A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Chloroplast division is driven by a macromolecular complex that assembles at the midplastid. The FtsZ ring (Z ring) is the central structure in this complex, and is composed of the functionally distinct cytoskeletal proteins FtsZ1 and FtsZ2. Recent studies in the heterologous Schizosaccharomyces pombe system showed that Arabidopsis FtsZ1 and FtsZ2 filaments have distinct assembly and turnover characteristics. To further analyze these FtsZs, we employed this system to compare the assembly and dynamic properties of FtsZ1 and FtsZ2 lacking their N- and/or C-termini with those of their full-length counterparts. Our data provide evidence that the N-terminus of FtsZ2 is critical for its structural dominance over FtsZ1, and that the N- and C-termini promote polymer bundling and turnover of both FtsZs and contribute to their distinct behaviors. We also assessed how ARC6 affects FtsZ2 filament dynamics, and found that it interacts with and stabilizes FtsZ2 filaments in S. pombe independent of its presumed Z-ring tethering function in planta. Finally, we generated FtsZ1-FtsZ2 coexpression constructs to facilitate reconstitution of more complex interaction networks. Our experiments yield new insight into factors influencing FtsZ behavior and highlight the utility of S. pombe for analyzing chloroplast FtsZs and their assembly regulators.

All mammalian-parasitic stages of a range of nematode species investigated (Brugia pahangi, Acanthocheilonema viteae, Strongyloides ratti, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi) labelled in a surface-restricted manner with the fluorescent lipid analogues 5-N-(octadecanoyl)aminofluorescein (AF18) or nitrobenzoxadiazole-cholesterol (NBD-chol), but failed to bind other similar probes. In contrast, the surfaces of the ‘pre-parasitic’ infective stages of these species had affinity for neither AF18 nor NBD-chol. This exclusion of lipid analogues changed rapidly upon exposure of the larvae to tissue culture conditions which mimic the mammalian tissue environment (e.g. RPMI 1640/37°C) such that the above probes could then insert into the surface layer of the larvae. The dauer larva of Caenorhabditis elegans also excluded the probes, but became permissive to labelling upon stimulation to emerge from the dauer state. The time taken for the surface transformation to occur ranged from less than 10 min in the vector-borne parasites to approximately 5 h in those which enter by the oral route, with direct skin-penetrators occupying an intermediate position. In all cases, the alteration proceeded too rapidly for it to have been associated with a moult. Fluorescence Recovery After Photobleaching (FRAP) studies of A. viteae larvae showed that approximately 50% of the AF18 probe was free to diffuse within the plane of the surface immediately after transformation. This is only a transitory state because AF18 was found to be highly restricted in its lateral diffusion on the surface of adult parasites. In the larvae of S. ratti, the change in affinity for AF18 was accompanied by the rapid shedding of an otherwise stable surface coat of polyanionic material, here visualized by labelling with fluorescein-conjugated cationized ferritin. Incubation of larvae in lipid-rich host serum during the induction of transformation inhibited subsequent labelling with AF 18. This possibly reflects competition for insertion sites and an in vivo propensity towards the acquisition of host lipid by invading parasites.