Single Cellular Organelles Research Articles

Mitochondrial Dysfunction: A Prelude to Neuropathogenesis of SARS-CoV-2.

The SARS-CoV-2 virus is notorious for its neuroinvasive capability, causing multiple neurological conditions. The neuropathology of SARS-CoV-2 is increasingly attributed to mitochondrial dysfunction of brain microglia cells. However, the changes in biochemical content of mitochondria that drive the progression of neuro-COVID remain poorly understood. Here we introduce a Raman microspectrometry approach that enables the molecular profiling of single cellular organelles to characterize the mitochondrial molecular makeup in the infected microglia cells. We found that microglia treated with either spike protein or heat-inactivated SARS-CoV-2 trigger a dramatic reduction in mtDNA content and an increase in phospholipid saturation levels. At the same time, no significant changes were detected in Golgi apparatus and in lipid droplets, the organelles that accommodate biogenesis and storage of lipids. We hypothesize that transformations in mitochondria are caused by increased synthesis of reactive oxygen species in these organelles. Our findings call for the development of mitochondria-targeted therapeutic approaches to limit neuropathology associated with SARS-CoV-2.

A Single-Organelle Optical Omics Platform for Cell Science and Biomarker Discovery.

Research in fundamental cell biology and pathology could be revolutionized by developing the capacity for quantitative molecular analysis of subcellular structures. To that end, we introduce the Ramanomics platform, based on confocal Raman microspectrometry coupled to a biomolecular component analysis algorithm, which together enable us to molecularly profile single organelles in a live-cell environment. This emerging omics approach categorizes the entire molecular makeup of a sample into about a dozen of general classes and subclasses of biomolecules and quantifies their amounts in submicrometer volumes. A major contribution of our study is an attempt to bridge Raman spectrometry with big-data analysis in order to identify complex patterns of biomolecules in a single cellular organelle and leverage discovery of disease biomarkers. Our data reveal significant variations in organellar composition between different cell lines. We also demonstrate the merits of Ramanomics for identifying diseased cells by using prostate cancer as an example. We report large-scale molecular transformations in the mitochondria, Golgi apparatus, and endoplasmic reticulum that accompany the development of prostate cancer. Based on these findings, we propose that Ramanomics datasets in distinct organelles constitute signatures of cellular metabolism in healthy and diseased states.

Prohibitin gene regulation in cancer and its possible therapeutic potential

Prohibitin (PHB), an evolutionary conserved gene, is mapped at the chromosomal location 17q21–q22 and is present with two isoforms, namely, PHB1 and PHB2. Both of these isoforms have their individual roles in tumor suppression and cell proliferation. The presence of these isoforms is not restricted to single cellular organelle but can be located in the nucleus, mitochondria, and cytosol. Importantly, loss of heterozygosity in the prohibitin gene has been noted in a significant number of different cancer types. Along with this, there are other mutations that contribute to tumorigenesis and cancer cell proliferation. Sporadic breast cancer, lung cancer, and prostate cancer are a few examples in which regulation of PHB plays a major role. PHB has shown to be both up-regulated and down-regulated depending on the type of cancer or disease. PHB acts as the biomarker for several types of cancers and is also seen as a potential therapeutic target. Along with cancer, PHB has also been seen to play an important role in other diseases such as neurological diseases, cardiac diseases, and renal diseases. The link between PHB and a plethora of diseases opens a new window in which PHB can be actually targeted in treatment as well as disease management. Here, we review the regulation of PHB in different types of cancers along with its significant interaction in other disorders.

BCAbox Algorithm Expands Capabilities of Raman Microscope for Single Organelles Assessment.

Raman microspectroscopy is a rapidly developing technique, which has an unparalleled potential for in situ proteomics, lipidomics, and metabolomics, due to its remarkable capability to analyze the molecular composition of live cells and single cellular organelles. However, the scope of Raman spectroscopy for bio-applications is limited by a lack of software tools for express-analysis of biomolecular composition based on Raman spectra. In this study, we have developed the first software toolbox for immediate analysis of intracellular Raman spectra using a powerful biomolecular component analysis (BCA) algorithm. Our software could be easily integrated with commercial Raman spectroscopy instrumentation, and serve for precise analysis of molecular content in major cellular organelles, including nucleoli, endoplasmic reticulum, Golgi apparatus, and mitochondria of either live or fixed cells. The proposed software may be applied in broad directions of cell science, and serve for further advancement and standardization of Raman spectroscopy.

Organic Nanoprobe Cocktails for Multilocal and Multicolor Fluorescence Imaging of Reactive Oxygen Species

Hypochlorite (ClO−) as a highly reactive oxygen species not only acts as a powerful “guarder” in innate host defense but also regulates inflammation‐related pathological conditions. Despite the availability of fluorescence probes for detection of ClO− in cells, most of them can only detect ClO− in single cellular organelle, limiting the capability to fully elucidate the synergistic effect of different organelles on the generation of ClO−. This study proposes a nanoprobe cocktail approach for multicolor and multiorganelle imaging of ClO− in cells. Two semiconducting oligomers with different π‐conjugation length are synthesized, both of which contain phenothiazine to specifically react with ClO− but show different fluorescent color responses. These sensing components are self‐assembled into the nanoprobes with the ability to target cellular lysosome and mitochondria, respectively. The mixture of these nanoprobes forms a nano‐cocktail that allows for simultaneous imaging of elevated level of ClO− in lysosome and mitochondria according to fluorescence color variations under selective excitation of each nanoprobe. Thus, this study provides a general concept to design probe cocktails for multilocal and multicolor imaging.

Simple non-fluorescent polarity labeling of microtubules for molecular motor assays

Transport of intracellular organelles along the microtubule cytoskeleton occurs in a bidirectional manner due to opposing activity of microtubule-associated motor proteins of the kinesin and dynein families. Regulation of this opposing activity and the resultant motion is believed to generate a polarized distribution of many organelles within the cell. The bidirectional motion can be reconstituted on in vitro assembled microtubules using organelles extracted from cells. This provides an opportunity to understand the regulation of intracellular transport through quantitative analysis of the motion of organelles in a controlled environment. Such analysis requires the use of polarity-labeled microtubules to resolve the plus and minus components of bidirectional motion. However, existing methods of in vitro microtubule polarity labeling are unsuitable for high-resolution recording of motion. Here we present a simple and reliable method that uses avidin-coated magnetic beads to prepare microtubules labeled at the minus end. The microtubule polarity can be identified without any need for fluorescence excitation. We demonstrate video-rate high-resolution imaging of single cellular organelles moving along plus and minus directions on labeled microtubules. Quantitative analysis of this motion indicates that these organelles are likely to be driven by multiple dynein motors in vivo.

Inhibition of Protein Trafficking by Coxsackievirus B3: Multiple Viral Proteins Target a Single Organelle

Despite replicating to very high titers, coxsackieviruses do not elicit strong CD8 T-cell responses, perhaps because antigen presentation is inhibited by virus-induced disruption of host protein trafficking. Herein, we evaluated the effects of three viral nonstructural proteins (2B, 2BC, and 3A) on intracellular trafficking. All three of these proteins inhibited secretion, to various degrees, and directly associated with the Golgi complex, causing trafficking proteins to accumulate in this compartment. The 3A protein almost completely ablated trafficking and secretion, by moving rapidly to the Golgi, and causing its disruption. Using an alanine-scanning 3A mutant, we show that Golgi targeting and disruption can be uncoupled. Thus, coxsackieviruses rely on the combined effects of several gene products that target a single cellular organelle to successfully block protein secretion during an infection. These findings have implications for viral pathogenesis.

Laser trapping and Raman spectroscopy of single cellular organelles in the nanometer range.

The laser trapping technique combined with near-infrared Raman (NIR) spectroscopy was used for the analysis of single cellular organelles in the nanometer range. The samples were synaptosomes, nerve-ending particles (about 500-700 nm in diameter) isolated from a neuron in a rat brain, dispersed in the phosphate buffer solution. The NIR laser Raman trapping (NIR-LRT) system trapped a single synaptosome without photochemical damage and provided a Raman spectrum of the sample with less fluorescence background. After the background subtraction from the Raman spectrum, two large peaks appeared, which are attributed to the peaks of the CH(2) deformation mode and the amide I mode. This indicates the laser-trapped synaptosomes include some types of lipids and proteins. The result demonstrates that the NIR-LRT system can determine biological molecules in single cellular organelles in the nanometer range. Further improvement of the detection sensitivity will enable us to get detailed information about the functions of single cellular organelles in the brain, which will be valuable for neuroscience.

Zur Pathomorphologie der Corticoidmyopathie. Ultrastrukturelle Veränderungen des Plasmalemms der Skelett- und Herzmuskelzelle im Vergleich zur intrazellulären Reaktion

Zur Pathomorphologie der Corticoidmyopathie. Ultrastrukturelle Veränderungen des Plasmalemms der Skelett- und Herzmuskelzelle im Vergleich zur intrazellulären Reaktion