The positioning mechanics of microtubule asters in Drosophila embryo explants.

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, we demonstrate that these asters likely generate an effective pushing force over short distances.

Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking.

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.

Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte.

In Drosophila melanogaster, the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the oocyte posterior end, while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. Past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, neither the molecular identity nor the nature of the signal is known. Here, we provide evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. We show that Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. Furthermore, we provide evidence that PFC contact maintains Par-1 and oskar mRNA localization and microtubule cytoskeleton polarity in the oocyte. Our observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.

Endoplasmic reticulum membranes are continuously required to maintain mitotic spindle size and forces.

Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the ER around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. We reveal that when centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected by ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.

Polarity Events in the Drosophila melanogaster Oocyte.

Cell polarity is a pre-requirement for many fundamental processes in animal cells, such as asymmetric cell division, axon specification, morphogenesis and epithelial tissue formation. For all these different processes, polarization is established by the same set of proteins, called partitioning defective (Par) proteins. During development in Drosophila melanogaster, decision making on the cellular and organism level is achieved with temporally controlled cell polarization events. The initial polarization of Par proteins occurs as early as in the germline cyst, when one of the 16 cells becomes the oocyte. Another marked event occurs when the anterior-posterior axis of the future organism is defined by Par redistribution in the oocyte, requiring external signaling from somatic cells. Here, we review the current literature on cell polarity events that constitute the oogenesis from the stem cell to the mature egg.

Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm.

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

Astral microtubule cross-linking safeguards uniform nuclear distribution in the Drosophila syncytium.

The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

Nuclear positioning during development: Pushing, pulling and flowing.

The positioning of the nucleus, the central organelle of the cell, is an active and regulated process crucially linked to cell cycle, differentiation, migration, and polarity. Alterations in positioning have been correlated with cell and tissue function deficiency and genetic or chemical manipulation of nuclear position is embryonic lethal. Nuclear positioning is a precursor for symmetric or asymmetric cell division which is accompanied by fate determination of the daughter cells. Nuclear positioning also plays a key role during early embryonic developmental stages in insects, such as Drosophila, where hundreds of nuclei divide without cytokinesis and are distributed within the large syncytial embryo at roughly regular spacing. While the cytoskeletal elements and the linker proteins to the nucleus are fairly well characterised, including some of the force generating elements driving nuclear movement, there is considerable uncertainty about the biophysical mechanism of nuclear positioning, while the field is debating different force models. In this review, we highlight the current body of knowledge, discuss cell context dependent models of nuclear positioning, and outline open questions.

Plk4 triggers autonomous de novo centriole biogenesis and maturation.

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.

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.

A cell-free system of Drosophila egg explants supporting native mitotic cycles.

Mitosis, in a broader sense, is an intracellular mechanical process that is fueled by chemical reactions and regulated by a complex protein interaction network. Research aimed at understanding mitosis in all these aspects is often limited to pharmaceutical treatment or genetic manipulation of single cells or entire tissues. These experimental models entail physical boundaries imposed by the cell membrane, making it extremely challenging to apply mechanical perturbations, or to introduce larger molecules such as peptides, proteins, or genetic transcripts in an acute and specific manner. Here, we present a cell-free experimental assay that is exploiting the properties of a large, multinucleated embryo cell. Drosophila, like almost all insects, initially develops as a syncytial embryo, the task of which is to replicate and distribute the genetic material quickly and regularly. We describe an experimental procedure that allows the isolation of nucleocytoplasm from single embryos that retains the developmental processes, most importantly the native mitotic progression of nuclei.

A single Drosophila embryo extract for the study of mitosis ex vivo.

Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.

Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo.

In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.

Reconstitution and quantification of dynamic microtubule end tracking in vitro using TIRF microscopy.

Several microtubule-associated proteins localize in living cells selectively to an extended region at the growing microtubule plus ends. Over the last years, these plus-end-tracking proteins, also called +TIPs, have attracted considerable interest because they are involved in a large variety of essential intracellular processes. GFP-labeled versions of EB proteins are also often used as markers for intracellular microtubule organization and dynamics. The mechanism of selective +TIP binding to the end region of growing microtubule was unknown. Recently, the phenomenon of end tracking was reconstituted in vitro from purified proteins, which allowed the identification of EB proteins as the minimal core of the plus-end-tracking system and the dissection of the molecular mechanism of end tracking by these proteins. This in vitro reconstitution has started to be widely used for several +TIPs and promises to provide mechanistic insight into the functioning of the dynamic +TIP network at growing microtubule ends. Here, we describe the purification of EB1 and CLIP-170, the total internal reflection fluorescence microscopy assay to observe dynamic end tracking in vitro, and the quantitative analysis of fluorescent +TIP comet shape and of single +TIP molecule turnover at growing microtubule ends.

Directional switching of the kinesin Cin8 through motor coupling.

Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end-directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end-directed motors, whereas they switched to plus-end-directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.

A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps.

During cell division, microtubules are arranged in a large bipolar structure, the mitotic spindle, to segregate the duplicated chromosomes. Antiparallel microtubule overlaps in the spindle center are essential for establishing bipolarity and maintaining spindle stability throughout mitosis. In anaphase, this antiparallel microtubule array is tightly bundled forming the midzone, which serves as a hub for the recruitment of proteins essential for late mitotic events. The molecular mechanism of midzone formation and the control of its size are not understood. Using an in vitro reconstitution approach, we show here that PRC1 autonomously bundles antiparallel microtubules and recruits Xklp1, a kinesin-4, selectively to overlapping antiparallel microtubules. The processive motor Xklp1 controls overlap size by overlap length-dependent microtubule growth inhibition. Our results mechanistically explain how the two conserved, essential midzone proteins PRC1 and Xklp1 cooperate to constitute a minimal protein module capable of dynamically organizing the core structure of the central anaphase spindle.

Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and +TIP dynamics.

Microtubule cytoskeleton function depends on the dynamic interplay of microtubules and various microtubule-binding proteins. To gain an understanding of cytoskeleton function at the molecular level, it is important to measure quantitatively how cytoskeletal proteins interact with each other in space and time. Here we describe fluorescence microscopy-based in vitro assays on chemically functionalized glass slides for the study of several aspects of microtubule cytoskeleton dynamics: single motor movements, dynamic microtubule plus-end tracking, antiparallel microtubule sliding by microtubule-crosslinking motors, and microtubule gliding by surface-immobilized motors. The combination of a passivating polyethylene glycol layer on the glass with covalently attached functional groups for selective protein capturing ensures excellent control of the surface properties and good preservation of protein activities in these assays. Common to all assays is that they can be performed in the presence of high concentrations of soluble proteins or even cell extract, which in combination with total internal reflection fluorescence microscopy allows the study of complex protein mixtures that were previously not accessible to quantitative imaging in vitro.

Obstacles on the microtubule reduce the processivity of Kinesin-1 in a minimal in vitro system and in cell extract.

Inside cells, a multitude of molecular motors and other microtubule-associated proteins are expected to compete for binding to a limited number of binding sites available on microtubules. Little is known about how competition for binding sites affects the processivity of molecular motors and, therefore, cargo transport, organelle positioning, and microtubule organization, processes that all depend on the activity of more or less processive motors. Very few studies have been performed in the past to address this question directly. Most studies reported only minor effects of crowding on the velocity of motors. However, a controversy appears to exist regarding the effect of crowding on motor processivity. Here, we use single-molecule imaging of mGFP-labeled minimal dimeric kinesin-1 constructs in vitro to study the effects of competition on kinesin's processivity. For competitors, we use kinesin rigor mutants as static roadblocks, minimal wild-type kinesins as motile obstacles, and a cell extract as a complex mixture of microtubule-associated proteins. We find that mGFP-labeled kinesin-1 detaches prematurely from microtubules when it encounters obstacles, leading to a strong reduction of its processivity, a behavior that is largely independent of the type of obstacle used here. Kinesin has a low probability to wait briefly when encountering roadblocks. Our data suggest, furthermore, that kinesin can occasionally pass obstacles on the protofilament track.

A multisegmental cross-bridge kinetics model of the myofibril.

Striated muscle is a mechanical system that develops force and generates power in serving vital activities in the body. Striated muscle is a complex biological system; a single mammalian muscle fibre contains up to hundred or even more myofibrils in parallel connected via an inter-myofibril filament network. In one single myofibril thousands of sarcomeres are lined up as a series of linear motors. We recently demonstrated that half-sarcomeres (hS) in a single myofibril operate non-uniformly. We outline a mathematical framework based on cross-bridge kinetics for the simulation of the force response and length change of individual hS in a myofibril. The model describes the muscle myofibril in contraction experiments under various conditions. The myofibril is modeled as a multisegmental mechanical system of hS models, which have active and viscoelastic properties. In the first approach, a two-state cross-bridge formalism relates the hS force to the chemical kinetics of ATP hydrolysis, as first described by Huxley [1957. Muscle structure and theories of contraction. Prog. Biophys. Mol. Biol. 7, 255-318]. Two possible types of biological variability are introduced and modeled. Numerical simulations of a myofibril composed of four to eight hS show a non-uniform hS length distribution and complex internal dynamics upon activation. We demonstrate that the steady-state approximation holds only in restricted time zones during activation. Simulations of myofibril contraction experiments that reproduce the classic steady-state force-length and force-velocity relationships, strictly constrained or "clamped" in either end-held isometric or isotonic contraction conditions, reveal a small but conspicuous effect of hS dynamics on force.