Internalization of α-synuclein oligomers into SH-SY5Y cells.

Aggregates of misfolded α-synuclein are a distinctive feature of Parkinson's disease. Small oligomers of α-synuclein are thought to be an important neurotoxic agent, and α-synuclein aggregates exhibit prion-like behavior, propagating misfolding between cells. α-Synuclein is internalized by both passive diffusion and active uptake mechanisms, but how uptake varies with the size of the oligomer is less clear. We explored how α-synuclein internalization into live SH-SY5Y cells varied with oligomer size by comparing the uptake of fluorescently labeled monomers to that of engineered tandem dimers and tetramers. We found that these α-synuclein constructs were internalized primarily through endocytosis. Oligomer size had little effect on their internalization pathway, whether they were added individually or together. Measurements of co-localization of the α-synuclein constructs with fluorescent markers for early endosomes and lysosomes showed that most of the α-synuclein entered endocytic compartments, in which they were probably degraded. Treatment of the cells with the Pitstop inhibitor suggested that most of the oligomers were internalized by the clathrin-mediated pathway.

Preparing Maize Synaptonemal Complex Spreads and Sequential Immunofluorescence and Fluorescence In Situ Hybridization.

Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180 bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.

Distribution and Co-localization of endosome markers in cells.

Clathrin mediated endocytosis is one pathway for internalization of extracellular nano materials into cells [1, 2]. In this pathway, proteins attached to receptors and the internalized materials are encapsulated in clathrin coated membrane vesicles that subsequently fuse with or transform into intracellular compartments (early and late endosomes) as their contents are being directed to the lysosomes for degradation. The following proteins are commonly used to mark the pathway at various stages: Rab5 (early endosome), Rab7 (late endosome), and LAMP-1 (lysosome). In this work, we studied the distribution and co-localization of these marker proteins in two cell lines (C2C12 and A549) to determine whether these markers are unique for specific endosome types or whether they can co-exist with other markers. We estimate the densities and sizes of the endosomes containing the three markers, as well as the number of marker antibodies attached to each endosome. We determine that the markers are not unique to one endosome type but that the extent of co-localization is different for the two cell types. In fact, we find endosomes that contain all three markers simultaneously. Our results suggest that the use of these proteins as specific markers for specific endosome types should be reevaluated. This was the first successful use of triple image cross correlation spectroscopy to qualitatively and quantitatively study the extent of interaction among three different species in cells and also the first experimental study of three-way interactions of clathrin mediated endocytic markers.

Meiotic Crossing Over in Maize Knob Heterochromatin.

There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relationship of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence in situ hybridization of the maize 180-bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs, pachytene chromosomes). MLH1 foci correspond to the location of recombination nodules (RNs) that mark sites of crossing over. We found that MLH1 foci occur at similar frequencies per unit length of SC in interstitial knobs and in the 1 µm segments of SC in euchromatin immediately to either side of interstitial knobs. These results indicate not only that crossing over occurs within knob heterochromatin, but also that crossing over is not suppressed in the context of SC length in maize knobs. However, because there is more DNA per unit length of SC in knobs compared to euchromatin, crossing over is suppressed (but not eliminated) in knobs in the context of DNA length compared to adjacent euchromatin.

Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers.

Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. In several organisms, mutants demonstrate that two molecularly distinct pathways produce COs. One pathway produces class I COs that exhibit interference (lowered probability of nearby COs), and the other pathway produces class II COs with little or no interference. However, the relative contributions, genomic distributions, and interactions of these two pathways are essentially unknown in nonmutant organisms because marker segregation only indicates that a CO has occurred, not its class type. Here, we combine the efficiency of light microscopy for revealing cellular functions using fluorescent probes with the high resolution of electron microscopy to localize and characterize COs in the same sample of meiotic pachytene chromosomes from wild-type tomato. To our knowledge, for the first time, every CO along each chromosome can be identified by class to unveil specific characteristics of each pathway. We find that class I and II COs have different recombination profiles along chromosomes. In particular, class II COs, which represent about 18% of all COs, exhibit no interference and are disproportionately represented in pericentric heterochromatin, a feature potentially exploitable in plant breeding. Finally, our results demonstrate that the two pathways are not independent because there is interference between class I and II COs.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome.

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.