The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

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Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix

The Journal of Cell Biology ◽

10.1083/jcb.140.4.821 ◽

1998 ◽

Vol 140 (4) ◽

pp. 821-829 ◽

Cited By ~ 160

Author(s):

Arthur Partikian ◽

Bence Ölveczky ◽

R. Swaminathan ◽

Yuxin Li ◽

A.S. Verkman

Keyword(s):

Green Fluorescent Protein ◽

Aqueous Phase ◽

Correlation Time ◽

Fluorescent Protein ◽

Rotational Correlation Time ◽

Mitochondrial Matrix ◽

Phase Diffusion ◽

The Matrix ◽

Green Fluorescent ◽

Metabolite Channeling

Abstract. It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100× objective (0.8-μm spot diam) gave half-times for fluorescence recovery of 15–19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2–3 × 10−7 cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid β-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 ± 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, ∼0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.

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Mmm1p, a Mitochondrial Outer Membrane Protein, Is Connected to Mitochondrial DNA (Mtdna) Nucleoids and Required for Mtdna Stability

The Journal of Cell Biology ◽

10.1083/jcb.152.2.401 ◽

2001 ◽

Vol 152 (2) ◽

pp. 401-410 ◽

Cited By ~ 157

Author(s):

Alyson E. Aiken Hobbs ◽

Maithreyan Srinivasan ◽

J. Michael McCaffery ◽

Robert E. Jensen

Keyword(s):

Mitochondrial Dna ◽

Outer Membrane ◽

Fluorescent Protein ◽

Mitochondrial Outer Membrane ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Saccharomyces Cerevisiae Mitochondria ◽

The Matrix ◽

Green Fluorescent

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p–green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.

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Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms

Journal of Water and Health ◽

10.2166/wh.2013.208 ◽

2013 ◽

Vol 11 (4) ◽

pp. 629-635 ◽

Cited By ~ 6

Author(s):

L. M. Schaefer ◽

V. S. Brözel ◽

S. N. Venter

Keyword(s):

Drinking Water ◽

Green Fluorescent Protein ◽

Health Risk ◽

Salmonella Typhimurium ◽

Fluorescent Protein ◽

Laboratory Scale ◽

Green Fluorescent ◽

In Situ Detection

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.

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The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

The Journal of Cell Biology ◽

10.1083/jcb.152.2.385 ◽

2001 ◽

Vol 152 (2) ◽

pp. 385-400 ◽

Cited By ~ 115

Author(s):

Patrick Heun ◽

Thierry Laroche ◽

M.K. Raghuraman ◽

Susan M. Gasser

Keyword(s):

Nuclear Envelope ◽

Chromatin Structure ◽

Fluorescent Protein ◽

Time Lapse ◽

Yeast Cells ◽

G1 Phase ◽

Nuclear Pore ◽

Origin Firing ◽

Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.721-732.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 721-732 ◽

Cited By ~ 216

Author(s):

Søren Møller ◽

Claus Sternberg ◽

Jens Bo Andersen ◽

Bjarke Bak Christensen ◽

Juan Luis Ramos ◽

...

Keyword(s):

Gene Expression ◽

Microbial Communities ◽

Pure Culture ◽

Fluorescent Protein ◽

Confocal Laser ◽

Specific Gene ◽

Community Members ◽

Metabolic Interactions ◽

Green Fluorescent

ABSTRACT Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of anAcinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and themeta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied inP. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from thePu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pmpromoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction ofPm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

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Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo

Blood Advances ◽

10.1182/bloodadvances.2018020156 ◽

2018 ◽

Vol 2 (20) ◽

pp. 2589-2606 ◽

Cited By ~ 1

Author(s):

Chris Moore ◽

Joanna L. Richens ◽

Yasmin Hough ◽

Deniz Ucanok ◽

Sunir Malla ◽

...

Keyword(s):

Cell Fate ◽

Fluorescent Protein ◽

Erythroid Differentiation ◽

Transcriptional Repressors ◽

Rna Seq ◽

Promote Cell Proliferation ◽

Cell Fate Decisions ◽

Maturation Defect ◽

Green Fluorescent

Abstract The transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebrafish, prRBCs express 2 of 3 zebrafish Gfi1/1b paralogs, Gfi1aa and Gfi1b. The recently identified zebrafish gfi1aa gene trap allele qmc551 drives erythroid green fluorescent protein (GFP) instead of Gfi1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of Gfi1b in gfi1aaqmc551 homozygous embryos. To study the transcriptome of the Gfi1aa/1b double-depleted cells, we performed an RNA-Seq experiment on GFP-positive prRBCs sorted from 20-hour-old embryos that were heterozygous or homozygous for gfi1aaqmc551, as well as wt or morphant for gfi1b. We subsequently confirmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combined, the data showed that in the absence of Gfi1aa, the synchronously developing prRBCs were delayed in activating late erythroid differentiation, as they struggled to suppress early erythroid and endothelial transcription programs. The latter highlighted the bipotent nature of the progenitors from which prRBCs arise. In the absence of Gfi1aa, Gfi1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed Gfi1aa-depleted prRBCs even further, showing that Gfi1aa and Gfi1b together set the pace for prRBC differentiation from hemangioblasts.

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Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI + ] by Causing Dissolution of the Prion Seeds

Eukaryotic Cell ◽

10.1128/ec.00300-13 ◽

2014 ◽

Vol 13 (5) ◽

pp. 635-647 ◽

Cited By ~ 38

Author(s):

Yang-Nim Park ◽

Xiaohong Zhao ◽

Yang-In Yim ◽

Horia Todor ◽

Robyn Ellerbrock ◽

...

Keyword(s):

Molecular Chaperone ◽

Fluorescent Protein ◽

Yeast Cells ◽

Yeast Prion ◽

Yeast Prions ◽

Daughter Cells ◽

Amyloid Aggregates ◽

Mother And Daughter ◽

Green Fluorescent ◽

Kinetics Of

ABSTRACT The [ PSI + ] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [ PSI + ], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [ PSI + ]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [ PSI + ] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [ PSI + ] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

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Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in Blastomyces dermatitidis Illuminates the Role of Septin in Morphogenesis and Sporulation

Eukaryotic Cell ◽

10.1128/ec.00401-06 ◽

2007 ◽

Vol 6 (8) ◽

pp. 1299-1309 ◽

Cited By ~ 24

Author(s):

T. Krajaejun ◽

G. M. Gauthier ◽

C. A. Rappleye ◽

T. D. Sullivan ◽

B. S. Klein

Keyword(s):

Green Fluorescent Protein ◽

Rna Interference ◽

Gene Silencing ◽

Rapid Detection ◽

Target Gene ◽

Fluorescent Protein ◽

Yeast Cells ◽

Blastomyces Dermatitidis ◽

Dimorphic Fungus ◽

Green Fluorescent

ABSTRACT A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

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