Mark H. Ellisman

Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.

Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominantly on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only approximately 15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.