Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales.

In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.

Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System.

Dorsal closure is a key process during Drosophila morphogenesis that models cell sheet movements in chordates, including neural tube closure, palate formation, and wound healing. Closure occurs midway through embryogenesis and entails circumferential elongation of lateral epidermal cell sheets that close a dorsal hole filled with amnioserosa cells. Signaling pathways regulate the function of cellular structures and processes, including Actomyosin and microtubule cytoskeletons, cell-cell/cell-matrix adhesion complexes, and endocytosis/vesicle trafficking. These orchestrate complex shape changes and movements that entail interactions between five distinct cell types. Genetic and laser perturbation studies establish that closure is robust, resilient, and the consequence of redundancy that contributes to four distinct biophysical processes: contraction of the amnioserosa, contraction of supracellular Actomyosin cables, elongation (stretching?) of the lateral epidermis, and zipping together of two converging cell sheets. What triggers closure and what the emergent properties are that give rise to its extraordinary resilience and fidelity remain key, extant questions.

DeepProjection: specific and robust projection of curved 2D tissue sheets from 3D microscopy using deep learning.

The efficient extraction of image data from curved tissue sheets embedded in volumetric imaging data remains a serious and unsolved problem in quantitative studies of embryogenesis. Here, we present DeepProjection (DP), a trainable projection algorithm based on deep learning. This algorithm is trained on user-generated training data to locally classify 3D stack content, and to rapidly and robustly predict binary masks containing the target content, e.g. tissue boundaries, while masking highly fluorescent out-of-plane artifacts. A projection of the masked 3D stack then yields background-free 2D images with undistorted fluorescence intensity values. The binary masks can further be applied to other fluorescent channels or to extract local tissue curvature. DP is designed as a first processing step than can be followed, for example, by segmentation to track cell fate. We apply DP to follow the dynamic movements of 2D-tissue sheets during dorsal closure in Drosophila embryos and of the periderm layer in the elongating Danio embryo. DeepProjection is available as a fully documented Python package.

Wound repair in sea urchin larvae involves pigment cells and blastocoelar cells.

Sea urchin larvae spend weeks to months feeding on plankton prior to metamorphosis. When handled in the laboratory they are easily injured, suggesting that in the plankton they are injured with some frequency. Fortunately, larval wounds are repaired through an efficient wound response with mesenchymal pigment cells and blastocoelar cells assisting as the epithelium closes. An injury to the epithelium leads to an immediate calcium transient that rapidly spreads around the entire larva and is necessary for activating pigment cell migration toward the wound. If calcium transport is blocked, the pigment cells fail to activate and remain in place. When activated, pigment cells initiate directed migration to the wound site from distances of at least 85 â€‹µm. Upon arrival at the wound site they participate in an innate immune response. Blastocoelar cells are recruited to the injury site as well, though the calcium transient is unnecessary for activating these cells. At the wound site, blastocoelar cells participate in several functions including remodeling the skeleton if it protrudes through the epithelium.

Mutations in Drosophila crinkled/Myosin VIIA disrupt denticle morphogenesis.

Actin filament crosslinking, bundling and molecular motor proteins are necessary for the assembly of epithelial projections such as microvilli, stereocilia, hairs, and bristles. Mutations in such proteins cause defects in the shape, structure, and function of these actin - based protrusions. One protein necessary for stereocilia formation, Myosin VIIA, is an actin - based motor protein conserved throughout phylogeny. In Drosophila melanogaster, severe mutations in the MyoVIIA homolog crinkled (ck) are "semi - lethal" with only a very small percentage of flies surviving to adulthood. Such survivors show morphological defects related to actin bundling in hairs and bristles. To better understand ck/MyoVIIA's function in bundled - actin structures, we used dominant female sterile approaches to analyze the loss of maternal and zygotic (M/Z) ck/MyoVIIA in the morphogenesis of denticles, small actin - based projections on the ventral epidermis of Drosophila embryos. M/Z ck mutants displayed severe defects in denticle morphology - actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, we demonstrated that both of the C - terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, we show that ck/MyoVIIA interacts genetically with dusky - like (dyl), a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane, and when mutated also disrupts normal denticle formation. Loss of either protein alone does not alter the localization of the other; however, loss of the two proteins together dramatically enhances the defects in denticle shape observed when either protein alone was absent. Our data indicate that ck/MyoVIIA plays a key role in the formation and/or organization of actin filament bundles, which drive proper shape of cellular projections.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure.

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca(2+) is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca(2+) leads to cell contraction. Furthermore, endogenous Ca(2+) fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca(2+) slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca(2+) and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.

Remodeling Tissue Interfaces and the Thermodynamics of Zipping during Dorsal Closure in Drosophila.

Dorsal closure during Drosophila embryogenesis is an important model system for investigating the biomechanics of morphogenesis. During closure, two flanks of lateral epidermis (with actomyosin-rich purse strings near each leading edge) close an eye-shaped opening that is filled with amnioserosa. At each canthus (corner of the eye) a zipping process remodels the tissue interfaces between the leading edges of the lateral epidermis and the amnioserosa. We investigated zipping dynamics and found that apposing leading edge cells come together at their apical ends and then square off basally to form a lateral junction. Meanwhile, the purse strings act as contractile elastic rods bent toward the embryo interior near each canthus. We propose that a canthus-localized force contributes to both bending the ends of the purse strings and the formation of lateral junctions. We developed a thermodynamic model for zipping based on three-dimensional remodeling of the tissue interfaces and the reaction dynamics of adhesion molecules in junctions and elsewhere, which we applied to zipping during unperturbed wild-type closure and to laser or genetically perturbed closure. We identified two processes that can contribute to the zipping mechanism, consistent with experiments, distinguished by whether amnioserosa dynamics do or do not augment canthus adhesion dynamics.

Minimal vertex model explains how the amnioserosa avoids fluidization during Drosophila dorsal closure.

Dorsal closure is a process that occurs during embryogenesis of Drosophila melanogaster . During dorsal closure, the amnioserosa (AS), a one-cell thick epithelial tissue that fills the dorsal opening, shrinks as the lateral epidermis sheets converge and eventually merge. During this process, both shape index and aspect ratio of amnioserosa cells increase markedly. The standard 2-dimensional vertex model, which successfully describes tissue sheet mechanics in multiple contexts, would in this case predict that the tissue should fluidize via cell neighbor changes. Surprisingly, however, the amnioserosa remains an elastic solid with no such events. We here present a minimal extension to the vertex model that explains how the amnioserosa can achieve this unexpected behavior. We show that continuous shrinkage of the preferred cell perimeter and cell perimeter polydispersity lead to the retention of the solid state of the amnioserosa. Our model accurately captures measured cell shape and orientation changes and predicts non-monotonic junction tension that we confirm with laser ablation experiments. Significance Statement: During embryogenesis, cells in tissues can undergo significant shape changes. Many epithelial tissues fluidize, i.e. cells exchange neighbors, when the average cell shape index increases above a threshold value, consistent with the standard vertex model. During dorsal closure in Drosophila melanogaster , however, the amnioserosa tissue remains solid even as the average cell shape index increases well above threshold. We introduce perimeter polydispersity and allow the preferred cell perimeters, usually held fixed in vertex models, to decrease linearly with time as seen experimentally. With these extensions to the standard vertex model, we capture experimental observations quantitatively. Our results demonstrate that vertex models can describe the behavior of the amnioserosa in dorsal closure by allowing normally fixed parameters to vary with time.

Notochord segmentation in zebrafish controlled by iterative mechanical signaling.

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting that these processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning.

Minimal vertex model explains how the amnioserosa avoids fluidization during Drosophila dorsal closure.

Dorsal closure is a process that occurs during embryogenesis of Drosophila melanogaster. During dorsal closure, the amnioserosa (AS), a one-cell thick epithelial tissue that fills the dorsal opening, shrinks as the lateral epidermis sheets converge and eventually merge. During this process, the aspect ratio of amnioserosa cells increases markedly. The standard 2-dimensional vertex model, which successfully describes tissue sheet mechanics in multiple contexts, would in this case predict that the tissue should fluidize via cell neighbor changes. Surprisingly, however, the amnioserosa remains an elastic solid with no such events. We here present a minimal extension to the vertex model that explains how the amnioserosa can achieve this unexpected behavior. We show that continuous shrink-age of the preferred cell perimeter and cell perimeter polydispersity lead to the retention of the solid state of the amnioserosa. Our model accurately captures measured cell shape and orientation changes and predicts non-monotonic junction tension that we confirm with laser ablation experiments.

Axial segmentation by iterative mechanical signaling.

In bony fishes, formation of the vertebral column, or spine, is guided by a metameric blueprint established in the epithelial sheath of the notochord. Generation of the notochord template begins days after somitogenesis and even occurs in the absence of somite segmentation. However, patterning defects in the somites lead to imprecise notochord segmentation, suggesting these processes are linked. Here, we reveal that spatial coordination between the notochord and the axial musculature is necessary to ensure segmentation of the zebrafish spine both in time and space. We find that the connective tissues that anchor the axial skeletal musculature, known as the myosepta in zebrafish, transmit spatial patterning cues necessary to initiate notochord segment formation, a critical pre-patterning step in spine morphogenesis. When an irregular pattern of muscle segments and myosepta interact with the notochord sheath, segments form non-sequentially, initiate at atypical locations, and eventually display altered morphology later in development. We determine that locations of myoseptum-notochord connections are hubs for mechanical signal transmission, which are characterized by localized sites of deformation of the extracellular matrix (ECM) layer encasing the notochord. The notochord sheath responds to the external mechanical changes by locally augmenting focal adhesion machinery to define the initiation site for segmentation. Using a coarse-grained mathematical model that captures the spatial patterns of myoseptum-notochord interactions, we find that a fixed-length scale of external cues is critical for driving sequential segment patterning in the notochord. Together, this work identifies a robust segmentation mechanism that hinges upon mechanical coupling of adjacent tissues to control patterning dynamics.

Cell ingression and apical shape oscillations during dorsal closure in Drosophila.

Programmed patterns of gene expression, cell-cell signaling, and cellular forces cause morphogenic movements during dorsal closure. We investigated the apical cell-shape changes that characterize amnioserosa cells during dorsal closure in Drosophila embryos with in vivo imaging of green-fluorescent-protein-labeled DE-cadherin. Time-lapsed, confocal images were assessed with a novel segmentation algorithm, Fourier analysis, and kinematic and dynamical modeling. We found two generic processes, reversible oscillations in apical cross-sectional area and cell ingression characterized by persistent loss of apical area. We quantified a time-dependent, spatially-averaged sum of intracellular and intercellular forces acting on each cell's apical belt of DE-cadherin. We observed that a substantial fraction of amnioserosa cells ingress near the leading edges of lateral epidermis, consistent with the view that ingression can be regulated by leading-edge cells. This is in addition to previously observed ingression processes associated with zipping and apoptosis. Although there is cell-to-cell variability in the maximum rate for decreasing apical area (0.3-9.5 µm(2)/min), the rate for completing ingression is remarkably constant (0.83 cells/min, r(2) > 0.99). We propose that this constant ingression rate contributes to the spatiotemporal regularity of mechanical stress exerted by the amnioserosa on each leading edge during closure.

Superresolution microscopy reveals actomyosin dynamics in medioapical arrays.

Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, superresolution approaches (grazing incidence structured illumination, GI-SIM, and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in Drosophila. In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved-some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue, show that medioapical arrays are tightly apposed to the plasma membrane and are continuous with meshworks of lamellar F-actin. Medioapical arrays thereby constitute modified cell cortex. In concert with other tagged array components, superresolution imaging of live specimens will offer new understanding of cortical architecture and function.

Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis.

Metazoan development involves a myriad of dynamic cellular processes that require cytoskeletal function. Nonmuscle myosin II plays essential roles in embryonic development; however, knowledge of its role in post-embryonic development, even in model organisms such as Drosophila melanogaster, is only recently being revealed. In this study, truncation alleles were generated and enable the conditional perturbation, in a graded fashion, of nonmuscle myosin II function. During wing development they demonstrate novel roles for nonmuscle myosin II, including in adhesion between the dorsal and ventral wing epithelial sheets; in the formation of a single actin-based wing hair from the distal vertex of each cell; in forming unbranched wing hairs; and in the correct positioning of veins and crossveins. Many of these phenotypes overlap with those observed when clonal mosaic analysis was performed in the wing using loss of function alleles. Additional requirements for nonmuscle myosin II are in the correct formation of other actin-based cellular protrusions (microchaetae and macrochaetae). We confirm and extend genetic interaction studies to show that nonmuscle myosin II and an unconventional myosin, encoded by crinkled (ck/MyoVIIA), act antagonistically in multiple processes necessary for wing development. Lastly, we demonstrate that truncation alleles can perturb nonmuscle myosin II function via two distinct mechanisms--by titrating light chains away from endogenous heavy chains or by recruiting endogenous heavy chains into intracellular aggregates. By allowing myosin II function to be perturbed in a controlled manner, these novel tools enable the elucidation of post-embryonic roles for nonmuscle myosin II during targeted stages of fly development.

Division of labor: subsets of dorsal-appendage-forming cells control the shape of the entire tube.

The function of an organ relies on its form, which in turn depends on the individual shapes of the cells that create it and the interactions between them. Despite remarkable progress in the field of developmental biology, how cells collaborate to make a tissue remains an unsolved mystery. To investigate the mechanisms that determine organ structure, we are studying the cells that form the dorsal appendages (DAs) of the Drosophila melanogaster eggshell. These cells consist of two differentially patterned subtypes: roof cells, which form the outward-facing roof of the lumen, and floor cells, which dive underneath the roof cells to seal off the floor of the tube. In this paper, we present three lines of evidence that reveal a further stratification of the DA-forming epithelium. Laser ablation of only a few cells in the anterior of the region causes a disproportionately severe shortening of the appendage. Genetic alteration through the twin peaks allele of tramtrack69 (ttk(twk)), a female-sterile mutation that leads to severely shortened DAs, causes no such shortening when removed from a majority of the DA-forming cells, but rather, produces short appendages only when removed from cells in the very anterior of the tube-forming tissue. Additionally we show that heterotrimeric G-protein function is required for DA morphogenesis. Like TTK69, Gbeta 13F is not required in all DA-forming follicle cells but only in the floor and leading roof cells. The different phenotypes that result from removal of Gbeta 13F from each region demonstrate a striking division of function between different DA-forming cells. Gbeta mutant floor cells are unable to control the width of the appendage while Gbeta mutant leading roof cells fail to direct the elongation of the appendage and the convergent-extension of the roof-cell population.

Identifying Key Genetic Regions for Cell Sheet Morphogenesis on Chromosome 2L Using a Drosophila Deficiency Screen in Dorsal Closure.

Cell sheet morphogenesis is essential for metazoan development and homeostasis of animal form - it contributes to developmental milestones including gastrulation, neural tube closure, heart and palate formation and to tissue maintenance during wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis and a model for cell sheet morphogenesis, is a remarkably robust process during which coordination of conserved gene expression patterns and signaling cascades regulate the cellular shape changes and movements. New 'dorsal closure genes' continue to be discovered due to advances in imaging and genetics. Here, we extend our previous study of the right arm of the 2nd chromosome to the left arm of the 2nd chromosome using the Bloomington deficiency kit's set of large deletions, which collectively remove 98.9% of the genes on the left arm of chromosome two (2L) to identify 'dorsal closure deficiencies'. We successfully screened 87.2% of the genes and identified diverse dorsal closure defects in embryos homozygous for 49 deficiencies, 27 of which delete no known dorsal closure gene. These homozygous deficiencies cause defects in cell shape, canthus formation and tissue dynamics. Within these deficiencies, we have identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on 2L that affect lateral epidermal cells. We will continue to identify novel 'dorsal closure genes' with further analysis. These forward genetic screens are expected to identify new processes and pathways that contribute to closure and links between pathways and structures already known to coordinate various aspects of closure.

Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure.

BACKGROUND: The morphogenic movements that characterize embryonic development require the precise temporal and spatial control of cell-shape changes. Drosophila dorsal closure is a well-established model for epithelial sheet morphogenesis, and mutations in more than 60 genes cause defects in closure. Closure requires that four forces, derived from distinct tissues, be precisely balanced. The proteins responsible for generating each of the forces have not been determined. RESULTS: We document dorsal closure in living embryos to show that mutations in nonmuscle myosin II (encoded by zipper; zip/MyoII) disrupt the integrity of multiple tissues during closure. We demonstrate that MyoII localization is distinct from, but overlaps, F-actin in the supracellular purse string, whereas in the amnioserosa and lateral epidermis each has similar, cortical distributions. In zip/MyoII mutant embryos, we restore MyoII function either ubiquitously or specifically in the leading edge, amnioserosa, or lateral epidermis and find that zip/MyoII function in any one tissue can rescue closure. Using a novel, transgenic mosaic approach, we establish that contractility of the supracellular purse string in leading-edge cells requires zip/MyoII-generated forces; that zip/MyoII function is responsible for the apical contraction of amnioserosa cells; that zip/MyoII is important for zipping; and that defects in zip/MyoII contractility cause the misalignment of the lateral-epidermal sheets during seam formation. CONCLUSIONS: We establish that zip/MyoII is responsible for generating the forces that drive cell-shape changes in each of the force-generating tissues that contribute to closure. This highly conserved contractile protein likely drives cell-sheet movements throughout phylogeny.

Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila.

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy (and references therein). Here, we show that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals. We demonstrate that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity.

Contractile protein biochemistry in the Pollard Lab in Baltimore.

We describe our search for the molecular mechanisms of cell motility with personal recollections of bucket biochemistry in Tom Pollards Lab at the Johns Hopkins, circa 1980.

Unified biophysical mechanism for cell-shape oscillations and cell ingression.

We describe a mechanochemical and percolation cascade that augments myosin's regulatory network to tune cytoskeletal forces. Actomyosin forces collectively generate cytoskeletal forces during cell oscillations and ingression, which we quantify by elastic percolation of the internally driven, cross-linked actin network. Contractile units can produce relatively large, oscillatory forces that disrupt crosslinks to reduce cytoskeletal forces. A (reverse) Hopf bifurcation switches contractile units to produce smaller, steady forces that enhance crosslinking and consequently boost cytoskeletal forces to promote ingression. We describe cell-shape changes and cell ingression in terms of intercellular force imbalances along common cell junctions.