Localization Of Organelles Research Articles

Automated 3D blastomere segmentation and injection for early-stage embryo

Automation of single biological cell surgery requires that the location of organelles and cell structures to be determined to permit automated processing to be carried out. In this work, z-stack images of mouse embryos are used to develop image processing algorithms to determine the position (x, y, z) coordinates of the embryo blastomeres. These images are processed utilising existing Matlab image processing algorithms. The embryos are transparent, allowing a series of images along the vertical cell axis to be obtained. Individual z-stack images are processed using 2D image processing steps to segment, then estimate the centroid (x, y) coordinates of the blastomeres in the 2D image. Successive processing of all z-stack images permits the centroid of the blastomeres to be determined in (x, y, z) coordinates. Image processing-based calibration allows a PZD micropipette to move to the computed centroid positions under closed-loop control. These algorithms are verified using z-stack images of mouse embryos at the two-blastomere stage.

Automated 3D blastomere segmentation and injection for early-stage embryo

Automation of single biological cell surgery requires that the location of organelles and cell structures to be determined to permit automated processing to be carried out. In this work, z-stack images of mouse embryos are used to develop image processing algorithms to determine the position (x, y, z) coordinates of the embryo blastomeres. These images are processed utilising existing Matlab image processing algorithms. The embryos are transparent, allowing a series of images along the vertical cell axis to be obtained. Individual z-stack images are processed using 2D image processing steps to segment, then estimate the centroid (x, y) coordinates of the blastomeres in the 2D image. Successive processing of all z-stack images permits the centroid of the blastomeres to be determined in (x, y, z) coordinates. Image processing-based calibration allows a PZD micropipette to move to the computed centroid positions under closed-loop control. These algorithms are verified using z-stack images of mouse embryos at the two-blastomere stage.

Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome.

ABSTRACTProtein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the accumulation of α-synuclein, the main component of Lewy bodies found in Parkinson's disease. Aggresomes are closely-related, cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organise the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to functional deficits and neuronal cell death in Parkinson's disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals.

Dissecting the localization of lipopolysaccharide-responsive and beige-like anchor protein (LRBA) in the endomembrane system

Introduction and objectivesLRBA deficiency is caused by loss of LRBA protein expression, due to either homozygous or compounds heterozygous mutations in LRBA. LRBA deficiency has been shown to affect vesicular trafficking and autophagy. To date, LRBA has been observed in the cytosol, Golgi apparatus and some lysosomes in LPS-stimulated murine macrophages. The objectives of the present study were to study the LRBA localization in organelles involved in vesicular traffic, phagocytosis, and autophagy in mononuclear phagocytes (MP). Materials and methodsWe analyzed LRBA colocalization with different endosomes markets using confocal microscopy in MP. We used the autophagy inhibitors to determine the role of LRBA in formation, maturation or degradation of the autophagosome. ResultsLRBA intracellular trafficking depends on the activity of the GTPase ADP ribosylation factor-1 (ARF) in MP. LRBA was identified in early, late endosomes but did not colocalize strongly with lysosomal markers. Although LRBA appears not to be recruited during the phagocytic cargo uptake, it greatly colocalized with the microtubule-associated protein 1A/1B-light chain 3 (LC3) under a steady state and this decreased after the induction of autophagy flux. Although the use of inhibitors of lysosome fusion did not restore the LRBA/LC3 colocalization, inhibitors of either early to late endosomes trafficking or PI3K pathway did. ConclusionsTaken together, our results show that LRBA is located in endomembrane system vesicles, mainly in the early and late endosomes. Although LRBA appears not to be involved in the phagocytic uptake, it is recruited in the early steps of the autophagy flux.

Fabrication of Multitargeting and pH-Regulated Nanocomposites for Antitumor Photodynamic Therapy Based on Triphenylphosphine and Graphene Oxide.

Targeted delivery of photosensitizer for antitumor photodynamic therapy has received much attention recently. In this research, a multitargeting graphene oxide (GO) nanosystem (GFFP-TPPa) was fabricated based on the magnetically targeted nano-Fe3O4, active targeted folic acid (FA), and mitochondria-targeted triphenylphosphine, aiming to provide an effective drug delivery system of photosensitizer for improved photodynamic therapy against human tumor. The effective photosensitizer release from GFFP-TPPa under mild acidic pH condition (such as tumor microenvironment) suggested our designed nanosystem could reduce cutaneous phototoxicity of nanoparticles (NPs) in normal cells environment, which was further verified by the observation that the fluorescence of GFFP-TPPa was significantly quenched compared with free PPa. Research of cellular morphological variation, uptaking test, and intracellular single oxygen detection has validated the effectiveness of GFFP-TPPa NPs against tumor cells and potential fluorescence imaging function. Moreover, GFFP-TPPa NPs have excellent superparamagnetic properties and FA-targeted property of NPs, which was verified by the tumor cellular uptake of GFFP-TPPa. Finally, the precise deliver of photosensitizer to mitochondria has been proved by Mito-Tracker Green organelle localization. Our research indicated that the fabricated nanosystem could be applied conveniently for PDT based on multitargetability.

Precise Quantitative Analysis of Cell Targeting by Particle-Based Agents Using Imaging Flow Cytometry and Convolutional Neural Network.

Understanding the intricacies of particle-cell interactions is essential for many applications such as imaging, phototherapy, and drug/gene delivery, because it is the key to accurate control of the particle properties for the improvement of their therapeutic and diagnostic efficiency. Recently, high-throughput methods have emerged for the detailed investigation of these interactions. For example, imaging flow cytometry (IFC) collects up to 60,000 images of cells per second (in 12 optical channels) and provides information about morphology and organelle localization in combination with fluorescence and side scatter intensity data. However, analysis of IFC data is extremely difficult to perform using conventional methods that calculate integral parameters or use mask-based object recognition. Here, we show application of a convolutional neural network (CNN) for precise quantitative analysis of particle targeting of cells using IFC data. CNN provides high-throughput object detection with almost human precision but avoids the subjective choice of image processing parameters that often leads to incorrect data interpretation. The method allows accurate counting of cell-bound particles with reliable discrimination from the nonbound particles in the field of view. The proposed method expands capabilities of spot counting applications (such as organelle counting, quantification of cell-cell and cell-bacteria interactions) and is going to be useful not only for high-throughput analysis of IFC data but also for other imaging techniques. © 2019 International Society for Advancement of Cytometry.

Deep learning for actinic keratosis classification

Classification of biological images plays a crucial role in many biological problems, e.g. recognition of cell phenotypes and maturation levels, localization of cell organelles and histopathological classification, and holds the potential to support early diagnosis, which is critical in disease prevention. In this paper, we tested different ensemble of canonical and deep classifiers to provide accurate identification of actinic keratosis (AK), one of the most common skin lesions that could degenerate into lethal squamous cell carcinomas. We used a clinical image dataset to build and test different ensembles of support vector machines trained by handcrafted descriptors and convolutional neural networks (CNNs) for which we experimented different learning rates, augmentation techniques (e.g. warping) and topologies. Our results show that the proposed ensemble obtains performance comparable to the state of the art. To reproduce the experiments reported in this paper, the MATLAB code of all the descriptors is available at https://github.com/LorisNanni.

Comprehensive analysis of key lncRNAs in ischemic stroke.

Ischemic stroke (IS) is a leading cause of mortality and disability worldwide. However, the treatments for ischemic stroke remained inadequate. The mechanisms underlying ischemic stroke are still not completely understood. the present study identified 19 lncRNAs related to stroke recovery by analyzing a public dataset GSE37587. A co-expression network included 24 lncRNAs, 1668 mRNAs and 3542 edges were constructed in the present study. Bioinformatics analysis showed these lncRNAs were involved in regulating multiple biological processes and pathways, such as mRNA nonsense-mediated decay, translation, cell-cell adhesion. Three lncRNAs, including DLEU1, LOC432369, and LOC338799, were identified as key lncRNAs in stroke. Bioinformatics showed DLEU1 was involved in regulating oxidative phosphorylation, and ubiquitin-mediated proteolysis. LOC432369 was associated with oxidative phosphorylation. LOC338799 was associated with clathrin-dependent endocytosis, the establishment of organelle localization and ribonucleoprotein complex assembly. We thought this study could provide useful information to understand the mechanisms underlying stroke progression.

Protein trafficking in plant cells: Tools and markers.

Eukaryotic cells consist of numerous membrane-bound organelles, which compartmentalize cellular materials to fulfil a variety of vital functions. In the post-genomic era, it is widely recognized that identification of the subcellular organelle localization and transport mechanisms of the encoded proteins are necessary for a fundamental understanding of their biological functions and the organization of cellular activity. Multiple experimental approaches are now available to determine the subcellular localizations and dynamics of proteins. In this review, we provide an overview of the current methods and organelle markers for protein subcellular localization and trafficking studies in plants, with a focus on the organelles of the endomembrane system. We also discuss the limitations of each method in terms of protein colocalization studies.

ER-localized Hrd1 ubiquitinates and inactivates Usp15 to promote TLR4-induced inflammation during bacterial infection

The special organelle-located MAVS, STING and TLR3 are important for clearing viral infections. Although TLR4 triggers NF-κB activation to produce pro-inflammatory cytokines for bacterial clearance, effectors with special organelle localization have not been identified. Here, we screened more than 280 E3 ubiquitin ligases and discovered that the endoplasmic reticulum-located Hrd1 regulates TLR4-induced inflammation during bacterial infection. Hrd1 interacts directly with the deubiquitinating enzyme Usp15. Unlike the classical function of Hrd1 in endoplasmic reticulum-associated degradation, Usp15 is not degraded but loses its deubiquitinating activity for IκBα deubiquitination, resulting in excessive NF-κB activation. Importantly, Hrd1 deficiency in macrophages protects mice against lipopolysaccharide-induced septic shock, and knockdown of Usp15 in Hrd1-knockout macrophages restores the reduced IL-6 production. This study proposes that there is crosstalk between Hrd1 and TLR4, thereby linking the endoplasmic reticulum-plasma membrane function during bacterial infection.

Mitochondria-targeted tri-triphenylphosphonium substituted meso-tetra(4-carboxyphenyl)porphyrin(TCPP) by conjugation with folic acid and graphene oxide for improved photodynamic therapy

Mitochondria are extensively researched as target sites to maximize photodynamic therapy (PDT) effects because they play crucial roles in metabolism. Here, a mitochondria targeting PDT agent, tri-triphenylphosphonium substituted meso-tetra(4-carboxyphenyl)porphyrin (TCPP-TPP) is prepared for the first time. Considering that many porphyrin derivatives are quick to aggregate, thereby reducing the PDT effect, our photosensitizer (PS) was loaded on a folic acid (FA) decorated graphene oxide (GO) nanosystem, called GF@TCPP-TPP, by electrostatic and [Formula: see text]–[Formula: see text] stacking or hydrophobic cooperative interactions, to improve the transportation of photosensitizers and enhance the therapeutic effect. Herein, we have performed a detailed study of photodynamic activity of GF@TCPP-TPP nanocomposites and evaluated their potential as a photosensitizer in PDT. An MTT assay showed that GF@TCPP-TPP inhibited HeLa cells in a concentration-dependent manner under light (650 ± 10 nm, 5 mW [Formula: see text] and 10 min), and presented remarkably improved PDT efficiency (IC[Formula: see text] g [Formula: see text] mL[Formula: see text] of equivalent TCPP-TPP) over free TCPP (IC[Formula: see text] after irradiation. Furthermore, our research indicated that Type I mechanisms (the generation of hydroxyl radicals) play a predominant role in the GF@TCPP-TPP induced PDT process. This coincides with the low singlet oxygen (1O[Formula: see text] quantum yield ([Formula: see text] [Formula: see text] 33.6%) in a DMF solution. Moreover, cell morphological changes after GF@TCPP-TPP PDT further demonstrated that GF@TCPP-TPP could induce damage and apoptotic cell death efficiently. In particular, precise delivery of photosensitizers to mitochondria was proven by organelle localization.

High-Throughput Isolation of Cell Protrusions with Single-Cell Precision for Profiling Subcellular Gene Expression.

Invading cancer cells extend cell protrusions, which guide cancer-cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single-cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high-throughput isolation of cell protrusions at single-cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell-protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single-cell precision for subsequent RNA-Seq analysis, which was further validated by RT-qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.

High‐Throughput Isolation of Cell Protrusions with Single‐Cell Precision for Profiling Subcellular Gene Expression

AbstractInvading cancer cells extend cell protrusions, which guide cancer‐cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single‐cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high‐throughput isolation of cell protrusions at single‐cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell‐protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single‐cell precision for subsequent RNA‐Seq analysis, which was further validated by RT‐qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.

Posttranslational regulation of PGC-1α and its implication in cancer metabolism.

Deregulation of cellular metabolism is well established in cancer. The mitochondria are dynamic organelles and act as the center stage for energy metabolism. Central to mitochondrial regulatory network is peroxisome proliferator‐activated receptor γ coactivator 1a (PGC‐1α), which serves as a master regulator of mitochondrial proliferation and metabolism. The activity and stability of PGC‐1α are subject to dynamic and versatile posttranslational modifications including phosphorylation, ubiquitination, methylation and acetylation in response to metabolic stress and other environmental signals. In this review, we describe the structure of PGC‐1α. Then, we discuss recent advances in the posttranslational regulatory machinery of PGC‐1α, which affects its transcriptional activity, stability and organelle localization. Furthermore, we address the important roles of PGC‐1α in tumorigenesis and malignancy. Finally, we also mention the clinical therapeutic potentials of PGC‐1α modulators. A better understanding of the elegant function of PGC‐1α in cancer progression could provide novel insights into therapeutic interventions through the targeting of PGC‐1α signaling.

Comprehensive transcriptome analysis of cerebral cavernous malformation across multiple species and genotypes.

The purpose of this study was to determine important genes, functions, and networks contributing to the pathobiology of cerebral cavernous malformation (CCM) from transcriptomic analyses across 3 species and 2 disease genotypes. Sequencing of RNA from laser microdissected neurovascular units of 5 human surgically resected CCM lesions, mouse brain microvascular endothelial cells, Caenorhabditis elegans with induced Ccm gene loss, and their respective controls provided differentially expressed genes (DEGs). DEGs from mouse and C. elegans were annotated into human homologous genes. Cross-comparisons of DEGs between species and genotypes, as well as network and gene ontology (GO) enrichment analyses, were performed. Among hundreds of DEGs identified in each model, common genes and 1 GO term (GO:0051656, establishment of organelle localization) were commonly identified across the different species and genotypes. In addition, 24 GO functions were present in 4 of 5 models and were related to cell-to-cell adhesion, neutrophil-mediated immunity, ion transmembrane transporter activity, and responses to oxidative stress. We have provided a comprehensive transcriptome library of CCM disease across species and for the first time to our knowledge in Ccm1/Krit1 versus Ccm3/Pdcd10 genotypes. We have provided examples of how results can be used in hypothesis generation or mechanistic confirmatory studies.

Development of an Imaging Pipeline to Model and Predict the Integrated Localization of Organelles in hiPSC-Derived Cardiomyocytes

Development of an Imaging Pipeline to Model and Predict the Integrated Localization of Organelles in hiPSC-Derived Cardiomyocytes

Dynamic organelle localization and cytoskeletal reorganization during preimplantation mouse embryo development revealed by live imaging of genetically encoded fluorescent fusion proteins.

SummaryLive imaging is one of the most powerful technologies for studying the behaviors of cells and molecules in living embryos. Previously, we established a series of reporter mouse lines in which specific organelles are labeled with various fluorescent proteins. In this study, we examined the localizations of fluorescent signals during preimplantation development of these mouse lines, as well as a newly established one, by time‐lapse imaging. Each organelle was specifically marked with fluorescent fusion proteins; fluorescent signals were clearly visible during the whole period of time‐lapse observation, and the expression of the reporters did not affect embryonic development. We found that some organelles dramatically change their sub‐cellular distributions during preimplantation stages. In addition, by crossing mouse lines carrying reporters of two distinct colors, we could simultaneously visualize two types of organelles. These results confirm that our reporter mouse lines can be valuable genetic tools for live imaging of embryonic development.

Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) for the Examination of Dental Hard Tissues.

This chapter describes laboratory protocols for TEM and SEM approaches allowing the examination of the dental hard tissues' constituents at the ultrastructural level. TEM has the highest resolution power to examine the cellular and extracellular matrix ultrastructure inside a given sample, detecting the presence, location, and quantification of organelles related to the metabolism of the cell type as well as membrane specializations. SEM allows the observation of the sample surface, for examining dimensional topography and distribution of exposed features.

High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels.

Aequorin, a 22kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.

Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation.

The tumor suppressor protein p53 plays an important role in the development and progression of colon cancer, and the subcellular organelle localization directly affects its function. Wogonin (5,7-dihydroxy-8-methoxyflavone), a mono-flavonoid extracted from root of Scutellaria baicalensis Georgi, possesses acceptable toxicity and has been used in colorectal cancer (CRC) chemoprevention in pre-clinical trials by oncologist. However, the underlying anti-colon cancer mechanisms of wogonin are not yet fully understood. In the present study, the effect of wogonin on the initiation and development of colitis-associated cancer through p53 nuclear translocation was explored. AOM-DSS CRC animal model and human CRC HCT-116 cell model were used to evaluate the in vivo and in vitro anti-colon cancer action of wogonin. We observed that wogonin showed a dramaticlly preventive effect on colon cancer. Our results showed that wogonin caused apoptotic cell death in human CRC HCT-116 cell through increased endoplasmic reticulum (ER) stress. Meanwhile, excessive ER stress facilitated the cytoplasmic localization of p53 through increasing phosphor-p53 at S315 and S376 sites, induced caspase-dependent apoptosis and inhibited autophagy. Furthermore, we verified the chemoprevention effect and toxicity of wogonin in vivo by utilizing an AOM-DSS colon cancer animal model. We found that wogonin not only reduced tumor multiplicity, preserved colon length to normal (6.79 ± 0.34 to 7.41 ± 0.56, P < 0.05) but also didn’t induce side effects on various organs. In conclusion, these results explain the anti-tumor effect of wogonin in CRC and suggest wogonin as a potential therapeutic candidate for the therapeutic strategy in CRC treatment.