scholarly journals Plug and play: Is “directed endosymbiosis” of chloroplasts possible?

2021 ◽  
Author(s):  
Karin Olszewski Shapiro
Keyword(s):  
Mammalian Cells ◽  
Photosynthetic Bacteria ◽  
Fluorescent Microscopy ◽  
Eukaryotic Cells ◽  
Foreign Protein ◽  
Zebrafish Embryos ◽  
Mammalian Mitochondria ◽  
Plant Chloroplast ◽  
Isolated Chloroplasts ◽  
Over Time

AbstractThe origin of mammalian mitochondria and plant chloroplasts is thought to be endosymbiosis. Millennia ago, a bacterium related to typhus-causing bacteria may have been consumed by a proto-eukaryote and over time evolved into an organelle inside eukaryotic cells, known as a mitochondrion. The plant chloroplast is believed to have evolved in a similar fashion from cyanobacteria. This project attempted to use “directed endosymbiosis” (my term) to investigate if chloroplasts can be taken up by a land animal and continue to function. It has been shown previously that mouse fibroblasts could incorporate isolated chloroplasts when co-cultured. Photosynthetic bacteria containing chloroplasts have been successfully injected into zebrafish embryos, mammalian cells, and ischemic rodent hearts. The photosynthetic alga Chlamydomonas reinhardtii (C. reinhardtii) has also been injected into zebrafish embryos. However, to the best of my knowledge, injection of isolated chloroplasts into a land animal embryo has not been attempted before.In four pilot experiments, solutions of chloroplasts in PBS were microinjected into Drosophila melanogaster (D. melanogaster) embryos to determine if the embryos would tolerate the foreign protein. Interestingly, results indicated that a portion of the D. melanogaster embryos appeared to tolerate the injections and survive to adulthood. To determine if chloroplasts had indeed been transferred, larvae were placed under fluorescent microscopy. Chlorophyll (serving as the reporter) was found to be present in several larvae and to decline in amount over time. To investigate if the chloroplasts still functioned, a radiotracer food intake assay was performed. It was hypothesized that if the chloroplasts were generating ATP (and possibly glucose), the larvae might need less food. Results indicated a decrease in intake, however this might have occurred for other reasons.

scholarly journals Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259837
Author(s):  
Zora Novakova ◽  
Daria Khuntsaria ◽  
Marketa Gresova ◽  
Jana Mikesova ◽  
Barbora Havlinova ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Eukaryotic Cells ◽  
Post Translational Modifications ◽  
Protoporphyrinogen Oxidase ◽  
Intracellular Targeting ◽  
Alternative Systems ◽  
Protein Functions ◽  
The Impact

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

Uptake of Isolated Chloroplasts by Mammalian Cells

Science ◽  
1969 ◽  
Vol 165 (3898) ◽  
pp. 1128-1131 ◽  
Author(s):  
M. M. k. Nass
Keyword(s):  
Mammalian Cells ◽  
Isolated Chloroplasts

scholarly journals Pexophagy: The Selective Degradation of Peroxisomes

2012 ◽  
Vol 2012 ◽  
pp. 1-18 ◽  
Author(s):  
Andreas Till ◽  
Ronak Lakhani ◽  
Sarah F. Burnett ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Current Knowledge ◽  
Cell Types ◽  
Model Systems ◽  
Eukaryotic Cells ◽  
Organelle Biogenesis ◽  
Selective Degradation ◽  
Future Challenges ◽  
Recent Developments ◽  
Distinct Features

Peroxisomes are single-membrane-bounded organelles present in the majority of eukaryotic cells. Despite the existence of great diversity among different species, cell types, and under different environmental conditions, peroxisomes contain enzymes involved inβ-oxidation of fatty acids and the generation, as well as detoxification, of hydrogen peroxide. The exigency of all eukaryotic cells to quickly adapt to different environmental factors requires the ability to precisely and efficiently control peroxisome number and functionality. Peroxisome homeostasis is achieved by the counterbalance between organelle biogenesis and degradation. The selective degradation of superfluous or damaged peroxisomes is facilitated by several tightly regulated pathways. The most prominent peroxisome degradation system uses components of the general autophagy core machinery and is therefore referred to as “pexophagy.” In this paper we focus on recent developments in pexophagy and provide an overview of current knowledge and future challenges in the field. We compare different modes of pexophagy and mention shared and distinct features of pexophagy in yeast model systems, mammalian cells, and other organisms.

scholarly journals PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

2014 ◽  
Vol 206 (7) ◽  
pp. 833-842 ◽  
Author(s):  
Antonio Espert ◽  
Pelin Uluocak ◽  
Ricardo Nunes Bastos ◽  
Davinderpreet Mangat ◽  
Philipp Graab ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Eukaryotic Cells ◽  
Aurora B ◽  
Safeguard Mechanism

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.

scholarly journals Divergent Regulation of Protein Synthesis in the Cytosol and Endoplasmic Reticulum Compartments of Mammalian Cells

2008 ◽  
Vol 19 (2) ◽  
pp. 623-632 ◽  
Author(s):  
Samuel B. Stephens ◽  
Christopher V. Nicchitta
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Steady State ◽  
Mammalian Cells ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Cellular Protein ◽  
Eukaryotic Cells ◽  
Global Protein Synthesis ◽  
Tightly Coupled

In eukaryotic cells, mRNAs encoding signal sequence-bearing proteins undergo translation-dependent trafficking to the endoplasmic reticulum (ER), thereby restricting secretory and integral membrane protein synthesis to the ER compartment. However, recent studies demonstrating that mRNAs encoding cytosolic/nucleoplasmic proteins are represented on ER-bound polyribosomes suggest a global role for the ER in cellular protein synthesis. Here, we examined the steady-state protein synthesis rates and compartmental distribution of newly synthesized proteins in the cytosol and ER compartments. We report that ER protein synthesis rates exceed cytosolic protein synthesis rates by 2.5- to 4-fold; yet, completed proteins accumulate to similar levels in the two compartments. These data suggest that a significant fraction of cytosolic proteins undergo synthesis on ER-bound ribosomes. The compartmental differences in steady-state protein synthesis rates correlated with a divergent regulation of the tRNA aminoacylation/deacylation cycle. In the cytosol, two pathways were observed to compete for aminoacyl-tRNAs—protein synthesis and aminoacyl-tRNA hydrolysis—whereas on the ER tRNA deacylation is tightly coupled to protein synthesis. These findings identify a role for the ER in global protein synthesis, and they suggest models where compartmentalization of the tRNA acylation/deacylation cycle contributes to the regulation of global protein synthesis rates.

Dynamics of autophagosome formation: a pulse and a sequence of waves

2014 ◽  
Vol 42 (5) ◽  
pp. 1389-1395 ◽  
Author(s):  
Nicholas T. Ktistakis ◽  
Eleftherios Karanasios ◽  
Maria Manifava
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Stress Conditions ◽  
Present Review ◽  
Eukaryotic Cells ◽  
Current View ◽  
Protein Assemblies ◽  
Autophagosome Formation ◽  
Flat Membrane ◽  
A Current

Autophagosomes form in eukaryotic cells in response to starvation or to other stress conditions brought about by the unwanted presence in the cytosol of pathogens, damaged organelles or aggregated protein assemblies. The uniqueness of autophagosomes is that they form de novo and that they are the only double-membraned vesicles known in cells, having arisen from flat membrane sheets which have expanded and self-closed. The various steps describing their formation as well as most of the protein and lipid components involved have been identified. Furthermore, the hierarchical relationships among the components are well documented, and the mechanistic rationale for some of these hierarchies has been revealed. In the present review, we try to provide a current view of the process of autophagosome formation in mammalian cells, emphasizing along the way gaps in our knowledge that need additional work.

scholarly journals Evolving mtDNA populations within cells

2019 ◽  
Vol 47 (5) ◽  
pp. 1367-1382 ◽  
Author(s):  
Iain G. Johnston ◽  
Joerg P. Burgstaller
Keyword(s):  
Mitochondrial Dna ◽  
Nonlinear Effects ◽  
Eukaryotic Cells ◽  
Change Over Time ◽  
Mean And Variance ◽  
Genetic Makeup ◽  
Population Processes ◽  
The Many ◽  
Cell Functionality ◽  
Over Time

Abstract Mitochondrial DNA (mtDNA) encodes vital respiratory machinery. Populations of mtDNA molecules exist in most eukaryotic cells, subject to replication, degradation, mutation, and other population processes. These processes affect the genetic makeup of cellular mtDNA populations, changing cell-to-cell distributions, means, and variances of mutant mtDNA load over time. As mtDNA mutant load has nonlinear effects on cell functionality, and cell functionality has nonlinear effects on tissue performance, these statistics of cellular mtDNA populations play vital roles in health, disease, and inheritance. This mini review will describe some of the better-known ways in which these populations change over time in different organisms, highlighting the importance of quantitatively understanding both mutant load mean and variance. Due to length constraints, we cannot attempt to be comprehensive but hope to provide useful links to some of the many excellent studies on these topics.

scholarly journals Sphingomyelin metabolism controls the shape and function of the Golgi cisternae

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Felix Campelo ◽  
Josse van Galen ◽  
Gabriele Turacchio ◽  
Seetharaman Parashuraman ◽  
Michael M Kozlov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Structural Organization ◽  
Structural Features ◽  
Membrane Curvature ◽  
Eukaryotic Cells ◽  
Golgi Cisterna ◽  
Golgi Membranes ◽  
And Function ◽  
Flat Cisternae

The flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae.

scholarly journals Quantities of Adenylate Homologues (ATP+ADP+AMP) Change over Time in Prokaryotic and Eukaryotic Cells

2019 ◽  
Vol 82 (12) ◽  
pp. 2088-2093 ◽  
Author(s):  
N. W. SMITH ◽  
J. J. SINDELAR ◽  
S. A. RANKIN
Keyword(s):  
Eukaryotic Cells ◽  
Metabolic State ◽  
Prokaryotic Cell ◽  
Rapid Assays ◽  
Time Periods ◽  
Food Residues ◽  
Quantitative Changes ◽  
Living Organisms ◽  
The Individual ◽  
Over Time

ABSTRACT Rapid assays for the assessment of the hygienic state of surfaces in food and medical industries include the use of technologies designed to detect the presence of the metabolite ATP. ATP is a critical metabolite and energy source for most living organisms; therefore, the presence of ATP can be an indicator of surface hygiene based on the presence of soil or food residues associated with inadequate cleaning. The concentrations of ATP vary based on an organism's metabolic state, thus potentially influencing the sensitivity of ATP-based assays. However, little has been published detailing the quantitative changes of ATP to the adenylate homologues ADP and AMP nor the quantitative and cumulative fate of these homologues over time as the metabolic state remains in flux. The objective of this study was to quantify the individual and cumulative (AXP) concentrations of these three adenylate homologues over defined time periods in selected eukaryotic tissue and prokaryotic cell cultures of significance to hygiene. ATP concentrations differed substantially across these selected variables of time and source. The 1- to 3-log reductions in ATP concentrations over time were highly affected by organism type. In general, ADP became the predominate adenylate in eukaryotic tissue, and AMP was the predominate adenylate in the prokaryotic cells at later time points in each study. Total AXP concentrations dropped in general, reflective primarily of the loss of ATP. The results of ATP-based techniques for hygiene surveillance will vary as a function of the amount of cellular material present and the metabolic state of such material. HIGHLIGHTS

Visualizing the intracellular membrane system of yeast cells using a laser scanning confocal microscope

1993 ◽  
Vol 51 ◽  
pp. 274-275
Author(s):  
Karen L. McCoy ◽  
Andrew G. Dillin ◽  
Ardythe A. McCracken
Keyword(s):  
Endoplasmic Reticulum ◽  
Yeast Cell ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Fluorescent Microscopy ◽  
Confocal Microscope ◽  
Yeast Cells ◽  
Intracellular Membrane ◽  
Laser Scanning Confocal Microscope

Progress has been made in developing new preparative techniques for electron microscopic visualization of the intracellular structures of yeast. In addition, development of the laser scanning confocal microscope (LSCM) has provided improved resolution for fluorescent microscopy. We asked whether the LSCM in combination with new preparative techniques could be used for comparable investigative research of the intracellular organizaton of the yeast cell.To investigate this possibility, a BioRad MRC600 LSCM equipped with a krypton/argon laser and integrated computer imaging capabilities, was used to study various dipliod strains of the yeast Saccharomyces cerevisiae. Cells were treated with the lipophilic, cationic fluorescent dye DiOC6 (3,3’-dihexyloxacarbocyanine iodide), which has been used to visualize intracellular membrane structures, and in particular the endoplasmic reticulum of mammalian cells and living yeast cells. Since one of our interests is the intracellular localization of proteins in the yeast cell, we utilized transformed yeast cells expressing a human gene encoding a protein that inappropriately accumulates in the endoplasmic reticulum (ER).

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