Albinism remains a major problem in cereal improvement programs that rely on doubled haploid (DH) technology, and the factors controlling the phenomenon are not well understood. Here we report on the positive influence of copper on the production of DH plants obtained through microspore embryogenesis (ME) in recalcitrant cultivars of barley (Hordeum vulgare L.). The presence of copper sulphate in the anther pre-treatment medium improved green DH plant regeneration from cultivars known to produce exclusively albino plants using classical procedures. In plastids, the effect of copper was characterized by a decrease in starch and a parallel increase in internal membranes. The addition of copper sulphate in the ME pre-treatment medium should enable breeders to exploit the genetic diversity of recalcitrant cultivars through DH technology. We examined programmed cell death (PCD) during microspore development to determine whether PCD may interfere with the induction of ME and/or the occurrence of albinism. By examining the fate of nuclei in various anther cell layers, we demonstrated that the kinetics of PCD in anthers differed between the barley cultivars Igri and Cork that show a low and a high rate of albinism, respectively. However, no direct correlation between PCD in the anther cell layers and the rate of albinism was observed and copper had no influence on the PCD kinetic in these cultivars. It was concluded that albinism following ME was not due to PCD in anthers, but rather to another unknown phenomenon that appears to specifically affect plastids during microspore/pollen development.
The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128-462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.
Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na+ and Cl- contents of the mitochondria and a strong decrease in mitochondrial K+ content. During the first step in apoptotic volume decrease (AVD), Na+ and Cl- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na+ and Cl- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K+ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K+ contents similar to those of cells entering apoptosis but lower Na+ and Cl- contents than both cells entering apoptosis and control cells.
Rationale: Numerous chemotherapeutic drugs that affect ribosome biogenesis in the nucleolus induce nucleolar stress. Improving our understanding of the effects of these drugs will require uncovering and comparing their impact on several biophysical parameters of the major cell compartments. Here, we quantified the water content and dry mass of cancerous cells treated with CX-5461, DRB or DAM to calculate macromolecular crowding and the volume occupied by free water, as well as elemental content. Methods: HeLa-H2B-GFP cells were treated with CX-5461, DRB or DAM. Water content and dry mass were measured in numerous regions of interest of ultrathin cryo-sections by quantitative scanning transmission electron microscope dark-field imaging and the elements quantified by energy dispersive X-ray spectrometry. The data were used to calculate macromolecular crowding and the volume occupied by free water in all cell compartments of control and treated cells. Hydrophobic and unfolded proteins were revealed by 8-Anilinonaphtalene-1-sulfonic acid (ANS) staining and imaging by two-photon microscopy. Immunolabeling of UBF, pNBS1 and pNF-κB was carried out and the images acquired with a confocal microscope for 3D imaging to address whether the localization of these proteins changes in treated cells. Results: Treatment with CX-5461, DRB or DAM induced completely different changes in macromolecular crowding and elemental content. Macromolecular crowding and elemental content were much higher in CX-5461-treated, moderately higher in DRB-treated, and much lower in DAM-treated cells than control cells. None of the drugs alone induced nucleolar ANS staining but it was induced by heat-shock of control cells and cells previously treated with DAM. UBF and pNBS1 were systematically co-localized in the nucleolus of CX-5461-and DAM-treated cells. pNF-κB only localized to the nucleolar caps of pre-apoptotic DAM-treated cells. Conclusion: We directly quantified water and ion content in cell compartments using cryo-correlative electron microscopy. We show that different chemotherapeutic nucleolar stress inducers result in distinctive, thus far-unrecognized changes in macromolecular crowding and elemental content which are known to modify cell metabolism. Moreover we were able to correlate these changes to the sensitivity of treated cells to heat-shock and the behavior of nucleolar pNBS1 and pNF-κB.
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