Plasmodium falciparum parasites exit the infected erythrocyte after haemolysis with saponin and streptolysin O.

Malaria is caused by unicellular parasites of the genus Plasmodium, which reside in erythrocytes during the clinically relevant stage of infection. To separate parasite from host cell material, haemolytic agents such as saponin are widely used. Previous electron microscopy studies on saponin-treated parasites reported both, parasites enclosed by the erythrocyte membrane and liberated from the host cell. These ambiguous reports prompted us to investigate haemolysis by live-cell time-lapse microscopy. Using either saponin or streptolysin O to lyse Plasmodium falciparum-infected erythrocytes, we found that ring-stage parasites efficiently exit the erythrocyte upon haemolysis. For late-stage parasites, we found that only approximately half were freed, supporting the previous electron microscopy studies. Immunofluorescence imaging indicated that freed parasites were surrounded by the parasitophorous vacuolar membrane. These results may be of interest for future work using haemolytic agents to enrich for parasite material.

Nanopore unitary permeability measured by electrochemical and optical single transporter recording.

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.

The mobility of phytochrome within protonemal tip cells of the moss Ceratodon purpureus, monitored by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a versatile tool for investigating the mobilities of fluorescent molecules in cells. In this article, we show that it is possible to distinguish between freely diffusing and membrane-bound forms of biomolecules involved in signal transduction in living cells. Fluorescence correlation spectroscopy was used to measure the mobility of phytochrome, which plays a role in phototropism and polarotropism in protonemal tip cells of the moss Ceratodon purpureus. The phytochrome was loaded with phycoerythrobilin, which is fluorescent only in the phytochrome-bound state. Confocal laser scanning microscopy was used for imaging and selecting the xy measuring position in the apical zone of the tip cell. Fluorescence correlation was measured at ancient z-positions in the cell. Analysis of the diffusion coefficients by nonlinear least-square fits showed a subcellular fraction of phytochrome at the cell periphery with a sixfold higher diffusion coefficient than in the core fraction. This phytochrome is apparently bound to the membrane and probably controls the phototropic and polarotropic response.