scholarly journals Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission

2020 ◽  
Author(s):  
David S. Guttery ◽  
Rajan Pandey ◽  
David J. P. Ferguson ◽  
Richard J. Wall ◽  
Declan Brady ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Reverse Genetics ◽  
Cell Imaging ◽  
Mitotic Division ◽  
Circular Ring ◽  
Live Cell ◽  
Gfp Fluorescence ◽  
Evolutionarily Conserved ◽  
And Function ◽  
Malaria Model

AbstractCells use fatty acids (FAs) for membrane biosynthesis, energy storage and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase – DEH – is a key component of very long chain FA (VLCFA) synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics and ultrastructure analysis using the mouse malaria model Plasmodium berghei. DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δdeh mutants showed a small but significant reduction in oocyst size compared to WT controls from day 10 post-infection onwards and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δdeh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER and plays a crucial role in sporogony.

scholarly journals Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission

2020 ◽  
Vol 3 (12) ◽  
pp. e202000879
Author(s):  
David S Guttery ◽  
Rajan Pandey ◽  
David JP Ferguson ◽  
Richard J Wall ◽  
Declan Brady ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Reverse Genetics ◽  
Cell Imaging ◽  
In Silico Analysis ◽  
Acid Synthesis ◽  
Mitotic Division ◽  
Circular Ring ◽  
Live Cell ◽  
Evolutionarily Conserved ◽  
And Function

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase—DEH—is a key component of very long chain fatty acid synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics, and ultrastructure analysis using the mouse malaria model Plasmodium berghei. DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δdeh mutants showed a small but significant reduction in oocyst size compared with WT controls from day 10 postinfection onwards, and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δdeh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER, and plays a crucial role in sporogony.

scholarly journals RNA-Selective, Live Cell Imaging Probes for Studying Nuclear Structure and Function

2006 ◽  
Vol 13 (6) ◽  
pp. 615-623 ◽  
Author(s):  
Qian Li ◽  
Yunkyung Kim ◽  
Joshua Namm ◽  
Amita Kulkarni ◽  
Gus R. Rosania ◽  
...  
Keyword(s):  
Nuclear Structure ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Structure And Function ◽  
Live Cell ◽  
Imaging Probes ◽  
And Function

scholarly journals Impaired mitochondrial–endoplasmic reticulum interaction and mitophagy in Miro1-mutant neurons in Parkinson’s disease

2020 ◽  
Vol 29 (8) ◽  
pp. 1353-1364 ◽  
Author(s):  
Clara Berenguer-Escuder ◽  
Dajana Grossmann ◽  
Paul Antony ◽  
Giuseppe Arena ◽  
Kobi Wasner ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Endoplasmic Reticulum ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Mitochondrial Dynamics ◽  
Live Cell ◽  
Calcium Handling ◽  
Imaging Results ◽  
And Function

Abstract Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson’s disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.

scholarly journals Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

RNA Biology ◽  
2018 ◽  
pp. 1-10 ◽  
Author(s):  
Laura Avogaro ◽  
Emmanuelle Querido ◽  
Myriam Dalachi ◽  
Michael F. Jantsch ◽  
Pascal Chartrand ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Dynamics And Function ◽  
And Function

scholarly journals HEAVY METAL POLLUTANT-INDUCED CYTOTOXICITY INVOLVES PERTURBATIONS OF MITOCHONDRIAL FUNCTION

2018 ◽  
Vol 23 (suppl_1) ◽  
pp. e38-e39
Author(s):  
Hussain Raza
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Growth Cone ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Structure And Function ◽  
Live Cell ◽  
Mitochondrial Potential ◽  
Vital Functions ◽  
And Function

Abstract BACKGROUND Environmental pollutants like heavy metals pose a tremendous risk to both animal and human health though the mechanisms underlying their cytotoxic actions at the cellular level remain largely unknown. The freshwater mollusc Lymnaea stagnalis carries hemocynin as its oxygen carrier whereby iron is replaced with copper. As such, this species is highly sensitive to its environmental copper contents and has been used as an important indicator of water quality. Studies have shown that miniscule levels of heavy metals (Pb, Ni, Cu, Co) dissolved in aqueous environments lead to detrimental effects on many of the Lymanea’s vital functions – including respiration and cardiac functions. OBJECTIVES The objective was to determine how heavy metal pulliutants target the cell and its vital functions and if the detirioration of cell motility and viability is a result of the reduced functioning and potential of the cells mitochondria. DESIGN/METHODS We tested the effects of Cu (EC20 as low as 1.8 ug L-1) on isolated blood cells (hemocytes) and neurons from Lymnaea. We sought to determine whether Cu2+ affected cellular viability, motility and neuronal growth cone movements involving cytoskeletal proteins such as actin and tubulin. Cells from the brain were harvested and plated on sigma coated dishes and left to incubate for 3 days to adjust to the environement. They were then stained with mitortracker dyes for mitochondrial potential and movement. The results were captured through live cell imaging and were later analyzed using neurite tracer. We demonstrate the effects of Cu on single mitochondrial movements, structure and function. RESULTS This study provides the first direct evidence that heavy metals such as Cu are indeed cytotoxic and that its detrimental effects on animal health likely involve perturbations of mitochondrial structure and function. Mitochondrial accumulation within the cell started detirioraiting within the first hour of live cell imaging. The mitochondria within the tested cells also started chainging how they cluster within an expanding growth cone. Mitochondrial density decreased substantially and clustered mitohochondira became more fragmeneted which is a positive indication of mitochndrial malfunction. The mitochondrial potential dropped substantially too, indicating that the mitochondria were unable to produce energy like they are suppose to in normal conditions. Finally, this also allowed us to show why heavy metal use in the dentistry profession and other medical professions where heavy metals are used can be linked to cell apoptosis and mitochondrial degradation. CONCLUSION Heavy metals indeed cause malfunction within the cells mitochondria and the reduced functioning disables the cells from fulfilling their associated functions. This was observed through the reduction of breathing, mastication, movement, and neuronal firing of the Lymnaea, which shows a direct realtionship to degraded mitochondria within these cells. Similar results were seen within the hemocytes, which demonstrates that their function of circulating and delivering oxygen was also impaired due to faulty mitochondria.

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