motor proteins exert stochastic forces between nuclei, actively driving their rearrangement. Yet, it is unknown how the stochastic forces are balanced to maintain nuclear order in light of increased nuclear density upon every round of divisions. Here, we investigate the nuclear arrangements in Drosophila melanogaster over the course of several nuclear divisions starting from interphase 11. We develop a theoretical model in which we distinguish long-ranged passive forces due to the nuclei as inclusions in the elastic matrix, namely the cytoplasm, and active, stochastic forces arising from the cytoskeletal dynamics mediated by motor proteins. We perform computer simulations and quantify the observed degree of orientational and spatial order of nuclei. Solely doubling the nuclear density upon nuclear division, the model predicts a decrease in nuclear order. Comparing results to experimental recordings of tracked nuclei, we make contradictory observations, finding an increase in nuclear order upon nuclear divisions. Our analysis of model parameters resulting from this comparison suggests that overall motor protein density as well as relative active-force amplitude has to decrease by a factor of about two upon nuclear division to match experimental observations. We therefore expect a dilution of cytoskeletal motors during the rapid nuclear division to account for the increase in nuclear order during syncytial embryo development. Experimental measurements of kinesin-5 cluster lifetimes support this theoretical finding.

Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer.

Epithelial cells are responsible for tissue homeostasis and form a barrier to maintain chemical gradients and mechanical integrity. Therefore, rapid wound closure is crucial for proper tissue function and restoring homeostasis. In this study, the mechanical properties of cells surrounding a single-cell wound are investigated during closure of the defect. The single-cell wound is induced in an intact layer using micropipette action and responses in neighboring cells are monitored with atomic force microscopy. Direct neighbors reveal a rise in the apparent pretension, which is dominated by cortical tension. The same effect was observed for a single-cell wound induced by laser ablation and during closure of a not fully confluent layer. Moreover, changes in the apparent pretension are far reaching and persist even in cells separated by three cell widths from the defect. This shows that epithelial cells respond to minimal wounds in a collective fashion by increased contractility with substantial reach.

Mechanical Properties of a Drosophila Larval Chordotonal Organ.

Proprioception is an integral part of the feedback circuit that is essential for locomotion control in all animals. Chordotonal organs perform proprioceptive and other mechanosensory functions in insects and crustaceans. The mechanical properties of these organs are believed to be adapted to the sensory functions, but had not been probed directly. We measured mechanical properties of a particular chordotonal organ-the lateral pentascolopidial (lch5) organ of Drosophila larvae-which plays a key role in proprioceptive locomotion control. We applied tension to the whole organ in situ by transverse deflection. Upon release of force, the organ displayed overdamped relaxation with two widely separated time constants, tens of milliseconds and seconds, respectively. When the muscles covering the lch5 organ were excised, the slow relaxation was absent, and the fast relaxation became faster. Interestingly, most of the strain in the stretched organ is localized in the cap cells, which account for two-thirds of the length of the entire organ, and could be stretched by ∼10% without apparent damage. In laser ablation experiments we found that cap cells retracted by ∼100 µm after being severed from the neurons, indicating considerable steady-state stress and strain in these cells. Given the fact that actin as well as myosin motors are abundant in cap cells, the results point to a mechanical regulatory role of the cap cells in the lch5 organ.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

The mechanical properties of early Drosophila embryos measured by high-speed video microrheology.

In early development, Drosophila melanogaster embryos form a syncytium, i.e., multiplying nuclei are not yet separated by cell membranes, but are interconnected by cytoskeletal polymer networks consisting of actin and microtubules. Between division cycles 9 and 13, nuclei and cytoskeleton form a two-dimensional cortical layer. To probe the mechanical properties and dynamics of this self-organizing pre-tissue, we measured shear moduli in the embryo by high-speed video microrheology. We recorded position fluctuations of injected micron-sized fluorescent beads with kHz sampling frequencies and characterized the viscoelasticity of the embryo in different locations. Thermal fluctuations dominated over nonequilibrium activity for frequencies between 0.3 and 1000 Hz. Between the nuclear layer and the yolk, the cytoplasm was homogeneous and viscously dominated, with a viscosity three orders of magnitude higher than that of water. Within the nuclear layer we found an increase of the elastic and viscous moduli consistent with an increased microtubule density. Drug-interference experiments showed that microtubules contribute to the measured viscoelasticity inside the embryo whereas actin only plays a minor role in the regions outside of the actin caps that are closely associated with the nuclei. Measurements at different stages of the nuclear division cycle showed little variation.

Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1.

The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes­pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps.

Function and dynamics of slam in furrow formation in early Drosophila embryo.

The Drosophila embryo undergoes a developmental transition in the blastoderm stage switching from syncytial to cellular development. The cleavage furrow, which encloses nuclei into cells, is a prominent morphological feature of this transition. It is not clear how the pattern of the furrow array is defined and how zygotic genes trigger the formation and invagination of interphase furrows. A key to these questions is provided by the gene slam, which has been previously implicated in controlling furrow invagination. Here we investigate the null phenotype of slam, the dynamics of Slam protein, and its control by the recycling endosome. We find that slam is essential for furrow invagination during cellularisation and together with nullo, for specification of the furrow. During cellularisation, Slam marks first the furrow, which is derived from the metaphase furrow of the previous mitosis. Slightly later, Slam accumulates at new furrows between daughter cells early in interphase. Slam is stably associated with the furrow canal except for the onset of cellularisation as revealed by FRAP experiments. Restriction of Slam to the furrow canal and Slam mobility during cellularisation is controlled by the recycling endosome and centrosomes. We propose a three step model. The retracting metaphase furrow leaves an initial mark. This mark and the border between corresponding daughter nuclei are refined by vesicular transport away from pericentrosomal recycling endosome towards the margins of the somatic buds. Following the onset of zygotic gene expression, Slam and Nullo together stabilise this mark and Slam triggers invagination of the cleavage furrow.