U. Serdar Tulu

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

Introduction In vivo imaging provides a window into the spatially complex, rapidly evolving physiology of the cell that structural imaging alone cannot. However, observing this physiology directly involves inevitable tradeoffs of spatial resolution, temporal resolution, and phototoxicity. This is especially true when imaging in three dimensions, which is essential to obtain a complete picture of many dynamic subcellular processes. Although traditional in vivo imaging tools, such as widefield and confocal microscopy, and newer ones, such as light-sheet microscopy, can image in three dimensions, they sacrifice substantial spatiotemporal resolution to do so and, even then, can often be used for only very limited durations before altering the physiological state of the specimen. Lattice light-sheet microscopy. An ultrathin structured light sheet (blue-green, center) excites fluorescence (orange) in successive planes as it sweeps through a specimen (gray) to generate a 3D image. The speed, noninvasiveness, and high spatial resolution of this approach make it a promising tool for in vivo 3D imaging of fast dynamic processes in cells and embryos, as shown here in five surrounding examples. Lattice light-sheet microscopy. An ultrathin structured light sheet (blue-green, center) excites fluorescence (orange) in successive planes as it sweeps through a specimen (gray) to generate a 3D image. The speed, noninvasiveness, and high spatial resolution of this approach make it a promising tool for in vivo 3D imaging of fast dynamic processes in cells and embryos, as shown here in five surrounding examples. Rationale To address these limitations, we developed a new microscope using ultrathin light sheets derived from two-dimensional (2D) optical lattices. These are scanned plane-by-plane through the specimen to generate a 3D image. The thinness of the sheet leads to high axial resolution and negligible photobleaching and background outside of the focal plane, while its simultaneous illumination of the entire field of view permits imaging at hundreds of planes per second even at extremely low peak excitation intensities. By implementing either superresolution structured illumination or by dithering the lattice to create a uniform light sheet, we imaged cells and small embryos in three dimensions, often at subsecond intervals, for hundreds to thousands of time points at the diffraction limit and beyond. Results We demonstrated the technique on 20 different biological processes spanning four orders of magnitude in space and time, including the binding kinetics of single Sox2 transcription factor molecules, 3D superresolution photoactivated localization microscopy of nuclear lamins, dynamic organelle rearrangements and 3D tracking of microtubule plus ends during mitosis, neutrophil motility in a collagen mesh, and subcellular protein localization and dynamics during embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. Throughout, we established the performance advantages of lattice light-sheet microscopy compared with previous techniques and highlighted phenomena that, when seen at increased spatiotemporal detail, may hint at previously unknown biological mechanisms. Conclusion Photobleaching and phototoxicity are typically reduced by one to two orders of magnitude relative to that seen with a 1D scanned Bessel beam or the point array scanned excitation of spinning disk confocal microscopy. This suggests that the instantaneous peak power delivered to the specimen may be an even more important metric of cell health than the total photon dose and should enable extended 3D observation of endogenous levels of even sparsely expressed proteins produced by genome editing. Improvements of similar magnitude in imaging speed and a twofold gain in axial resolution relative to confocal microscopy yield 4D spatiotemporal resolution high enough to follow fast, nanoscale dynamic processes that would otherwise be obscured by poor resolution along one or more axes of spacetime. Last, the negligible background makes lattice light-sheet microscopy a promising platform for the extension of all methods of superresolution to larger and more densely fluorescent specimens and enables the study of signaling, transport, and stochastic self-assembly in complex environments with single-molecule sensitivity. From single molecules to embryos in living color Animation defines life, and the three-dimensional (3D) imaging of dynamic biological processes occurring within living specimens is essential to understand life. However, in vivo imaging, especially in 3D, involves inevitable tradeoffs of resolution, speed, and phototoxicity. Chen et al. describe a microscope that can address these concerns. They used a class of nondiffracting beams, known as 2D optical lattices, which spread the excitation energy across the entire field of view while simultaneously eliminating out-of-focus excitation. Lattice light sheets increase the speed of image acquisition and reduce phototoxicity, which expands the range of biological problems that can be investigated. The authors illustrate the power of their approach using 20 distinct biological systems ranging from single-molecule binding kinetics to cell migration and division, immunology, and embryonic development. Science, this issue 10.1126/science.1257998 A new microscope allows three-dimensional imaging of living systems at very high resolution in real time. 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.

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.

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.

Triggering a Cell Shape Change by Exploiting Preexisting Actomyosin Contractions

A Time and a Place The onset of morphogenetic cell shape changes is thought to be triggered by initiation of actomyosin contractions. Roh-Johnson et al. (p. 1232, published online 9 February; see the Perspective by Razzell and Martin) have now discovered in both Caenorhabditis elegans and Drosophila embryos that the actomyosin contractions driving morphogenesis run constitutively, only being engaged to trigger cell shape changes at a specific time during development. Morphogenesis in developing worms and flies harnesses ongoing cortical motility. 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.

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–α-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.

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 μm2 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.

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 α‐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.

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

Laser microsurgery and computer tracking of embryo structures indicate that the morphogenetic process of Drosophila dorsal closure requires only forces generated by the amnioserosa tissue. Forces generated by both “zipping” of epidermal tissue at the canthi corners and the resulting actomyosin purse string curvature are not necessary for closure.

Stable expression of fluorescently tagged proteins for studies of mitosis in mammalian cells.

Stable expression of fluorescently tagged proteins for studies of mitosis in mammalian cells

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.