Biomedical subjects

Janet M Shaw

Publications and source records attributed to Janet M Shaw.

13 recordsLinked to original sources

PubMed2006

The novel F-box protein Mfb1p regulates mitochondrial connectivity and exhibits asymmetric localization in yeast.

Although it is clear that mitochondrial morphogenesis is a complex process involving multiple proteins in eukaryotic cells, little is known about regulatory molecules that modulate mitochondrial network formation. Here, we report the identification of a new yeast mitochondrial morphology gene called MFB1 (YDR219C). MFB1 encodes an F-box protein family member, many of which function in Skp1-Cdc53/Cullin-F-box protein (SCF) ubiquitin ligase complexes. F-box proteins also act in non-SCF complexes whose functions are not well understood. Although cells lacking Mfb1p contain abnormally short mitochondrial tubules, Mfb1p is not essential for known pathways that determine mitochondrial morphology and dynamics. Mfb1p is peripherally associated with the mitochondrial surface. Coimmunoprecipitation assays reveal that Mfb1p interacts with Skp1p in an F-box-dependent manner. However, Mfb1p does not coimmunoprecipitate with Cdc53p. The F-box motif is not essential for Mfb1p-mediated mitochondrial network formation. These observations suggest that Mfb1p acts in a complex lacking Cdc53p required for mitochondrial morphogenesis. During budding, Mfb1p asymmetrically localizes to mother cell mitochondria. By contrast, Skp1p accumulates in the daughter cell cytoplasm. Mfb1p mother cell-specific asymmetry depends on the F-box motif, suggesting that Skp1p down-regulates Mfb1p mitochondrial association in buds. We propose that Mfb1p operates in a novel pathway regulating mitochondrial tubular connectivity.

Amino Acid Motifs↗

PubMed2006

Dimeric Dnm1-G385D interacts with Mdv1 on mitochondria and can be stimulated to assemble into fission complexes containing Mdv1 and Fis1.

Interactions between yeast Dnm1p, Mdv1p, and Fis1p are required to form fission complexes that catalyze division of the mitochondrial compartment. During the formation of mitochondrial fission complexes, the Dnm1p GTPase self-assembles into large multimeric complexes on the outer mitochondrial membrane that are visualized as punctate structures by fluorescent labeling. Although it is clear that Fis1p.Mdv1p complexes on mitochondria are required for the initial recruitment of Dnm1p, it is not clear whether Dnm1p puncta assemble before or after this recruitment step. Here we show that the minimum oligomeric form of cytoplasmic Dnm1p is a dimer. The middle domain mutant protein Dnm1G385Dp forms dimers in vivo but fails to assemble into punctate structures. However, this dimeric mutant stably interacts with Mdv1p on the outer mitochondrial membrane, demonstrating that assembly of stable Dnm1p multimers is not required for Dnm1p-Mdv1p association or for mitochondrial recruitment of Dnm1p. Dnm1G385Dp is reported to be a terminal dimer in vitro. We describe conditions that allow assembly of Dnm1G385Dp into functional fission complexes on mitochondria in vivo. Using these conditions, we demonstrate that multimerization of Dnm1p is required to promote reorganization of Mdv1p from a uniform mitochondrial localization into punctate fission complexes. Our studies also reveal that Fis1p is present in these assembled fission complexes. Based on our results, we propose that Dnm1p dimers are initially recruited to the membrane via interaction with Mdv1p.Fis1p complexes. These dimers then assemble into multimers that subsequently promote the reorganization of Mdv1p into punctate fission complexes.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Development and reproducibility of the bone loading history questionnaire.

PURPOSE: To develop an historical bone loading questionnaire that assesses loads applied to the skeleton using bone loading units and to determine its reproducibility. Additional purposes were to correlate bone loading units with areal bone mineral density (aBMD) and to determine whether low levels of bone loading increase the odds for low aBMD in healthy premenopausal women. METHODS: Premenopausal women (N=80; age: 31+/-7.7 yr) completed the newly developed bone loading history questionnaire (BLHQ) twice within 4-6 wk to establish reproducibility. aBMD (g.cm) of the whole body, lumbar spine (L2-4), and right hip (proximal femur) were assessed with dual energy x-ray absorptiometry (DXA) in the women. RESULTS: The questionnaire demonstrated good test-retest reliability in premenopausal women using intraclass correlation coefficients (spine r=0.89, P<0.001; hip r=0.92, P<0.001). Total spine bone loading exposure (r=0.338; P=0.002) and hip bone loading exposure (r=0.317; P=0.004) were significantly and independently correlated with femoral neck aBMD, after adjusting for BMI. Logistic regression analyses revealed increased odds for low femoral neck aBMD among those in the lowest tertile for recent hip bone loading exposure after adjusting for older ages, oral contraceptive use, low calcium intake, and an overweight BMI (OR=3.62; CI=1.09-11.94; P=0.035). CONCLUSION: Historical bone loading activity can be reliably assessed and is moderately associated with femoral neck aBMD. This questionnaire could help to identify women with low femoral neck aBMD prior to menopause.

Absorptiometry, Photon↗

PubMed2005

The role of Fis1p-Mdv1p interactions in mitochondrial fission complex assembly.

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)-like fold and a short NH(2)-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p-Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH(2)-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes.

Mitochondria form dynamic tubular networks that continually change their shape and move throughout the cell. In eukaryotes, these organellar gymnastics are controlled by numerous pathways that preserve proper mitochondrial morphology and function. The best understood of these are the fusion and fission pathways, which rely on conserved GTPases and their binding partners to regulate organelle connectivity and copy number in healthy cells and during apoptosis. In budding yeast, mitochondrial shape is also maintained by proteins acting in the tubulation pathway. Novel proteins and pathways that control mitochondrial dynamics continue to be discovered, indicating that the mechanisms governing this organelle's behavior are more sophisticated than previously appreciated. Here we review recent advances in the field of mitochondrial dynamics and highlight the importance of these pathways to human health.

Apoptosis↗

PubMed2004

Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway.

Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. We show that Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. We demonstrate that Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells (Fransson et al., 2003. J. Biol. Chem. 278:6495-6502), Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

Actins↗

PubMed2004

Importance of mitochondrial dynamics during meiosis and sporulation.

Opposing fission and fusion events maintain the yeast mitochondrial network. Six proteins regulate these membrane dynamics during mitotic growth-Dnm1p, Mdv1p, and Fis1p mediate fission; Fzo1p, Mgm1p, and Ugo1p mediate fusion. Previous studies established that mitochondria fragment and rejoin at distinct stages during meiosis and sporulation, suggesting that mitochondrial fission and fusion are required during this process. Here we report that strains defective for mitochondrial fission alone, or both fission and fusion, complete meiosis and sporulation. However, visualization of mitochondria in sporulating cultures reveals morphological defects associated with the loss of fusion and/or fission proteins. Specifically, mitochondria collapse to one side of the cell and fail to fragment during presporulation. In addition, mitochondria are not inherited equally by newly formed spores, and mitochondrial DNA nucleoid segregation defects give rise to spores lacking nucleoids. This nucleoid inheritance defect is correlated with an increase in petite spore colonies. Unexpectedly, mitochondria fragment in mature tetrads lacking fission proteins. The latter finding suggests either that novel fission machinery operates during sporulation or that mechanical forces generate the mitochondrial fragments observed in mature spores. These results provide evidence of fitness defects caused by fission mutations and reveal new phenotypes associated with fission and fusion mutations.

Cell Respiration↗

PubMed2004

Bone mineral density of olympic-level female winter sport athletes.

PURPOSE: To compare areal bone mineral density (aBMD) of female winter sport athletes to healthy controls of similar age and body mass index (BMI). METHODS: Areal BMD (g x cm(-2)) of the whole body, lumbar spine (L2-L4), and right proximal femur were assessed by dual energy x-ray absorptiometry in athletes (N = 40; age: 26.1 +/- 5.7 yr; ht: 165.6 +/- 0.1 cm; wt: 63.0 +/- 6.5 kg; BMI: 23.0 +/- 1.9 kg x m(-2)) involved in speed skating (N = 9), snowboarding (N = 13), freestyle skiing (N = 3), biathlon (N = 8), bobsleigh, skeleton, luge (N = 7), and controls (N = 21; age: 26.0 +/- 5.1 yr; ht: 165.8 +/- 0.1 cm; wt: 62.8 +/- 5.9 kg; BMI: 22.9 +/- 1.3 kg x m(-2)). RESULTS: Using independent t-tests, athletes had lower fat mass, percent body fat, and higher lean mass than controls (P < 0.001). Areal BMD was higher in athletes than controls for all skeletal sites (P </= 0.007). With lean tissue mass as a covariate (ANCOVA), differences in aBMD remained significant for most skeletal sites (P </= 0.016). Menstrual history, mean daily calcium intake, and oral contraceptive use were not associated with aBMD in the athletic group. CONCLUSION: Results show that female winter sport athletes have greater aBMD compared with controls of similar age and BMI. Most aBMD differences remained significant after adjusting for lean tissue mass, and athletes with a history of oligo- and/or amenorrhea had similar aBMD than their eumenorrheic counterparts. This is the first study to examine aBMD in winter sport athletes. The results support the hypothesis that the loading characteristics of intense winter sport participation have osteogenic potential.

Adult↗

PubMed2003

Mmm1p spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation.

In the yeast Saccharomyces cerevisiae, the integral membrane protein Mmm1p is required for maintenance of mitochondrial morphology and retention of mitochondrial DNA (mtDNA). Mmm1p localizes to discrete foci on mitochondria that are adjacent to mtDNA nucleoids in the matrix, raising the possibility that this protein plays a direct role in organizing, replicating, or segregating mtDNA. Although Mmm1p has been shown to cross the outer membrane with its C terminus facing the cytoplasm, the location of the N terminus has not been resolved. Here we show that Mmm1p spans both the outer and inner mitochondrial membranes, exposing its N terminus to the matrix. Surprisingly, deletion of the N-terminal extension decreased steady-state levels of the Mmm1 protein but did not affect mitochondrial morphology or mtDNA maintenance. Moreover, expression of Neurospora crassa MMM1, which naturally lacks a long N-terminal extension, substituted for loss of Mmm1p in budding yeast. These results indicate that the matrix-exposed portion of Mmm1p is not essential for mtDNA nucleoid maintenance. Additional studies revealed that the transmembrane segment and C-terminal domain of Mmm1p are required for foci formation and mitochondrial targeting, respectively. Our data suggest that the double membrane-spanning topology of Mmm1p at the membrane contact site is critical for formation of tubular mitochondria.

Intracellular Membranes↗

PubMed2003

Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin-actin interactions in budding yeast.

The evolutionarily conserved Mdm20 protein (Mdm20p) plays an important role in tropomyosin-F-actin interactions that generate actin filaments and cables in budding yeast. However, Mdm20p is not a structural component of actin filaments or cables, and its exact function in cable stability has remained a mystery. Here, we show that cells lacking Mdm20p fail to N-terminally acetylate Tpm1p, an abundant form of tropomyosin that binds and stabilizes actin filaments and cables. The F-actin-binding activity of unacetylated Tpm1p is reduced severely relative to the acetylated form. These results are complemented by the recent report that Mdm20p copurifies with one of three acetyltransferases in yeast, the NatB complex. We present genetic evidence that Mdm20p functions cooperatively with Nat3p, the catalytic subunit of the NatB acetyltransferase. These combined results strongly suggest that Mdm20p-dependent, N-terminal acetylation of Tpm1p by the NatB complex is required for Tpm1p association with, and stabilization of, actin filaments and cables.

Acetylation↗

PubMed2003

A fuzzy mitochondrial fusion apparatus comes into focus.

Membrane fusion is fundamental to eukaryotic life. Unlike the predominant intracellular fusion machineries that fuse compartments bounded by a single membrane, the mitochondrial fusion machinery must sequentially fuse the outer and inner mitochondrial membranes. These coordinated fusion events rely on a transmembrane GTPase that is known as fuzzy onions or Fzo. Recent studies have revealed that Fzo has an evolutionarily conserved role in mitochondrial fusion, and they take the first strides in determining the molecular nature of such a role.

Amino Acid Motifs↗

PubMed2002

Mitochondrial dynamics and division in budding yeast.

Mitochondria adopt a variety of different shapes in eukaryotic cells, ranging from multiple, small compartments to elaborate tubular networks. The establishment and maintenance of different mitochondrial morphologies depends, in part, on the equilibrium between opposing fission and fusion events. Recent studies in yeast, flies, worms and mammalian cells indicate that three high-molecular-weight GTPases control mitochondrial membrane dynamics. One of these is a dynamin-related GTPase that acts on the outer mitochondrial membrane to regulate fission. Recently, genetic approaches in budding yeast have identified additional components of the fission machinery. These and other new findings suggest a common mechanism for membrane fission events that has been conserved and adapted during eukaryotic evolution.

Cell Cycle↗

PubMed2002

Mitochondrial GFA2 is required for synergid cell death in Arabidopsis.

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.

Amino Acid Sequence↗