Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals.

Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components.

Effects of tick saliva on the migratory and invasive activity of Saos-2 osteosarcoma and MDA-MB-231 breast cancer cells.

In previous studies we showed that tick saliva modulates the migratory activity of cells involved in the wound healing response. Since cell migration is a prerequisite for tumor invasion and metastasis, we examined the effects of tick saliva on the migratory and invasive activity of Saos-2 osteosarcoma and MDA-MB-231 (MB-231) breast cancer cells and the potential signaling pathways that may be affected. Saliva inhibited basal and agonist-induced Saos-2 and MB-231 migration and invasion through a matrigel-coated filter. In the Saos-2 cells, saliva suppressed epidermal growth factor (EGF)-activation of Akt/Protein Kinase B, however, only basal extracellular signal-regulated kinase (ERK) activity was affected in MB-231 cells. EGF receptor (EGFR) overexpression masked the effect of saliva on MB-231 cells, but its ability to inhibit MB-231 migration was enhanced by the EGFR inhibitor PD 168393 and MEK inhibitor U0126. Our data indicate that the mechanisms ticks have evolved to regulate the wound healing response have generalized effects on the migratory and invasive activities of metastatic cancer cells.