scholarly journals The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

1997 ◽  
Vol 137 (1) ◽  
pp. 141-153 ◽  
Author(s):  
Greg J. Hermann ◽  
Edward J. King ◽  
Janet M. Shaw
Keyword(s):  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Mitochondrial Inheritance ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Polarized Transport ◽  
Synthetically Lethal ◽  
Mutant Phenotypes ◽  
Actin Cables

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

scholarly journals GCS1, an Arf Guanosine Triphosphatase-activating Protein in Saccharomyces cerevisiae, Is Required for Normal Actin Cytoskeletal Organization In Vivo and Stimulates Actin Polymerization In Vitro

1999 ◽  
Vol 10 (3) ◽  
pp. 581-596 ◽  
Author(s):  
Ira J. Blader ◽  
M. Jamie T. V. Cope ◽  
Trevor R. Jackson ◽  
Adam A. Profit ◽  
Angela F. Greenwood ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Synthetic Lethality ◽  
Null Alleles ◽  
Gene Encoding ◽  
Amino Terminal ◽  
Guanosine Triphosphatase

Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate binding protein, centaurin α, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin α is Gcs1p, the product of theGCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin α, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novelGCS1 disruption strain (gcs1Δ) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Δ was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 andSLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 andSAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 693-709 ◽  
Author(s):  
T S Karpova ◽  
M M Lepetit ◽  
J A Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Actin Binding ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Synthetic Lethal ◽  
Capping Protein ◽  
Colony Color ◽  
Actin Cables

Abstract Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

scholarly journals ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells

Blood ◽  
2016 ◽  
Vol 127 (5) ◽  
pp. 582-595 ◽  
Author(s):  
Marwan Kwok ◽  
Nicholas Davies ◽  
Angelo Agathanggelou ◽  
Edward Smith ◽  
Ceri Oldreive ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Synthetic Lethality ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Selective Cytotoxicity ◽  
Key Points ◽  
Synthetically Lethal

Key PointsATR inhibition is synthetically lethal to TP53- or ATM-defective CLL cells. ATR targeting induces selective cytotoxicity and chemosensitization in TP53- or ATM-defective CLL cells in vitro and in vivo.

scholarly journals Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

2004 ◽  
Vol 3 (6) ◽  
pp. 1574-1588 ◽  
Author(s):  
R. Martin ◽  
A. Walther ◽  
J. Wendland
Keyword(s):  
Candida Albicans ◽  
Heavy Chain ◽  
Time Lapse ◽  
Yeast Cells ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Mutant Strains ◽  
Mutant Phenotypes

ABSTRACT Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1.

scholarly journals The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton.

1996 ◽  
Vol 134 (1) ◽  
pp. 117-132 ◽  
Author(s):  
V Moreau ◽  
A Madania ◽  
R P Martin ◽  
B Winson
Keyword(s):  
Actin Cytoskeleton ◽  
Genetic Interaction ◽  
Lucifer Yellow ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Gene Encoding ◽  
Membrane Growth ◽  
Mutant Phenotypes ◽  
Mutant Cells

Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2-H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament-associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis.

Synthetic-lethal interactions between ATR inhibition and functional genetic and proteomic biomarkers to predict responses in endometriosis-associated cancers.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18000-e18000
Author(s):  
Oren Gilad ◽  
Dansu Li ◽  
Erin George ◽  
Rakesh Chettier ◽  
Fiona Simpkins ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
In Vivo Studies ◽  
Multiple Cancer ◽  
Driver Genes ◽  
Synthetic Lethal ◽  
Cancer Driver ◽  
Synthetic Lethal Interactions ◽  
Synthetically Lethal

e18000 Background: Endometriosis is a common gynecologic disorder proven to be a precursor to several cancer types. We developed a potent and selective inhibitor (ATRN-119) of a critical DNA damage response (DDR) protein kinase: the ataxia telangiectasia and Rad3-related protein (ATR). Treatment with ATRN-119 is synthetically lethal with multiple cancer-associated changes in DDR pathways, representing a new and effective strategy to treat cancer. The objective of this study is to evaluate the overlap of DDR genes that respond to ATRN-119 and those mutated in endometriosis. Methods: We sequenced the exomes of 2,932 unrelated women with surgically-confirmed endometriosis (GERMLINE) and 274 tissue blocks containing endometriosis lesions (LESION). DNA was extracted using standard methods. Missense and truncation variants were analyzed. These data were compared to analysis of a whole proteome screen for factors that respond to exposure to ATRN-119 and may influence responsiveness to treatment. Factors observed in both methods were considered high-priority biomarker candidates and were experimentally tested for synthetic lethality with ATRN-119 treatment. Results: Analysis of endometriosis patients found 89% of the LESION samples had 2 or more DDR mutations vs 83% of the GERMLINE samples. There is an excess of DDR mutations per sample in LESION (5.5 mutations) vs GERMLINE (3.89 mutations) [p = 4.66x10-6, Mann Whitney test]. In parallel, we identified 92 genes as protein responders to ATRN-119 treatment. Mutations in 21 of these 92 genes show nominal association with surgical endometriosis (p < 0.05). However, of these responsive genes, 18 are known TIER 1 cancer-driver genes and well-characterized mutations were found in three dominant genes in the LESION tissue (ATM, DDB1, and ARID1A). Overall 20% of the patients who’s LESION we examined subsequently developed an endometriosis-associated cancer. Both in vitro and in vivo studies confirmed synthetic-lethal interactions between ATRN-119 treatment and alteration of these genes. Conclusions: The overlap between DDR genes responding to ATRN-119 and those mutated in endometriosis-associated cancer suggest that genetic markers underlying response and resistance will be critical to extend the use of these drugs while increasing efficacy and minimizing toxicities. Furthermore, our data support the inclusion of endometriosis-associated cancer patients in planned ATRN-119 clinical trials.

scholarly journals Dependence of ORC Silencing Function on NatA-Mediated Nα Acetylation in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (23) ◽  
pp. 10300-10312 ◽  
Author(s):  
Antje Geissenhöner ◽  
Christoph Weise ◽  
Ann E. Ehrenhofer-Murray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Modification ◽  
Origin Recognition Complex ◽  
Synthetic Lethality ◽  
Large Subunit ◽  
Functional Link ◽  
Repressive Chromatin ◽  
N Terminus ◽  
Synthetically Lethal

ABSTRACT Nα acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures. Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing. Orc1p was Nα acetylated in vivo by NatA. Mutations that abrogated its ability to be acetylated caused strong telomeric derepression. Thus, Nα acetylation of Orc1p represents a protein modification that modulates chromatin function in S. cerevisiae. Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Δ was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Δ and SUM1-1 required the Orc1 N terminus. We also found Sir3p to be acetylated by NatA. In summary, we propose a model by which Nα acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression.

scholarly journals The myosin-related motor protein Myo2 is an essential mediator of bud-directed mitochondrial movement in yeast

2011 ◽  
Vol 194 (3) ◽  
pp. 473-488 ◽  
Author(s):  
Johannes Förtsch ◽  
Eric Hummel ◽  
Melanie Krist ◽  
Benedikt Westermann
Keyword(s):  
Synthetic Lethality ◽  
Motor Protein ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Gene Encoding ◽  
Mitochondrial Movement ◽  
Isolated Mitochondria ◽  
Mitochondrial Distribution ◽  
Synthetically Lethal ◽  
Guanosine Triphosphatase

The inheritance of mitochondria in yeast depends on bud-directed transport along actin filaments. It is a matter of debate whether anterograde mitochondrial movement is mediated by the myosin-related motor protein Myo2 or by motor-independent mechanisms. We show that mutations in the Myo2 cargo binding domain impair entry of mitochondria into the bud and are synthetically lethal with deletion of the YPT11 gene encoding a rab-type guanosine triphosphatase. Mitochondrial distribution defects and synthetic lethality were rescued by a mitochondria-specific Myo2 variant that carries a mitochondrial outer membrane anchor. Furthermore, immunoelectron microscopy revealed Myo2 on isolated mitochondria. Thus, Myo2 is an essential and direct mediator of bud-directed mitochondrial movement in yeast. Accumulating genetic evidence suggests that maintenance of mitochondrial morphology, Ypt11, and retention of mitochondria in the bud contribute to Myo2-dependent inheritance of mitochondria.

scholarly journals N-myristoyltransferase inhibition is synthetic lethal in MYC-deregulated cancers

2021 ◽  
Author(s):  
Gregor A. Lueg ◽  
Monica Faronato ◽  
Andrii Gorelik ◽  
Andrea Goya Grocin ◽  
Eva Caamano-Gutierrez ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Complex I ◽  
Protein Modification ◽  
Large Scale ◽  
Synthetic Lethality ◽  
Cancer Cell Line ◽  
New Paradigm ◽  
Synthetic Lethal ◽  
Synthetically Lethal

Human N-myristoyltransferases (NMTs) catalyze N-terminal protein myristoylation, a modification regulating membrane trafficking and interactions of >100 proteins. NMT is a promising target in cancer, but a mechanistic rationale for targeted therapy remains poorly defined. Here, large-scale cancer cell line screens against a panel of NMT inhibitors (NMTi) were combined with systems-level analyses to reveal that NMTi is synthetic lethal with deregulated MYC. Synthetic lethality is mediated by post-transcriptional failure in mitochondrial respiratory complex I protein synthesis concurrent with loss of myristoylation and degradation of complex I assembly factor NDUFAF4, followed by mitochondrial dysfunction specifically in MYC-deregulated cancer cells. NMTi eliminated MYC-deregulated tumors in vivo without overt toxicity, providing a new paradigm in which targeting a constitutive co-translational protein modification is synthetically lethal in MYC-deregulated cancers.

scholarly journals The Yeast Dynamin-like Protein, Mgm1p, Functions on the Mitochondrial Outer Membrane to Mediate Mitochondrial Inheritance

1999 ◽  
Vol 144 (4) ◽  
pp. 711-720 ◽  
Author(s):  
Kelly A. Shepard ◽  
Michael P. Yaffe
Keyword(s):  
Mitochondrial Genome ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Mitochondrial Inheritance ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Molecular Weight Complex ◽  
Mitochondrial Distribution ◽  
Mutant Phenotypes

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37°C, while loss of the mitochondrial genome occurred after 4–24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

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