Intracellular localization of samarium in the lactating mammary gland cells: ultrastructural and microanalytical study

The frequent use of some rare earths in the medical and industrial domains make us worry about their intracellular behavior into the body. Reason for which we have investigated the subcellular localization of one of these elements, the samarium, in the mammary gland of lactating female wistar rats using two very sensitive methods of observation and microanalysis, the transmission electron microscopy and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits in the lactating mammary glandular epithelial cell lysosomes of the samarium-treated rats, but no loaded lysosomes were observed in those of control rats. The microanalytical study allowed both the identification of the chemical species present in those deposits as samarium isotopes ((152) Sm(+)) and the cartography of its distribution. Our results confirm the previous ones showing that lysosomes of the glandular epithelial cells are the site of the intracellular concentration of foreign elements such as gallium. The intralysosomal deposits observed in the mammary glandular cells of the samarium-treated rats are similar in their form and density to those observed with the same element in other varieties of cells, such as liver, bone marrow, and spleen cells. Our ultrastructural and microanalytical results and those obtained in previous studies allow deducing that the intralysosomal deposits are very probably composed of an insoluble samarium phosphate salt.     

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze‐substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high‐pressure frozen and aldehyde‐fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.     

The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review

Background: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN). Objective: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy. Data sources: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015–2020 (92%). Conclusion: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.     

Impact of neurite alignment on organelle motion

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, highlighting the need for a deeper understanding of the mechanisms involved. Here, we use an experimental methodology that leads neurites of differentiated PC12 cells into either one of two configurations: a one-dimensional configuration, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a flat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion than the situation in which the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly, we found that the swelling-induced mobility change affected each of the (sub-/super-)diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might in part be controlled by the extracellular matrix.     

Live‐Cell Imaging with Water‐Soluble Aminophenoxazinone Dyes Synthesised through Laccase Biocatalysis

Aminophenoxazinone dyes with variable water solubilities were assayed for the first time in a live‐cell imaging application. Among a library of ten sulfonylated chromophores, one compound gave excellent results as an endocytic marker, showing a precise subcellular distribution. The compound was compared to four commercial vital tracers, including Lucifer Yellow. The first laccase‐mediated regioselective synthesis of a diphosphorylated 2‐aminophenoxazinone dye was also described. This compound, water‐soluble at 10^?2 M , displayed modest fluorescence properties and the ability to complex Mg²⁺ and Ca²⁺ cations, therefore giving fluorescence quenching.     

Unbiased linear property estimation for spheres,from sections exhibiting overprojection and truncation

In the biological sciences, stereological techniques are frequently used to infer changes in structural parameters (volume fraction, for example) between samples from different populations or subject to differing treatment regimes. Non-homogeneity of these parameters is virtually guaranteed, both between experimental animals and within the organ under consideration. A two-stage strategy is then desirable, the first stage involving unbiased estimation of the required parameter, separately for each experimental unit, the latter being defined as a subset of the organ for which homogeneity can reasonably be assumed. In the second stage, these point estimates are used as data inputs to a hierarchical analysis of variance, to distinguish treatment effects from variability between animals, for example. Techniques are therefore required for unbiased estimation of parameters from potentially small numbers of sample profiles. This paper derives unbiased estimates of linear properties in one special case—the sampling of spherical particles by transmission microscopy, when the section thickness is not negligible and the resulting circular profiles are subject to lower truncation. The derivation uses the general integral equation formulation of Nicholson (1970); the resulting formulae are simplified, algebraically, and their efficient computation discussed. Bias arising from variability in slice thickness is shown to be negligible in typical cases. The strategy is illustrated for data examining the effects, on the secondary lysosomes in the digestive cells, of exposure of the common mussel to hydrocarbons. Prolonged exposure, at 30 μg 1^?1 total oil-derived hydrocarbons, is seen to increase the average volume of a lysosome, and the volume fraction that lysosomes occupy, but to reduce their number.     

Red AIE Luminogens with Tunable Organelle Specific Anchoring for Live Cell Dynamic Super Resolution Imaging

Lysosomes and mitochondria play an important role in maintaining cell homeostasis. Visualizing the long-term activities of lysosomes and mitochondria on the nanometer scale in live cells is essential for further understanding their functions but remains challenging due to the limitations of existing fluorescent probes, such as aggregation-caused quenching (ACQ) effect, limited signal-to-noise ratio from fluorescence “always on” in the process of targeting organelle and poor photobleaching resistance. Herein, two efficient red-emitting aggregation-induced emission (AIE) luminogens are reported, which showed “off-on” fluorescence characteristic and specific lysosomes as well as mitochondria targeting capability. Owing to their AIE characteristics, a Stokes’ shift larger than 100 nm, good biocompatibility, and excellent photostability, the AIE luminogens have been successfully utilized for high fidelity imaging of lysosomes and mitochondria. By virtue of these two probes, stimulated emission depletion (STED) images of dynamic lysosomal fusion and mitochondrial fission with a high resolution of 65.6 nm are obtained. Furthermore, the interactions between lysosomes and mitochondria in the process of mitophagy are recorded. This study also provides practical guidance for designing specific organelle targeting probes to support live cell dynamic super-resolution imaging.     

Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma

Changes in zinc content and dysregulated zinc homeostatic mechanisms have been recognized in several solid malignancies such as prostate cancer, breast cancer, or pancreatic cancer. Moreover, it has been shown that zinc serum and/or tissue levels are altered in melanoma with varying effects on melanoma development and biology. This study was conducted to explore the effects of acute increases of intracellular zinc in a set of melanoma tissue explants obtained from clinical samples. Measurements of their zinc content showed an extant heterogeneity in total and free intracellular zinc pools associated with varying biological behavior of individual cells, e.g., autophagy levels and propensity to cell death. Use of zinc pyrithione elevated intracellular zinc in a short time frame which resulted in marked changes in mitochondrial activity and lysosomes. These alterations were accompanied by significantly enhanced autophagy flux and subsequent cell demise in the absence of typical apoptotic cell death markers. The present results show for the first time that acutely increased intracellular zinc in melanoma cells specifically enhances their autophagic activity via mitochondria and lysosomes which leads to autophagic cell death. While biologically relevant, this discovery may contribute to our understanding and exploration of zinc in relation to autophagy as a means of controlling melanoma growth and survival.     

Localization of metals in cells of pterygote insects

Insects maintain the equilibrium of their internal milieu by diffusion mechanisms, but more frequently by a substantial storage of metals in the cells of numerous organs: digestive tract, Malpighian tubules, fat body, integument, and genital organs. This storage implies the precipitation, of numerous cations (Ca, Mg, K, Mn, Fe, Zn, Cu) in a structure called the spherocrystal, which originates from the endoplasmic reticulum-Golgi complex: elements precipitate on a glycosaminoglycan nucleus in thin peripheral strata. Some spherocrystals contain exclusively mineral compounds, frequently phosphates, whereas others may contain organic compounds such as urates. In some species mineralized lysosomes store Ca, Fe, Zn, and Cu. When fed additional metals found in the environment, insects such as cockroach and ant are able to stay alive and to trap the metals (Cd or Pb, for example) in the peripheral strata of spherocrystals; the cytoplasm is not altered. It seems that these insects are able to resist exposures to high levels of toxic metals. The lysosomes are able to retain toxic heavy metals (Cd or Hg, for example) within metallothionein-like proteins.     

Tuning Cerium(IV)‐Assisted Hydrolysis of Phosphatidylcholine Liposomes under Mildly Acidic and Neutral Conditions

With the goal of designing a lysosomal phospholipase mimic, we optimized experimental variables to enhance Ce^IV‐assisted hydrolysis of phosphatidylcholine (PC) liposomes. Our best result was obtained with the chelating agent bis–tris propane (BTP). Similar to the hydrolytic enzyme, Ce^IV‐assisted hydrolysis of PC phosphate ester bonds was higher at lysosomal pH (～4.8) compared to pH 7.2. In the presence of BTP, the average cleavage yield at ～pH 4.8 and 37 °C was: 67±1 %, 5.7‐fold higher than at ～pH 7.2 and roughly equivalent to the percent of phospholipid found on the metal‐accessible exo leaflet of small liposomes. No Ce^IV precipitation was observed. When BTP was absent, there was significant turbidity, and the amount of cleavage at ～pH 4.8 (69±1 %) was 2.1‐fold higher than the yield obtained at ～pH 7.2. Our results show that BTP generates homogenous solutions of Ce^IV that hydrolyze phosphatidylcholine with enhanced selectivity for lysosomal pH.