47 research outputs found
Regulation and quantification of cellular mitochondrial morphology and content
Mitochondria play a key role in signal transduction, redox homeostasis and cell survival, which extends far beyond their classical functioning in ATP production and energy metabolism. In living cells, mitochondrial content (āmitochondrial massā) depends on the cell-controlled balance between mitochondrial biogenesis and degradation. These processes are intricately linked to changes in net mitochondrial morphology and spatiotemporal positioning (āmitochondrial dynamicsā), which are governed by mitochondrial fusion, fission and motility. It is becoming increasingly clear that mitochondrial mass and dynamics, as well as its ultrastructure and volume, are mechanistically linked to mitochondrial function and the cell. This means that proper quantification of mitochondrial morphology and content is of prime importance in understanding mitochondrial and cellular physiology in health and disease. This review first presents how cellular mitochondrial content is regulated at the level of mitochondrial biogenesis, degradation and dynamics. Next we discuss how mitochondrial dynamics and content can be analyzed with a special emphasis on quantitative live-cell microscopy strategies.acceptedVersio
Mitochondrial DNA mutations affect calcium handling in differentiated neurons
Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeostasis. We therefore examined calcium responses in the neurons derived from various ācybridā embryonic stem cell lines carrying different mitochondrial DNA mutations. Brief (ā¼50 ms), focal glutamatergic stimuli induced a transient rise in intracellular calcium concentration, which was visualized by bulk loading the cells with the calcium dye, Oregon Green BAPTA-1. Calcium entered the neurons through N-methyl-d-aspartic acid and voltage-gated calcium channels, as has been described in many other neuronal classes. Intriguingly, while mitochondrial mutations did not affect the calcium transient in response to single glutamatergic stimuli, they did alter the responses to repeated stimuli, with each successive calcium transient decaying ever more slowly in mitochondrial mutant cell lines. A train of stimuli thus caused intracellular calcium in these cells to be significantly elevated for many tens of seconds. These results suggest that calcium-handling deficits are likely to contribute to the pathological phenotype seen in patients with mitochondrial DNA mutations
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Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing.
Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium
Regulation and quantification of cellular mitochondrial morphology and content
Mitochondria play a key role in signal transduction, redox homeostasis and cell survival, which extends far beyond their classical functioning in ATP production and energy metabolism. In living cells, mitochondrial content (āmitochondrial massā) depends on the cell-controlled balance between mitochondrial biogenesis and degradation. These processes are intricately linked to changes in net mitochondrial morphology and spatiotemporal positioning (āmitochondrial dynamicsā), which are governed by mitochondrial fusion, fission and motility. It is becoming increasingly clear that mitochondrial mass and dynamics, as well as its ultrastructure and volume, are mechanistically linked to mitochondrial function and the cell. This means that proper quantification of mitochondrial morphology and content is of prime importance in understanding mitochondrial and cellular physiology in health and disease. This review first presents how cellular mitochondrial content is regulated at the level of mitochondrial biogenesis, degradation and dynamics. Next we discuss how mitochondrial dynamics and content can be analyzed with a special emphasis on quantitative live-cell microscopy strategies
Controlling TāCell Activation with Synthetic Dendritic Cells Using the Multivalency Effect
Artificial
antigen-presenting cells (aAPCs) have recently gained
a lot of attention. They efficiently activate T cells and serve as
powerful replacements for dendritic cells in cancer immunotherapy.
Focusing on a specific class of polymer-based aAPCs, so-called synthetic
dendritic cells (sDCs), we have investigated the importance of multivalent
binding on T-cell activation. Using antibody-functionalized sDCs,
we have tested the influence of polymer length and antibody density.
Increasing the multivalent character of the antibody-functionalized
polymer lowered the effective concentration required for T-cell activation.
This was evidenced for both early and late stages of activation. The
most important effect observed was the significantly prolonged activation
of the stimulated T cells, indicating that multivalent sDCs sustain
T-cell signaling. Our results highlight the importance of multivalency
for the design of aAPCs and will ultimately allow for better mimics
of natural dendritic cells that can be used as vaccines in cancer
treatment