The versatile electron microscope : an ultrastructural overview of autophagy

Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectively known as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two blockface imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electron tomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).Peer reviewe

Magnetic nanoparticles as MRI contrast agents for the diagnosis of Alzheimer’s disease

Background Nanoparticle‐based magnetic contrast agents have opened the potential for Magnetic Resonance Imaging (MRI) to be used for early non‐invasive diagnosis of Alzheimer’s disease (AD). Current methods for clinical diagnosis in the early stages of the disease, such as Positron Emission Tomography imaging of amyloid build‐up, are limited by their availability and cost. The aim of this research is to develop a novel non‐toxic amyloid targeted nanoparticle which can successfully permeate the blood brain barrier and bind amyloid plaques resulting in enhanced contrast in the MR image and improved diagnostic sensitivity. Methods Targeted iron nanoparticles were assessed using a U‐251 cell line to determine their in vitro toxicity. Transmission electron microscopy was used to determine the movement of the nanoparticles within the cell and in vitro binding to amyloid fibrils. APPSwe/PSEN1 mice were treated with increasing doses of targeted and non‐targeted nanoparticles to evaluate acute in vivo toxicity, in addition to nanoparticle biodistribution and MRI contrast enhancement. Results The novel targeted nanoparticles have demonstrated no significant in vitro toxicity and electron microscopy results show their movement through the endocytic cycle within the cell, demonstrating an effective degradation and clearance pathway (Figure). No acute toxicity was observed in the animal model. In addition, immunohistochemistry demonstrated nanoparticles to co‐localise with plaques on ex vivo brain sections. Conclusion The present work shows promising preliminary results in the development of a targeted non‐invasive method of early AD diagnosis using contrast enhanced MRI

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.Peer reviewe

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.</p

Unlocking the ultrastructure of colorectal cancer cells in vitro using selective staining

AIM: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties

Ultrastructural relationship of the phagophore with surrounding organelles

<div><p>Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.</p></div

Optimising Extracellular Vesicle Metabolomic Methodology for Prostate Cancer Biomarker Discovery

Conventional diagnostic tools for prostate cancer (PCa), such as prostate-specific antigen (PSA), transrectal ultrasound (TRUS), digital rectal examination (DRE), and tissue biopsy face, limitations in individual risk stratification due to invasiveness or reliability issues. Liquid biopsy is a less invasive and more accurate alternative. Metabolomic analysis of extracellular vesicles (EVs) holds a promise for detecting non-genetic alterations and biomarkers in PCa diagnosis and risk assessment. The current research gap in PCa lies in the lack of accurate biomarkers for early diagnosis and real-time monitoring of cancer progression or metastasis. Establishing a suitable approach for observing dynamic EV metabolic alterations that often occur earlier than being detectable by other omics technologies makes metabolomics valuable for early diagnosis and monitoring of PCa. Using four distinct metabolite extraction approaches, the metabolite cargo of PC3-derived large extracellular vesicles (lEVs) was evaluated using a combination of methanol, cell shearing using microbeads, and size exclusion filtration, as well as two fractionation chemistries (pHILIC and C18 chromatography) that are also examined. The unfiltered methanol–microbeads approach (MB-UF), followed by pHILIC LC-MS/MS for EV metabolite extraction and analysis, is effective. Identified metabolites such as L-glutamic acid, pyruvic acid, lactic acid, and methylmalonic acid have important links to PCa and are discussed. Our study, for the first time, has comprehensively evaluated the extraction and separation methods with a view to downstream sample integrity across omics platforms, and it presents an optimised protocol for EV metabolomics in PCa biomarker discovery