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.

A Novel Osteotomy Preparation Technique to Preserve Implant Site Viability and Enhance Osteogenesis.

The preservation of bone viability at an osteotomy site is a critical variable for subsequent implant osseointegration. Recent biomechanical studies evaluating the consequences of site preparation led us to rethink the design of bone-cutting drills, especially those intended for implant site preparation. We present here a novel drill design that is designed to efficiently cut bone at a very low rotational velocity, obviating the need for irrigation as a coolant. The low-speed cutting produces little heat and, consequently, osteocyte viability is maintained. The lack of irrigation, coupled with the unique design of the cutting flutes, channels into the osteotomy autologous bone chips and osseous coagulum that have inherent osteogenic potential. Collectively, these features result in robust, new bone formation at rates significantly faster than those observed with conventional drilling protocols. These preclinical data have practical implications for the clinical preparation of osteotomies and alveolar bone reconstructive surgeries.

Human Tendon-Derived Collagen Hydrogel Significantly Improves Biomechanical Properties of the Tendon-Bone Interface in a Chronic Rotator Cuff Injury Model.

PURPOSE: Poor healing of the tendon-bone interface (TBI) after rotator cuff (RTC) tears leads to high rates of recurrent tear following repair. Previously, we demonstrated that an injectable, thermoresponsive, type I collagen-rich, decellularized human tendon-derived hydrogel (tHG) improved healing in an acute rat Achilles tendon injury model. The purpose of this study was to investigate whether tHG enhances the biomechanical properties of the regenerated TBI in a rat model of chronic RTC injury and repair. METHODS: Tendon hydrogel was prepared from chemically decellularized human cadaveric flexor tendons. Eight weeks after bilateral resection of supraspinatus tendons, repair of both shoulders was performed. One shoulder was treated with a transosseous suture (control group) and the other was treated with a transosseous suture plus tHG injection at the repair site (tHG group). Eight weeks after repair, the TBIs were evaluated biomechanically, histologically, and via micro-computed tomography (CT). RESULTS: Biomechanical testing revealed a larger load to failure, higher stiffness, higher energy to failure, larger strain at failure, and higher toughness in the tHG group versus control. The area of new cartilage formation was significantly larger in the tHG group. Micro-CT revealed no significant difference between groups in bone morphometry at the supraspinatus tendon insertion, although the tHG group was superior to the control. CONCLUSIONS: Injection of tHG at the RTC repair site enhanced biomechanical properties and increased fibrocartilage formation at the TBI in a chronic injury model. CLINICAL RELEVANCE: Treatment of chronic RTC injuries with tHG at the time of surgical treatment may improve outcomes after surgical repair.

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.

Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure in Drosophila.

Drosophila's dorsal closure provides an excellent model system with which to analyze biomechanical processes during morphogenesis. During native closure, the amnioserosa, flanked by two lateral epidermal sheets, forms an eye-shaped opening with canthi at each corner. The dynamics of amnioserosa cells and actomyosin purse strings in the leading edges of epidermal cells promote closure, whereas the bulk of the lateral epidermis opposes closure. Canthi maintain purse string curvature (necessary for their dorsalward forces), and zipping at the canthi shortens leading edges, ensuring a continuous epithelium at closure completion. We investigated the requirement for intact canthi during closure with laser dissection approaches. Dissection of one or both canthi resulted in tissue recoil and flattening of each purse string. After recoil and a temporary pause, closure resumed at approximately native rates until slowing near the completion of closure. Thus the amnioserosa alone can drive closure after dissection of one or both canthi, requiring neither substantial purse string curvature nor zipping during the bulk of closure. How the embryo coordinates multiple, large forces (each of which is orders of magnitude greater than the net force) during native closure and is also resilient to multiple perturbations are key extant questions.

Triggering a cell shape change by exploiting preexisting actomyosin contractions.

Apical constriction changes cell shapes, driving critical morphogenetic events, including gastrulation in diverse organisms and neural tube closure in vertebrates. Apical constriction is thought to be triggered by contraction of apical actomyosin networks. We found that apical actomyosin contractions began before cell shape changes in both Caenorhabitis elegans and Drosophila. In C. elegans, actomyosin networks were initially dynamic, contracting and generating cortical tension without substantial shrinking of apical surfaces. Apical cell-cell contact zones and actomyosin only later moved increasingly in concert, with no detectable change in actomyosin dynamics or cortical tension. Thus, apical constriction appears to be triggered not by a change in cortical tension, but by dynamic linking of apical cell-cell contact zones to an already contractile apical cortex.

Photoactivatable green fluorescent protein-tubulin.

Direct observations of live cells expressing fluorescently tagged tubulin have led to important advances in our understanding of mitosis. A limitation of this approach is that all of the cells' microtubules are fluorescent and thus observation of the behavior of specific subsets of microtubules is precluded. To address this problem, we have tagged tubulin with a photoactivatable variant of green fluorescent protein (PA-GFP), thereby allowing one to follow the behavior of a subset of tagged molecules in the cell. Here, we describe methods to tag and express proteins with PA-GFP, locally photoactivate the recombinant protein and record the dynamic behavior of the photoactivated molecules in live cells. Use of photoactivatable proteins is a powerful approach to examine dynamic processes, including spindle formation, in diverse cells.

Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient.

Dorsal closure in Drosophila is a model system for cell sheet morphogenesis and wound healing. During closure two sheets of lateral epidermis move dorsally to close over the amnioserosa and form a continuous epidermis. Forces from the amnioserosa and actomyosin-rich, supracellular purse strings at the leading edges of these lateral epidermal sheets drive closure. Purse strings generate the largest force for closure and occur during development and wound healing throughout phylogeny. We use laser microsurgery to remove some or all of the purse strings from developing embryos. Free edges produced by surgery undergo characteristic responses as follows. Intact cells in the free edges, which previously had no purse string, recoil away from the incision and rapidly assemble new, secondary purse strings. Next, recoil slows, then pauses at a turning point. Following a brief delay, closure resumes and is powered to completion by the secondary purse strings. We confirm that the assembly of the secondary purse strings requires RhoA. We show that alpha-actinin alternates with nonmuscle myosin II along purse strings and requires nonmuscle myosin II for its localization. Together our data demonstrate that purse strings are renewable resources that contribute to the robust and resilient nature of closure.

Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint.

During mitosis, the motor molecule cytoplasmic dynein plays key direct and indirect roles in organizing microtubules (MTs) into a functional spindle. At this time, dynein is also recruited to kinetochores, but its role or roles at these organelles remain vague, partly because inhibiting dynein globally disrupts spindle assembly [1-4]. However, dynein can be selectively depleted from kinetochores by disruption of ZW10 [5], and recent studies with this approach conclude that kinetochore-associated dynein (KD) functions to silence the spindle-assembly checkpoint (SAC) [6]. Here we use dynein-antibody microinjection and the RNAi of ZW10 to explore the role of KD in chromosome behavior during mitosis in mammals. We find that depleting or inhibiting KD prevents the rapid poleward motion of attaching kinetochores but not kinetochore fiber (K fiber) formation. However, after kinetochores attach to the spindle, KD is required for stabilizing kinetochore MTs, which it probably does by generating tension on the kinetochore, and in its absence, chromosome congression is defective. Finally, depleting KD reduces the velocity of anaphase chromosome motion by approximately 40%, without affecting the rate of poleward MT flux. Thus, in addition to its role in silencing the SAC, KD is important for forming and stabilizing K fibers and in powering chromosome motion.

Molecular requirements for kinetochore-associated microtubule formation in mammalian cells.

In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly. In addition, microtubule formation at kinetochores has also been observed, most recently under physiological conditions in live cells. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-beta or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture.

Quantification of microtubule nucleation, growth and dynamics in wound-edge cells.

Mammalian cells develop a polarized morphology and migrate directionally into a wound in a monolayer culture. To understand how microtubules contribute to these processes, we used GFP-tubulin to measure dynamic instability and GFP-EB1, a protein that marks microtubule plus-ends, to measure microtubule growth events at the centrosome and cell periphery. Growth events at the centrosome, or nucleation, do not show directional bias, but are equivalent toward and away from the wound. Cells with two centrosomes nucleated approximately twice as many microtubules/minute as cells with one centrosome. The average number of growing microtubules per microm2 at the cell periphery is similar for leading and trailing edges and for cells containing one or two centrosomes. In contrast to microtubule growth, measurement of the parameters of microtubule dynamic instability demonstrate that microtubules in the trailing edge are more dynamic than those in the leading edge. Inhibition of Rho with C3 transferase had no detectable effect on microtubule dynamics in the leading edge, but stimulated microtubule turnover in the trailing edge. Our data demonstrate that in wound-edge cells, microtubule nucleation is non-polarized, in contrast to microtubule dynamic instability, which is highly polarized, and that factors in addition to Rho contribute to microtubule stabilization.

Centrosome maturation: measurement of microtubule nucleation throughout the cell cycle by using GFP-tagged EB1.

Understanding how cells regulate microtubule nucleation during the cell cycle has been limited by the inability to directly observe nucleation from the centrosome. To view nucleation in living cells, we imaged GFP-tagged EB1, a microtubule tip-binding protein, and determined rates of nucleation by counting the number of EB1-GFP comets emerging from the centrosome over time. Nucleation rate increased 4-fold between G(2) and prophase and continued to rise through anaphase and telophase, reaching a maximum of 7 times interphase rates. We tested several models for centrosome maturation, including gamma-tubulin recruitment and increased centrosome size. The centrosomal concentration of gamma-tubulin reached a maximum at metaphase, and centrosome size increased through anaphase, whereas nucleation remained high through telophase, implying the presence of additional regulatory processes. Injection of anti-gamma-tubulin antibodies significantly blocked nucleation during metaphase but was less effective during anaphase, suggesting that a nucleation mechanism independent of gamma-tubulin contributes to centrosome function after metaphase.

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport.

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.