Localizing mRNAs Encoding Mitochondrial Proteins in Yeast by Fluorescence Microscopy and Subcellular Fractionation

2017 ◽  
pp. 197-216
Author(s):  
Dmitry Zabezhinsky ◽  
Hannah Sperber ◽  
Jeffrey E. Gerst
Keyword(s):  
Fluorescence Microscopy ◽  
Subcellular Fractionation ◽  
Mitochondrial Proteins

scholarly journals Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA.

1997 ◽  
Vol 8 (7) ◽  
pp. 1233-1242 ◽  
Author(s):  
J Nunnari ◽  
W F Marshall ◽  
A Straight ◽  
A Murray ◽  
J W Sedat ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescence Microscopy ◽  
Fluorescent Dyes ◽  
Mitochondrial Protein ◽  
Mitochondrial Proteins ◽  
Dimensional Structure ◽  
Wide Field ◽  
Protein Markers ◽  
Protein Marker ◽  
Mitochondrial Transmission

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.

The human SIRT3 protein deacetylase is exclusively mitochondrial

2008 ◽  
Vol 411 (2) ◽  
pp. 279-285 ◽  
Author(s):  
Helen M. Cooper ◽  
Johannes N. Spelbrink
Keyword(s):  
Cellular Stress ◽  
Mitochondrial Protein ◽  
Subcellular Fractionation ◽  
Precursor Protein ◽  
Mitochondrial Proteins ◽  
Lysine Acetylation ◽  
Crucial Importance ◽  
Mitochondrial Localization ◽  
Genome Databases ◽  
Protein Deacetylase

It has recently been suggested that perhaps as many as 20% of all mitochondrial proteins are regulated through lysine acetylation while SIRT3 has been implicated as an important mitochondrial protein deacetylase. It is therefore of crucial importance that the mitochondrial localization of potential protein deacetylases is unambiguously established. Although mouse SIRT3 was recently shown to be mitochondrial, HsSIRT3 (human SIRT3) was reported to be both nuclear and mitochondrial and to relocate from the nucleus to the mitochondrion upon cellular stress. In the present study we show, using various HsSIRT3 expression constructs and a combination of immunofluorescence and careful subcellular fractionation, that in contrast with earlier reports HsSIRT3 is exclusively mitochondrial. We discuss possible experimental explanations for these discrepancies. In addition we suggest, on the basis of the analysis of public genome databases, that the full-length mouse SIRT3 protein is a 37 kDa mitochondrial precursor protein contrary to the previously suggested 29 kDa protein.

Vimentin supports mitochondrial morphology and organization

2008 ◽  
Vol 410 (1) ◽  
pp. 141-146 ◽  
Author(s):  
Ho Lam Tang ◽  
Hong Lok Lung ◽  
Ka Chun Wu ◽  
Anh-Huy Phan Le ◽  
Ho Man Tang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Fluorescence Microscopy ◽  
Intermediate Filaments ◽  
Mitochondrial Function ◽  
Subcellular Fractionation ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Structural Support ◽  
Important Relationship ◽  
A Cell

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Localization of Immune Complexes in the Basement Membrane of the Ductuli Efferentes of the Rhesus Monkey

1980 ◽  
Vol 38 ◽  
pp. 456-457
Author(s):  
D. Marsh
Keyword(s):  
Basement Membrane ◽  
Fluorescence Microscopy ◽  
Rhesus Monkey ◽  
Immune Complex ◽  
Immune Complexes ◽  
Vas Deferens ◽  
Frozen Sections ◽  
Efferent Ducts ◽  
Complex Deposition ◽  
Sperm Antibodies

As a result of vasectomy, spermatozoa are confined to the epididymis and vas deferens, where they degenerate, releasing antigens that enter the circulation or are engulfed by macrophages. Multiple antigens of the sperm can elicit production of autoantibodies; circulating anti-sperm antibodies are found in a large percentage of vasectomized men, indicating the immunogenicity of the sperm. The increased prevalence of macrophages in the liomen of the rhesus monkey testicular efferent ducts after vasectomy led to further study of this region. Frozen sections were used for evaluation of immunopathological status by fluorescence microscopy with fluorescein-conjugated antibody. Subsequent granular deposits of immune complexes were revealed by positive immunofluorescence staining for complement. The immune complex deposition in the basement membrane surrounding the efferent ducts implies that this region is involved in antigen leakage (Fig. 1).

On the Role of Transferrin in 67Ga Uptake by Tumor Cells

1988 ◽  
Vol 27 (04) ◽  
pp. 151-153
Author(s):  
P. Thouvenot ◽  
F. Brunotte ◽  
J. Robert ◽  
L. J. Anghileri
Keyword(s):  
Specific Binding ◽  
Subcellular Fractionation ◽  
Animal Blood ◽  
Tumor Bearing ◽  
Cell Structures ◽  
The Difference ◽  
Sarcoma Cells ◽  
In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

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