Cytocompatibility of Graphene Monolayer and Its Impact on Focal Cell Adhesion, Mitochondrial Morphology and Activity in BALB/3T3 Fibroblasts

This study investigates the effect of graphene scaffold on morphology, viability, cytoskeleton, focal contacts, mitochondrial network morphology and activity in BALB/3T3 fibroblasts and provides new data on biocompatibility of the “graphene-family nanomaterials”. We used graphene monolayer applied onto glass cover slide by electrochemical delamination method and regular glass cover slide, as a reference. The morphology of fibroblasts growing on graphene was unaltered, and the cell viability was 95% compared to control cells on non-coated glass slide. There was no significant difference in the cell size (spreading) between both groups studied. Graphene platform significantly increased BALB/3T3 cell mitochondrial activity (WST-8 test) compared to glass substrate. To demonstrate the variability in focal contacts pattern, the effect of graphene on vinculin was examined, which revealed a significant increase in focal contact size comparing to control-glass slide. There was no disruption in mitochondrial network morphology, which was branched and well connected in relation to the control group. Evaluation of the JC-1 red/green fluorescence intensity ratio revealed similar levels of mitochondrial membrane potential in cells growing on graphene-coated and uncoated slides. These results indicate that graphene monolayer scaffold is cytocompatible with connective tissue cells examined and could be beneficial for tissue engineering therapy.

Download Full-text

CED-9 and EGL-1: A Duo Also Regulating Mitochondrial Network Morphology

Molecular Cell ◽

10.1016/j.molcel.2006.03.003 ◽

2006 ◽

Vol 21 (6) ◽

pp. 730-732 ◽

Cited By ~ 7

Author(s):

Jérôme Estaquier ◽

Damien Arnoult

Keyword(s):

Mitochondrial Network ◽

Network Morphology

Download Full-text

Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology

Scientific Reports ◽

10.1038/s41598-020-75899-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ali Rohani ◽

Jennifer A. Kashatus ◽

Dane T. Sessions ◽

Salma Sharmin ◽

David F. Kashatus

Keyword(s):

Single Cell ◽

High Throughput ◽

Data Science ◽

Structural Information ◽

Morphological Changes ◽

Mitochondrial Morphology ◽

Single Cell Level ◽

Mitochondrial Network ◽

Cell Level ◽

Wide Range

Abstract Mitochondria are highly dynamic organelles that can exhibit a wide range of morphologies. Mitochondrial morphology can differ significantly across cell types, reflecting different physiological needs, but can also change rapidly in response to stress or the activation of signaling pathways. Understanding both the cause and consequences of these morphological changes is critical to fully understanding how mitochondrial function contributes to both normal and pathological physiology. However, while robust and quantitative analysis of mitochondrial morphology has become increasingly accessible, there is a need for new tools to generate and analyze large data sets of mitochondrial images in high throughput. The generation of such datasets is critical to fully benefit from rapidly evolving methods in data science, such as neural networks, that have shown tremendous value in extracting novel biological insights and generating new hypotheses. Here we describe a set of three computational tools, Cell Catcher, Mito Catcher and MiA, that we have developed to extract extensive mitochondrial network data on a single-cell level from multi-cell fluorescence images. Cell Catcher automatically separates and isolates individual cells from multi-cell images; Mito Catcher uses the statistical distribution of pixel intensities across the mitochondrial network to detect and remove background noise from the cell and segment the mitochondrial network; MiA uses the binarized mitochondrial network to perform more than 100 mitochondria-level and cell-level morphometric measurements. To validate the utility of this set of tools, we generated a database of morphological features for 630 individual cells that encode 0, 1 or 2 alleles of the mitochondrial fission GTPase Drp1 and demonstrate that these mitochondrial data could be used to predict Drp1 genotype with 87% accuracy. Together, this suite of tools enables the high-throughput and automated collection of detailed and quantitative mitochondrial structural information at a single-cell level. Furthermore, the data generated with these tools, when combined with advanced data science approaches, can be used to generate novel biological insights.

Download Full-text

Constitutive regulation of mitochondrial morphology by Aurora A kinase depends on a predicted cryptic targeting sequence at the N-terminus

Open Biology ◽

10.1098/rsob.170272 ◽

2018 ◽

Vol 8 (6) ◽

pp. 170272 ◽

Cited By ~ 9

Author(s):

Rhys Grant ◽

Ahmed Abdelbaki ◽

Alessia Bertoldi ◽

Maria P. Gavilan ◽

Jörg Mansfeld ◽

...

Keyword(s):

Cancer Progression ◽

Mitochondrial Fraction ◽

Mitochondrial Morphology ◽

Aurora A Kinase ◽

Mitochondrial Network ◽

Aurora A ◽

Mitochondrial Targeting ◽

Cell Extracts ◽

N Terminus ◽

Different Levels

Aurora A kinase (AURKA) is a major regulator of mitosis and an important driver of cancer progression. The roles of AURKA outside of mitosis, and how these might contribute to cancer progression, are not well understood. Here, we show that a fraction of cytoplasmic AURKA is associated with mitochondria, co-fractionating in cell extracts and interacting with mitochondrial proteins by reciprocal co-immunoprecipitation. We have also found that the dynamics of the mitochondrial network are sensitive to AURKA inhibition, depletion or overexpression. This can account for the different mitochondrial morphologies observed in RPE-1 and U2OS cell lines, which show very different levels of expression of AURKA. We identify the mitochondrial fraction of AURKA as influencing mitochondrial morphology, because an N-terminally truncated version of the kinase that does not localize to mitochondria does not affect the mitochondrial network. We identify a cryptic mitochondrial targeting sequence in the AURKA N-terminus and discuss how alternative conformations of the protein may influence its cytoplasmic fate.

Download Full-text

Royal Society: On Hendry's Crystals

Journal of Cell Science ◽

10.1242/jcs.s2-4.15.241 ◽

1864 ◽

Vol s2-4 (15) ◽

pp. 241-246

Author(s):

WILLIAM HENDRY

Keyword(s):

Royal Society ◽

Chemical Nature ◽

Glass Slide ◽

Thin Glass ◽

Similar Treatment ◽

Glass Cover

The author stated that four years since, in attempting to substitute fusion by the blowpipe for cement, in fixing their glass covers to slides, he noticed masses of crystals produced in the covers after the treatment, and believing them to be unkown, he named them after himself. To obtain the crytals he heats a thin glass cover on a piece of mica, over a spirit-lamp, holding both with forceps; then quickly turning them to the side of the flame, applies a blowpipe, withdrawing the cover to the apex of the flame for a few moments. An examination with a 1 or ½inch objective will then show the crystals. Similar results were ottseryed in a thin glass slide, after a similar treatment, when examined with a 1/12th objective. Specimens were sent with the paper, and the author suggests that it would be desirable to ascertain the chemical nature of the crystals, whether a silicate of lead or soda.

Download Full-text

Precise expression of Fis1 is important for glucose responsiveness of beta cells

Journal of Endocrinology ◽

10.1530/joe-16-0111 ◽

2016 ◽

Vol 230 (1) ◽

pp. 81-91 ◽

Cited By ~ 11

Author(s):

Julia Schultz ◽

Rica Waterstradt ◽

Tobias Kantowski ◽

Annekatrin Rickmann ◽

Florian Reinhardt ◽

...

Keyword(s):

Insulin Secretion ◽

Beta Cells ◽

Pancreatic Beta Cells ◽

Mitochondrial Dynamics ◽

Mitochondrial Morphology ◽

Mitochondrial Network ◽

Primary Mouse ◽

Glucose Stimulated Insulin Secretion ◽

Glucose Responsiveness ◽

Precise Expression

Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.

Download Full-text

Endophilin B1 is required for the maintenance of mitochondrial morphology

The Journal of Cell Biology ◽

10.1083/jcb.200407046 ◽

2004 ◽

Vol 166 (7) ◽

pp. 1027-1039 ◽

Cited By ~ 178

Author(s):

Mariusz Karbowski ◽

Seon-Yong Jeong ◽

Richard J. Youle

Keyword(s):

Mitochondrial Membrane ◽

Fatty Acyl ◽

Membrane Dynamics ◽

Mitochondrial Morphology ◽

Outer Mitochondrial Membrane ◽

Mitochondrial Network ◽

Truncated Protein ◽

The Matrix ◽

Membrane Compartment ◽

Mitochondrial Distribution

We report that a fatty acyl transferase, endophilin B1, is required for maintenance of mitochondrial morphology. Down-regulation of this protein or overexpression of endophilin B1 lacking the NH2-terminal lipid-modifying domain causes striking alterations of the mitochondrial distribution and morphology. Dissociation of the outer mitochondrial membrane compartment from that of the matrix, and formation of vesicles and tubules of outer mitochondrial membrane, was also observed in both endophilin B1 knockdown cells and after overexpression of the truncated protein, indicating that endophilin B1 is required for the regulation of the outer mitochondrial membrane dynamics. We also show that endophilin B1 translocates to the mitochondria during the synchronous remodeling of the mitochondrial network that has been described to occur during apoptosis. Double knockdown of endophilin B1 and Drp1 leads to a mitochondrial phenotype identical to that of the Drp1 single knockdown, a result consistent with Drp1 acting upstream of endophilin B1 in the maintenance of morphological dynamics of mitochondria.

Download Full-text

Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency

AJP Cell Physiology ◽

10.1152/ajpcell.00104.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. C881-C890 ◽

Cited By ~ 121

Author(s):

Werner J. H. Koopman ◽

Henk-Jan Visch ◽

Sjoerd Verkaart ◽

Lambertus W. P. J. van den Heuvel ◽

Jan A. M. Smeitink ◽

...

Keyword(s):

Form Factor ◽

Aspect Ratio ◽

Complex I ◽

Mitochondrial Morphology ◽

Nadh:Ubiquinone Oxidoreductase ◽

Mitochondrial Network ◽

Mitochondrial Structure ◽

Complex I Deficiency ◽

Complex I Activity ◽

Human Complex

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

Download Full-text