Subcellular Imaging by TEM and Immunolabeling

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

We have used hyperbaric freezing, freeze-substitution, electron microscopy, and immunogold labeling to address several fundamental cell biological processes including chemotaxis, adhesion, motility, and macroautophagy. High-pressure freezing and immunolabeling were used to investigate chemotactic signal amplification in Dictyostelium discoideum cells. We found that the asymmetrical distribution of adenylyl cyclase (ACA) at the back of Dictyostelium discoideum cells, which is an essential determinant of their ability to migrate in a head-to-tail fashion, requires vesicular trafficking. This trafficking results in a local accumulation of ACA-containing intracellular vesicles and involves intact actin, microtubule networks, and de novo protein synthesis. We have also shown that migrating cells leave behind ACA-containing vesicles, likely secreted as multivesicular bodies and presumably involved in the formation of head-to-tail arrays of migrating cells. It is proposed that similar compartmentalization and shedding mechanisms exist in mammalian cells during embryogenesis, wound healing, neuron growth, and metastasis. Electron micrographs revealed cells with a polarized ultrastructure, with a centrally located nucleus, and various types of vesicles concentrated toward the back of the cell. Immunogold labeling visualized the distribution of ACA. Gold labeling was particularly dense at the plasma membrane and in extracellular as well as intracellular vesicles. Quantification of gold particles showed seven-fold enrichment at the plasma membrane relative to the nuclear membrane. Intracellularly, the gold labeling was particularly dense in three specific locations: on highly tubular, membranous structures that cluster around the centriole and on multivesicular bodies. The tubular structures are believed to be part of the biosynthetic pathway that feeds into the vesicular pool of the ACA. Ultrastructural electron microscopy studies were also performed to investigate the role of integrins in cell-cell adhesion and cell motility. Integrins are heterodimeric cell adhesion receptors consisting of an alpha- and a beta-subunit. Integrins not only play an important role in cell-extracellular matrix (ECM) adhesion or cell-cell adhesion but also mediate signal transduction in a bidirectional mode by relaying extracellular signals into cells or receiving intracellular signals to modulate cell-ECM or cell-cell adhesion and cell motility. The maturation of beta-1 is critical for the trafficking of this protein to the cell surface and for the activity of beta-1 integrins in binding to ECM proteins. We have identified the alkaline ceramidase 2 in the Golgi complex of HeLa cells as a regulator of beta-1 maturation through control of the sphingosine generation. Another process, macroautophagy, which is an important response to multiple cellular stresses, was also investigated. Macroautophagy has been reported to induce cell death in some cell types when subjected to anti-cancer therapies, but it may provide a survival mechanism in other cells. For example, it is found that inhibiting activity of the heat shock protein Hsp90 can induce macroautophagy in human cancer cell lines and that autophagy is the survival pathway of those cells when treated with Hsp90 inhibitors. Ultrathin sections of HeLa cells were examined by transmission electron microscopy at an accelerating voltage 120 kV. Electron micrographs revealed the formation of double membrane vesicle structures attributable to autophagosomes in cells treated with Hsp90 inhibitors. Experiments are currently being performed to compare conventional immuno-labeling with the Tokuyasu method, whereby antigenicity is better preserved by immuno-labeling thawed cryosections of frozen sucrose-embedded cells and tissues to study cellular processes.