Clustering and Protein Dynamics ofDrosophila melanogasterTelomeres

Abstract

Telomeres are obligatory chromosomal landmarks that demarcate the ends of linear chromosomes to distinguish them from broken ends and can also serve to organize the genome. In both budding and fission yeast, they cluster at the periphery of the nucleus, potentially to establish a compartment of silent chromatin. To gain insight into telomere organization in higher organisms, we investigated their distribution in interphase nuclei of Drosophila melanogaster. We focused on the syncytial blastoderm, an excellent developmental stage for live imaging due to the synchronous division of the nuclei at this time. We followed the EGFP-labeled telomeric protein HOAP in vivo and found that the 16 telomeres yield four to six foci per nucleus, indicative of clustering. Furthermore, we confirmed clustering in other somatic tissues. Importantly, we observed that HOAP signal intensity in the clusters increases in interphase, potentially due to loading of HOAP to newly replicated telomeres. To determine the rules governing clustering, we used in vivo imaging and fluorescence in situ hybridization to test several predictions. First, we inspected mutant embryos that develop as haploids and found that clustering is not mediated by associations between homologs. Second, we probed specifically for a telomere of novel sequence and found strong evidence against DNA sequence identity and homology as critical factors. Third, we ruled out predominance of intrachromosomal interactions by marking both ends of a chromosome. Based on these results, we propose that clustering is independent of sequence and is likely maintained by an as yet undetermined factor.

MTV, an ssDNA Protecting Complex Essential for Transposon-Based Telomere Maintenance in Drosophila

“…In live interphase cells from larval diploid tissues, GFP-Tea forms foci reminiscent of interphase localization of other telomeric proteins such as HOAP and HipHop (Fig 1H and 1I; ref [20; 21]). In live larval polytene cells from the salivary gland, GFP-Tea forms 5–6 distinct strips in the nucleus likely representing telomeres on euchromatic chromosome arms (Fig 1G).…”

“…To monitor HOAP and HipHop, we used previously characterized antibodies and a previously made gfp-cav locus, which encodes HOAP [6; 20; 21]. For Ver, we constructed a ver locus with an N-terminally tagged egfp gene using SIRT ( gfp-ver , Supplemental Materials).…”

The Deadbeat Paternal Effect of Uncapped Sperm Telomeres on Cell Cycle Progression and Chromosome Behavior in Drosophila melanogaster

“…To determine if Ddbt was telomeric, we coexpressed mCherry-Ddbt and GFP-K81 and observed identical patterns in canoe-stage nuclei ( Figure 3B). Because large K81 foci represent aggregated telomeres (Wesolowska et al 2013), we selected the smallest focus in each spermatid nucleus for a precise comparison of signal location (Figure 3, B and C). Analysis of the smallest foci of mCherry-Ddbt and GFP-K81 from spermatid nuclei yielded a Pearson correlation coefficient of 0.92 6 0.04 (n = 100 nuclei).…”

Protection ofDrosophilachromosome ends through minimal telomere capping

“…To confirm our observation, we quantified HOAP signals in spermatid nuclei. We and others have previously observed that telomeres cluster in spermatid nuclei and in various somatic cell types (Dubruille et al, 2010;Wesolowska et al, 2013). We thus first evaluated the clustering in spermatid nuclei of 5′K81-GFP::hiphop flies by counting the number of foci in individual nuclei.…”