Stephan W. Grill
Dresden University of Technology
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Featured researches published by Stephan W. Grill.
Science | 2014
Bi-Chang Chen; Wesley R. Legant; Kai Wang; Lin Shao; Daniel E. Milkie; Michael W. Davidson; Chris Janetopoulos; Xufeng S. Wu; John A. Hammer; Zhe Liu; Brian P. English; Yuko Mimori-Kiyosue; Daniel P. Romero; Alex T. Ritter; Jennifer Lippincott-Schwartz; Lillian K. Fritz-Laylin; R. Dyche Mullins; Diana M. Mitchell; Joshua N. Bembenek; Anne-Cécile Reymann; Ralph Böhme; Stephan W. Grill; Jennifer T. Wang; Geraldine Seydoux; U. Serdar Tulu; Daniel P. Kiehart; Eric Betzig
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.
Nature | 2001
Stephan W. Grill; Pierre Gönczy; Ernst H. K. Stelzer; Anthony A. Hyman
Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior–posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.
Cell | 2003
Matthew Kirkham; Thomas Müller-Reichert; Karen Oegema; Stephan W. Grill; Anthony A. Hyman
Centrosomes consist of a centriole pair surrounded by pericentriolar material (PCM). Previous work suggested that centrioles are required to organize PCM to form a structurally stable organelle. Here, we characterize SAS-4, a centriole component in Caenorhabditis elegans. Like tubulin, SAS-4 is incorporated into centrioles during their duplication and remains stably associated thereafter. In the absence of SAS-4, centriole duplication fails. Partial depletion of SAS-4 results in structurally defective centrioles that contain reduced levels of SAS-4 and organize proportionally less PCM. Thus, SAS-4 is a centriole-associated component whose amount dictates centrosome size. These results provide novel insight into the poorly understood role of centrioles as centrosomal organizers.
Nature | 2010
Mirjam Mayer; Martin Depken; Justin S. Bois; Frank Jülicher; Stephan W. Grill
Asymmetric cell divisions are essential for the development of multicellular organisms. To proceed, they require an initially symmetric cell to polarize. In Caenorhabditis elegans zygotes, anteroposterior polarization is facilitated by a large-scale flow of the actomyosin cortex, which directs the asymmetry of the first mitotic division. Cortical flows appear in many contexts of development, but their underlying forces and physical principles remain poorly understood. How actomyosin contractility and cortical tension interact to generate large-scale flow is unclear. Here we report on the subcellular distribution of cortical tension in the polarizing C. elegans zygote, which we determined using position- and direction-sensitive laser ablation. We demonstrate that cortical flow is associated with anisotropies in cortical tension and is not driven by gradients in cortical tension, which contradicts previous proposals. These experiments, in conjunction with a theoretical description of active cortical mechanics, identify two prerequisites for large-scale cortical flow: a gradient in actomyosin contractility to drive flow and a sufficiently large viscosity of the cortex to allow flow to be long-ranged. We thus reveal the physical requirements of large-scale intracellular cortical flow that ensure the efficient polarization of the C. elegans zygote.
Nature | 2007
Eric A. Galburt; Stephan W. Grill; Anna Wiedmann; Lucyna Lubkowska; Jason Choy; Eva Nogales; Mikhail Kashlev; Carlos Bustamante
RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 ± 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t-3/2 power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 ± 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.
Science | 2012
Martin Behrndt; Guillaume Salbreux; Pedro Campinho; Robert Hauschild; Felix Oswald; Julia Roensch; Stephan W. Grill; Carl-Philipp Heisenberg
Embryonic Cell Sorting and Movement Differential cell adhesion has long been thought to drive cell sorting. Maître et al. (p. 253, published online 23 August) show that cell sorting in zebrafish gastrulation is triggered by differences in the ability of cells to modulate cortex tension at cell-cell contacts, thereby controlling contact expansion. Cell adhesion functions in this process by mechanically coupling the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. In zebrafish epiboly the enveloping cell layer (EVL)—a surface epithelium formed at the animal pole of the gastrula—gradually spreads over the entire yolk cell to engulf it at the end of gastrulation. Behrndt et al. (p. 257) show that an actomyosin ring connected to the epithelial margin triggers EVL spreading both by contracting around its circumference and by generating a pulling force through resistance against retrograde actomyosin flow. Contraction of an actomyosin ring drives epithelial morphogenesis during embryonic development. Contractile actomyosin rings drive various fundamental morphogenetic processes ranging from cytokinesis to wound healing. Actomyosin rings are generally thought to function by circumferential contraction. Here, we show that the spreading of the enveloping cell layer (EVL) over the yolk cell during zebrafish gastrulation is driven by a contractile actomyosin ring. In contrast to previous suggestions, we find that this ring functions not only by circumferential contraction but also by a flow-friction mechanism. This generates a pulling force through resistance against retrograde actomyosin flow. EVL spreading proceeds normally in situations where circumferential contraction is unproductive, indicating that the flow-friction mechanism is sufficient. Thus, actomyosin rings can function in epithelial morphogenesis through a combination of cable-constriction and flow-friction mechanisms.
Science | 2011
Nathan W. Goehring; Philipp Khuc Trong; Justin S. Bois; Debanjan Chowdhury; Ernesto M. Nicola; Anthony A. Hyman; Stephan W. Grill
Patterning of Caenorhabditis elegans zygotes involves passive as well as active mechanisms. In the Caenorhabditis elegans zygote, a conserved network of partitioning-defective (PAR) polarity proteins segregates into an anterior and a posterior domain, facilitated by flows of the cortical actomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arise from transient cortical flows remain unclear. We present evidence that PAR polarity arises from coupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusion system. By inducing transient PAR segregation, advection serves as a mechanical trigger for the formation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passive advective transport in an active and flowing material may be a general mechanism for mechanochemical pattern formation in developmental systems.
Nature Reviews Molecular Cell Biology | 2011
Jonathon Howard; Stephan W. Grill; Justin S. Bois
Nearly 60 years ago, Alan Turing showed theoretically how two chemical species, termed morphogens, diffusing and reacting with each other can generate spatial patterns. Diffusion plays a crucial part in transporting chemical signals through space to establish the length scale of the pattern. When coupled to chemical reactions, mechanical processes — forces and flows generated by motor proteins — can also define length scales and provide a mechanochemical basis for morphogenesis. forces and flows generated by motor proteins — can also define length scales and provide a mechanochemical basis for morphogenesis.
Cell | 2004
Heather A. Hess; Jens-Christian Röper; Stephan W. Grill; Michael R. Koelle
Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. We found that mutating a regulator of G protein signaling, RGS-7, resulted in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galphao-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, we found that RIC-8 stimulates GTP binding by Galphao and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galphao, demonstrating that Galphao passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector.
Development | 2011
Louis C. Leung; Abigail V. Klopper; Stephan W. Grill; William A. Harris; Caren Norden
Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics – the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.