Junctions of polytene chromosomes and the inner nuclear membrane

Abstract

The results of the scanning electron microscopy are summarised in the table. Ectoderm ceils from the hydroxyureatreated embryos were different from ectoderm cells from normal embryos, since after 3 rain in culture they showed no filopodia (figure 3). After 0.5 h in culture the cells remained featureless and had not flattened onto the glass substrate, and after 3 h the cells had not formed pseudopodia. Endoderm cells from the hydroxyurea-treated embryos were also different from endoderm cells from normal embryos, since they were featureless at all times of culture and did not show pseudopodia at 3 h. Since it was possible that cells were featureless because they were moribund, cell viability was determined by trypan blue exclusion. Cells were prepared as for electron microscopy, and after 3 rain and 3 h of culture they were exposed to 1% trypan blue in dissociation medium for 10 rain. The results confirmed that at least 87%, and usually 95%, of the cells from both control embryos and hydroxyurea-treated embryos remained viable. Thus cells from embryos which were prevented by hydroxyurea treatment from undergoing normal morphogenesis did not show the features and changes in vitro shown by cells from normal embryos. 3. Cells cultured in hydroxyurea: An additional experiment was performed to determine whether hydroxyurea had any direct effect on the appearance of Xenopus embryonic cells.Ectoderm cells were dissected from normal embryos, and dissociated and cultured in the presence of 10 -2 M hydroxyurea. Cells after 3 rain in culture with hydroxyurea (that had been exposed to hydroxyurea during dissociation) were seen to be different from cells from both normal and hydroxyurea-treated embryos -many ectoderm cells showed smooth 'blebs' or 'bulges' (figure 4). After 0.5 h in culture in the presence of hydroxyurea many of the cells were featureless, and a few still showed 'blebs'; after 3 h more of the cells were featureless (table). This finding might suggest that hydroxyurea has 2 effects on cells from Xenopus early embryos; a more immediate effect which produces 'blebs', followed by a longer-term effect which prevents the formation of surface features such as filopodia and pseudopodia. These results suggest that cells from embryos which are prevented from undergoing normal morphogenesis at gastrulation show a deficiency in their properties in vitro, and are compatible with the idea that the study of cells isolated from early embryos and cultured in vitro may increase our understanding of the mechanisms and controls of morphogenesis.1 Acknowledgments. We wish to thank Mr C. Veltkamp for his help with the electron microscopy, and Mrs J. Clumpus for technical assistance.Summary. Polytene chromosomes in salivary gland nuclei from chironomid larvae of defined stages are connected along their entire length, by some 100 chromatin fibers, to the inner nuclear membrane.Chromatin forms structural attachments on the inside of the nuclear envelope, which may serve to hold the interphase chromosomes in fixed positions relative to...

Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster.

“…However, several regions that are associated with the NE in the embryo, such as 23A, 34A, and 87B, are not in particularly frequent contact with the NE in polytene nuclei (Hochstrasser et al, 1986), suggesting that these associations may be lost during polytenization. This is consistent with studies of NE-chromosome contact in polytene chromosomes of Chironomus and Acricotopus (Quick, 1980) in which a progressive loss of NE contact was found to accompany polytenization.…”

“…Associations between chromatin and the nuclear envelope (NE) have been observed cytologically for many years (DuPraw, 1965;Murray and Davies, 1979;Quick, 1980;Hochstrasser et al, 1986;Loidl, 1990;Paddy et al, 1990;Belmont et al, 1993). It has been proposed that these chromatin-NE interactions may play a role in a variety of processes, including organization of the interphase nucleus (Comings, 1980), gene regulation (Blobel, 1985;Hutchison and Weintraub, i 1996 by The American Society for Cell Biology 1985; Palladino et al 1993), chromatin condensation , nuclear reassembly (reviewed in Wiese and Wilson, 1993), and meiotic homologue pairing (reviewed in Loidl, 1990).…”

Gene expression and nuclear architecture during development and differentiation

“…There is, however, substantial evidence that NE localization does in fact involve an actual physical interaction between chromatin and the NE. This has been most clearly demonstrated for polytene chromosomes, whose large size has allowed a variety of mechanical approaches to be applied in order to show that the chromosomes really are physically adhering to the inner face of the NE at discrete sites (Quick, 1980;Hill and Whytock, 1993;Skaer et al, 1976). For interphase chromatin in non-polytene cells, direct mechanical manipulation is not feasible, but passive observations of chromatin motion in vivo have clearly indicated that NE-associated loci are more highly constrained that internally located loci, suggesting that it is the NE localized sites that are physically tethered and the interior sites that are forced into their position by a process of physical exclusion (Heun et al, 2001;Chubb et al, 2002).…”

Order and Disorder in the Nucleus

“…ability of polytene chromosomes to interact with the nuclear envelope was in several ways: by visualizing thin fibers running from chromosome bands to the nuclear envelope [34]; by microdissection experiments in which chromosomes pulled out of ruptured nuclei stuck tenaciously to the nuclear envelope remnant [35]; and by electrophoresis of chromosomes inside living cells [36]. More recently, direct analysis of chromatin motion has shown that nuclear envelopeassociated loci are more highly constrained, providing direct evidence that they are physically tethered to the envelope during interphase [29,30].…”