As Clear as a Cell: Researchers See Deeper into Tissue than Ever Before

Researchers have long sought to visualize neural projections and other features of animal brains by imaging them using light microscopes. Connectomics studies strive to elucidate the connections among nerve cells, but brain is dense, and gray matter is tremendously light-scattering. So scientists have not been able to peer as deeply into brain tissue as is necessary to view its complex connections and structures without cutting brain tissue into thin sections. The slicing causes damage that makes it even more difficult to reconstruct exactly how nervous system structures are interconnected. Issues with light scattering have also limited studies of microvasculature, developmental structures, and other features in mouse embryos and other tissues. Traditional methods of retrograde or anterograde labeling have been able to provide high specificity of connection circuitry, but at the price of losing the organizational context of the network. In addition, there is a limit to the practical number of labels that can be used at once. Even multicolor methods have required serial section reconstruction.

Structure of the Golgi and Distribution of Reporter Molecules at 20°C Reveals the Complexity of the Exit Compartments

Incubating cells at 20°C blocks transport out of the Golgi complex and amplifies the exit compartments. We have used the 20°C block, followed by EM tomography and serial section reconstruction, to study the structure of Golgi exit sites in NRK cells. The dominant feature of Golgi structure in temperature-blocked cells is the presence of large bulging domains on the three trans-most cisternae. These domains extend laterally from the stack and are continuous with “cisternal” domains that maintain normal thickness and alignment with the other stacked Golgi cisternae. The bulging domains do not resemble the perpendicularly extending tubules associated with the trans-cisternae of control cells. Such tubules are completely absent in temperature-blocked cells. The three cisternae with bulging domains can be identified as trans by their association with specialized ER and the presence of clathrin-coated buds on the trans-most cisterna only. Immunogold labeling and immunoblots show a significant degradation of a medial- and a trans-Golgi marker with no evidence for their redistribution within the Golgi or to other organelles. These data suggest that exit from the Golgi occurs directly from three trans-cisternae and that specialized ER plays a significant role in trans-Golgi function.

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589–601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP–Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

A transient association of gamma-tubulin at the midbody is required for the completion of cytokinesis during the mammalian cell division

gamma-Tubulin, a relatively new member of the tubulin gene family, is localized primarily at the centrosome throughout the mammalian cell cycle and may play a key role in nucleation of cellular microtubule assembly. A transient association of gamma-tubulin at the cytoplasmic bridge of telophase mammalian cells, the midbody, is recently documented. Using immunogold electron microscopy and serial section reconstruction analysis, we show here that the transiently associated midbody gamma-tubulin is localized at the minus ends of microtubules in the midbody structure. Using antisense RNA methods we also demonstrate that a selective depletion of transiently associated midbody gamma-tubulin causes an abortive cytokinesis due to a failure in the morphogenesis of the midbody structure.

Oblique section 3-D reconstruction from thin sectioned biological specimens

Three dimensional (3-D) image reconstruction of plastic embedded and sectioned tissue has become an important tool for imaging complex biological structures in situ. Most 3-D imaging methods combine multiple views of tilted specimens which leads to radiation damage and mass loss. On the other hand oblique section 3-D reconstruction (OSR) can produce a 3-D image from a single view because an oblique section through a 2- or 3-D periodic specimen contains 3-D information which can be folded into a 3-D image. The resolution achievable in an OSR depends on section thickness and is lowest in the direction perpendicular to the section plane. In OSR there is no missing cone or wedge in the 3-D image because of tilt angle restrictions, but finite section thickness limits the vertical resolution. Two OSR methodologies have been developed, one called Crystallographic Serial Section Reconstruction or CSSR, and another called Super Lattice Reconstruction or SLR.