Adam T. Hammond

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. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.

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.

Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF)

Abstract. We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette, pREMI-Z, was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha, we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (pdd). Many previously described PEX genes were obtained from the mutant collection, as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry out genome-wide mutagenesis screens.

Potent effects of low levels of MHC class II-associated invariant chain on CD4+ T cell development.

Invariant chain (Ii)-negative mice exhibit defects in MHC class II assembly and transport that results in reduced levels of surface class II, altered antigen presentation, and inefficient positive selection of CD4+ T cells. Many CD4+ T cells that do mature in Ii-negative mice express a cell surface phenotype consistent with aberrant positive selection or peripheral activation. Reconstitution of these mice with low levels of either the p31 or p41 form of Ii does not restore transport of the bulk of class II or class II surface expression, but surprisingly does restore positive selection as measured by numbers and surface phenotype of CD4+ T cells. Thus, an Ii-dependent process, independent of effects on class II surface density, appears to be required for normal positive selection of CD4+ T cells.

LAP3, a novel plant protein required for pollen development, is essential for proper exine formation

We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in β-propeller enzymes. Insertion mutations in LAP3 lead to male sterility. To investigate possible roles for LAP3 in pollen development, we assayed the metabolite profile of anther tissues containing developing pollen grains and found that the lap3-2 defect leads to a broad range of metabolic changes. The largest changes were seen in levels of a straight-chain hydrocarbon nonacosane and in naringenin chalcone, an obligate compound in the flavonoid biosynthesis pathway.

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. Here, we used an experimental strategy that allowed us to differentiate different pollen populations in mixed pollinations to ask: (1) What pollen performance traits differed between Col and Ler accessions that direct nonrandom mating? (2) Is there evidence of interference competition? METHODS We used genetically marked pollen that can be visualized colorimetrically to quantify pollen performance of single populations of pollen in mixed pollinations. We used this and other assays to measure pollen viability, germination, tube growth, patterns of fertilization, and seed abortion. Finally, we assessed interference competition. RESULTS In mixed pollinations on Col pistils, Col pollen sired significantly more seeds than Ler pollen. Col pollen displayed higher pollen viability, faster and greater pollen germination, and faster pollen tube growth. We saw no evidence of nonrandom seed abortion. Finally, we found interference competition occurs in mixed pollinations. CONCLUSION The lack of differences in postzygotic processes coupled with direct observation of pollen performance traits indicates that nonrandom mating in Arabidopsis thaliana is prezygotic, due mostly to differential pollen germination and pollen tube growth rates. Finally, this study unambiguously demonstrates the existence of interference competition.

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. The crosslinking reaction stabilizes cellular structures during subsequent incubation and wash steps, usually without compromising antigenicity. This method reproducibly yields high-quality images of endomembrane compartments and cytoskeletal elements.

Raising the Speed Limits for 4D Fluorescence Microscopy: Raising the Speed Limits

Three‐dimensional time‐lapse (4D) fluorescence microscopy is becoming a routine experimental tool. This article summarizes current technologies, and describes a new method for speeding image acquisition during 4D confocal microscopy.

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. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.