Analysis of cytokinesis by electron microscopy.

Following up on a chapter on the Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis (MCB 79, 101-119), we present an adaptation of our established protocol for the ultrastructural analysis of either permeabilized or injected embryonic systems. We prepared both drug-treated early C. elegans embryos and fluorescently labeled sea urchin embryos of Lytechinus pictus for ultrastructural studies on animal cytokinesis. Here we focus on the initial preparation steps of postmitotic embryos for high-pressure freezing and subsequent electron microscopy with an emphasis on electron tomography. The advantages and limitations of our extended protocol will be discussed.

A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia.

Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, we identified DCX-EMAP, a unique member of the EMAP family (echinoderm-microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. Our results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification.

Electron microscopy of the early Caenorhabditis elegans embryo.

The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser-induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time-resolved fixation to arrest development at specific stages. The second method uses high-pressure freezing of whole worms followed by freeze-substitution (HPF-FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high-resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF-FS and electron tomography. This procedure combines the advantages of time-resolved fixation and superior ultrastructural preservation by high-pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.

Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure.

Caenorhabditis elegans is one of the most important genetic systems used in current biological research. Increasingly, these genetics-based research projects are including ultrastructural analyses in their attempts to understand the molecular basis for cell function. Here, we present and review state-of-the-art methods for both ultrastructural analysis and immunogold localization in C. elegans. For the initial cryofixation, high-pressure freezing is the method of choice, and in this article we describe two different strategies to prepare these nematode worms for rapid freezing. The first method takes advantage of transparent, porous cellulose capillary tubes to contain the worms, and the second packs the worms in E. coli and/or yeast paste prior to freezing. The latter method facilitates embedding of C. elegans in a thin layer of resin so individual worms can be staged, selected and precisely orientated for serial sectioning followed by immunolabelling or electron tomography.

Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex.

Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.

Structural changes at microtubule ends accompanying GTP hydrolysis: information from a slowly hydrolyzable analogue of GTP, guanylyl (alpha,beta)methylenediphosphonate.

Microtubules are dynamic polymers that interconvert between periods of slow growth and fast shrinkage. The energy driving this nonequilibrium behavior comes from the hydrolysis of GTP, which is required to destabilize the microtubule lattice. To understand the mechanism of this destabilization, cryo-electron microscopy was used to compare the structure of the ends of shrinking microtubules assembled in the presence of either GTP or the slowly hydrolyzable analogue guanylyl (alpha,beta)methylenediphosphonate (GMPCPP). Depolymerization was induced by cold or addition of calcium. With either nucleotide, we have observed curled oligomers at the ends of shrinking microtubules. However, GDP oligomers were consistently more curved than GMPCPP oligomers. This difference in curvature between depolymerizing GDP and GMPCPP protofilaments suggests that GTP hydrolysis is accompanied by an increase in curvature of the protofilaments, thereby destabilizing the lateral interactions between tubulin subunits in the microtubule lattice.

STM of metal embedded and coated DNA and DNA-protein complexes.

Bare and Pt/Ir/C-coated DNA has been analysed using scanning tunnelling microscopy (STM). To achieve reproducible imaging of bare DNA on mica ethanol/air-dried molecules were embedded in Pt/C. By peeling the metal film off the mica, the previously mica-exposed side of the Pt/C-film with the embedded DNA molecules was accessible for STM analysis. By applying this replica/anchoring technique only hollow trenches in the metal film, and not the DNA itself, could be visualized. The gaps averaged 3.1 nm (+/- 0.9 nm) wide and 1 nm (+/- 0.5 nm) deep. Using scanning force microscopy it could be confirmed that the DNA remained in the Pt/C film during the peel-off procedure. For STM, DNA fragments were also coated with 0.7-1 nm Pt/Ir/C. Owing to the high Z-resolution the STM samples were coated at a high elevation angle (65 degrees), thereby minimizing the problem of self-shadowing. Coating by Pt/Ir/C allowed routine imaging and quantitative analysis of both ethanol/air- and freeze-dried DNA under atmospheric conditions. After ethanol/air drying measured values for DNA width and height were 5.1 nm (+/- 1.8 nm) and 0.9 nm (+/- 0.2 nm), respectively. Freeze-dried DNA averaged 4.2 nm (+/- 1.3 nm) wide and 1.1 nm (+/- 0.1 nm) high. A Pt/Ir/C-coating was also applied to visualize DNA-protein interaction using STM.

Microscopic analysis of DNA and DNA-protein assembly by transmission electron microscopy, scanning tunneling microscopy and scanning force microscopy.

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA supercoiling were analyzed using electron microscopy. In parallel, this Mg2+/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or in liquid by scanning force microscopy. An advantage of such a correlative approach is that parallel imaging can reveal complementary information. The benefit of such a combined approach in analysis of protein-induced DNA bending is discussed.