Daniel P. Romero

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.

A conserved secondary structure for telomerase RNA

The RNA moiety of the ribonucleoprotein enzyme telomerase contains the template for telomeric DNA synthesis. We present a secondary structure model for telomerase RNA, derived by a phylogenetic comparative analysis of telomerase RNAs from seven tetrahymenine ciliates. The telomerase RNA genes from Tetrahymena malaccensis, T. pyriformis, T. hyperangularis, T. pigmentosa, T. hegewishii, and Glaucoma chattoni were cloned, sequenced, and compared with the previously cloned RNA gene from T. thermophila and with each other. To define secondary structures of these RNAs, homologous complementary sequences were identified by the occurrence of covariation among putative base pairs. Although their primary sequences have diverged rapidly overall, a strikingly conserved secondary structure was identified for all these telomerase RNAs. Short regions of nucleotide conservation include a block of 22 totally conserved nucleotides that contains the telomeric templating region.

A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium.

Paramecium telomeric DNA consists largely of a random distribution of TTGGGG and TTTGGG repeats. Given the precise nature of other ciliate telomerases, it has been postulated that there are two distinct types of the Paramecium enzyme, each synthesizing perfect telomeric repeats: one with a template RNA that specifies the addition of TTTGGG and the second dictating the synthesis of TTGGGG repeats. We have cloned and sequenced telomerase RNA genes from Paramecium tetraurelia, P. primaurelia, P. multimicronucleatum, and P. caudatum. Surprisingly, a single gene encodes telomerase RNA in all four species, although an apparently nontranscribed pseudogene is also present in the genome of P. primaurelia. The overall lengths of the telomerase RNAs range between 202 and 209 nucleotides, and they can be folded into a conserved secondary structure similar to that derived for other ciliate RNAs. All Paramecium telomerase RNAs examined include a template specific for the synthesis of TTGGGG telomeric repeats, which has not been posttranscriptionally edited to account for the conventional synthesis of TTTGGG repeats. On the basis of these results, possible mechanisms for the synthesis of variable telomeric repeats by Paramecium telomerase are discussed.

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P.tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P.caudatum telomerase RNA can participate in telomere synthesis when expressed in the P.tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P.tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P.tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P.caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.

A β-Tubulin Mutation Selectively Uncouples Nuclear Division and Cytokinesis in Tetrahymena thermophila

ABSTRACT The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell—the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant β-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.

Phylogenetic relationships amongst tetrahymenine ciliates inferred by a comparison of telomerase RNAs

The phylogenetic relationships between ciliate species in the suborder Tetrahymenina, order Hymenostomata, was investigated by comparing their telomerase RNA (TER) sequences. This relatively small RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyses the synthesis of telomeric DNA. Despite a relatively rapid rate of primary sequence divergence, conserved functional and structural elements within TERs facilitate the accurate alignment of truly homologous nucleotides. The tetrahymenine phylogeny derived from distance analysis of TER sequences is largely consistent with those based on rRNA and histone sequences.

Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.

Nested Genes CDA12 and CDA13 Encode Proteins Associated with Membrane Trafficking in the Ciliate Tetrahymena thermophila

ABSTRACT We describe a novel pair of nested genes, CDA12 and CDA13, from Tetrahymena thermophila. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and their neighboring Golgi networks. The expression of antisense CDA12 RNA in transformants produces defects in cytokinesis, macronuclear segregation, and the processing of pinosomes to downstream compartments. Antisense CDA13 RNA expression produces a conjugation phenotype, resulting in the failure of mating pairs to separate, as well as failures in postconjugation cytokinesis and macronuclear fission. This study offers insight into the membrane trafficking events linking endosome and Golgi network activities, cytokinesis, and karyokinesis and the unique membrane-remodeling events that accompany conjugation in the ciliate T. thermophila. We also highlight an unusual aspect of genome organization in Tetrahymena, namely, the existence of nested, antisense genes.

Circular rDNA replicons persist in Tetrahymena thermophila transformants synthesizing GGGGTC telomeric repeats.

Site‐directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site‐directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.

The alpha subunit of initiation factor 2 is phosphorylated in vivo in the yeast Saccharomyces cerevisiae

The phosphorylation state of the alpha subunit of initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae has been determined by two-dimensional gel electrophoresis and autoradiography of lysates from cultures grown under a variety of conditions. The alpha subunit was maintained in a phosphorylated state during logarithmic growth on fermentable and nonfermentable carbon sources, during starvation for an essential amino acid, during heat shock, during stationary phase, and during sporulation. Only when cells were starved for a carbon source for 2 h in 1 M sorbitol was eIF-2 alpha isolated in the nonphosphorylated state. This is in contrast with the studies in rabbit reticulocyte lysates, in which arrested protein synthesis was correlated with a relative increase in the extent of phosphorylation of eIF-2 alpha.