A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects.

Pollen performance traits reveal prezygotic nonrandom mating and interference competition in Arabidopsis thaliana.

Premise: The lack of ability to measure pollen performance traits in mixed pollinations has been a major hurdle in understanding the mechanisms of differential success of compatible pollen donors. In previous work, we demonstrated that nonrandom mating between two accessions of Arabidopsis thaliana, Columbia (Col) and Landsberg (Ler), is mediated by the male genotype. Despite these genetic insights, it was unclear at what stage of reproduction these genes were acting.

High-quality immunofluorescence of cultured cells.

Immunofluorescence microscopy of cultured cells often gives poor preservation of delicate structures. We have obtained dramatically improved results with a simple modification of a standard protocol. Cells growing on a coverslip are rapidly dehydrated in a cold organic solvent and then are rehydrated in a solution containing a homobifunctional crosslinker.

Lipid segregation and IgE receptor signaling: a decade of progress.

Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment. Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation.

Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells.

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT).

De novo formation of transitional ER sites and Golgi structures in Pichia pastoris.

Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo.

Paz2 and 13 other PAZ gene products regulate vacuolar engulfment of peroxisomes during micropexophagy.

Background: In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded through direct engulfment by the vacuole in a process known as micropexophagy, but the mechanism of micropexophagy is not known.

Results: To gain molecular insights into micropexophagy, we used fluorescence time-lapse microscopy, coupled with gene-tagging mutagenesis to isolate P. pastoris mutants defective in micropexophagy.