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PubMed1989

The capacity of normal and talpid3 mutant fowl myogenic cells to migrate in quail limb buds.

Talpid3 is a recessive lethal mutant of the fowl. It has been shown previously that, in vitro, talpid3 limb mesenchyme cells are more adhesive and less mobile than normal cells. It is therefore of interest to investigate the effect of the gene on cell movement in vivo, in the limb bud itself, in cells in which it is known to occur in normal embryos. Myogenic cells, which normally migrate into the limb bud from the somites, continue to move distalwards when grafted into the limb bud at a later stage. Blocks of normal or talpid3 limb mesenchyme containing myogenic cells were transplanted into quail limb buds in ovo. Since quail cells are histologically distinguishable from chick cells the progress of myogenic cell movement 5 days after transplantation could be observed. In 10 out of 14 cases normal myogenic cells migrated extensively in a proximo-distal direction within the limb bud of the quail host. In contrast, only 2 out of 11 talpid3 transplants showed a moderate degree of distalwards movement.

Animals↗

PubMed2002

Distribution and possible function of an adrenomedullin-like peptide in the developing chick limb bud.

Adrenomedullin (AM) is a multifunctional peptide that exhibits discrete domains of expression during mouse embryogenesis consistent with a role in regulating growth and differentiation during morphogenesis. Here we report that AM immunoreactivity is present at high levels throughout the apical ectodermal ridge (AER) of the chick limb bud as the AER is directing the outgrowth and patterning of underlying limb mesoderm. Immunostaining is particularly strong along the surfaces of the contiguous cells of the AER. AM immunoreactivity attenuates as the AER regresses and is absent from the distal apical ectoderm of stage 20 limbless mutant limb buds which fail to develop an AER. To explore the possible role of AM in AER activity, we examined the effect of exogenous AM and an AM inhibitor on the in vitro morphogenesis of limb mesoderm, cultured in the presence and absence of the AER. Although exogenous AM cannot substitute for the AER in promoting outgrowth of limb mesoderm in vitro, a specific AM antagonist, AM(22-52), impairs the outgrowth and proliferation of limb mesoderm cultured in the presence of the AER. This is consistent with the possibility that inhibition of endogenous AM activity in the AER impairs the ability of the AER to promote limb morphogenesis. Taken together, these studies suggest that an AM-like molecule may function in an autocrine fashion to regulate some aspect of AER activity.

Adrenomedullin↗

PubMed1998

Endocytic internalization in yeast and animal cells: similar and different.

The internalization step of endocytosis has been the focus of several laboratories during the last forty years. Unlike some other budding events in the cell, many fundamental questions regarding the molecular machinery involved in the mechanism of budding itself still remain unsolved. Over the last few years the general picture of the field has quickly evolved from the originally simplistic view which postulated that clathrin polymerization is the major force driving budding at the plasma membrane. Refinement of the assays and molecular markers to measure endocytosis in animal cells has shown that other factors in addition to the clathrin coat are required and that endocytosis can also take place through clathrin-independent mechanisms. At the same time, recent introduction of genetic approaches to study endocytosis has accelerated the identification of molecules required for this process. The isolation of endocytosis mutants in budding yeast has been especially fruitful in this respect. Preliminary comparison of the results obtained in yeast and animal cells did not seem to coincide, but further progress in both systems now suggests that part of the divergence originally seen may be due to the particular experimental approaches used rather than fundamental differences in endocytic mechanisms. In this review we present a short historical overview on the advances made in yeast and animal cells regarding the study of endocytosis, underlining both emerging similarities and still interesting differences.

Endocytosis↗

PubMed2006

Revealing hidden relationships among yeast genes involved in chromosome segregation using systematic synthetic lethal and synthetic dosage lethal screens.

The vast accumulation of knowledge from genome sequencing projects and studies with model organisms has presented a remarkable challenge to biologists: to understand the functions of thousands of highly conserved genes and how they work together to regulate fundamental cellular processes. This challenge is compounded by the inescapable reality that most genes are 'buffered' by other genes that contribute to the same biological processes, limiting the impact of phenotypic studies with single mutants. In budding yeast, functional genomic methods have been developed for the systematic application of established genetic techniques. In particular, the Synthetic Genetic Array (SGA) method allows genome-wide synthetic lethal (SL) and synthetic dosage lethal (SDL) screens thus enabling an unbiased survey of genetic interactions. We have used genes encoding components of the yeast kinetochore as a biological testbed for assaying the utility of SGA-based SL and SDL screens for revealing new pathways and genes involved in chromosome segregation. We identified 211 nonessential deletion mutants that were unable to tolerate either overexpression or loss of function of kinetochore genes. Our study uncovered a wealth of relationships between gene products that functionally interact with the kinetochore, and also highlighted the value of performing genome-wide screens with both hypomorphic and hypermorphic alleles of query genes. Here, we will highlight our recent kinetochore SGA genomic screens, in the broader context of applying complementary genetic screening approaches in the systematic exploration of biological pathways or functional complexes.

Cell Division↗

PubMed2002

Mutant membrane protein of the budding yeast spindle pole body is targeted to the endoplasmic reticulum degradation pathway.

Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.

Carrier Proteins↗

PubMed2004

Polyalanine expansion in HOXA13: three new affected families and the molecular consequences in a mouse model.

Polyalanine expansions in two of three large imperfect trinucleotide repeats encoded by the first exon of HOXA13 have been reported in hand-foot-genital syndrome (HFGS). Here we report additional families with expansions in the third repeat of 11 and 12 alanine residues, the latter being the largest expansion reported. We also report a patient with a novel, de novo 8-alanine expansion in the first large repeat. Thus, expansions in all three large HOXA13 polyalanine repeats can cause HFGS. To determine the molecular basis for impaired HOXA13 function, we performed homologous recombination in ES cells in mice to expand the size of the third largest polyalanine tract by 10 residues (HOXA13(ALA28)). Mutant mice were indistinguishable from Hoxa13 null mice. Mutant limb buds had normal steady-state Hoxa13 RNA expression, normal mRNA splicing and reduced levels of steady-state protein. In vitro translation efficiency of the HOXA13(ALA28) protein was normal. Thus, loss of function is secondary to a reduction in the in vivo abundance of the expanded protein likely due to degradation.

Animals↗

PubMed2002

Ubc9 is essential for viability of higher eukaryotic cells.

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.

Animals↗

PubMed1991

Feedback control of mitosis in budding yeast.

We have investigated the feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. We isolated budding yeast mutants sensitive to the anti-microtubule drug benomyl. Mitotic arrest-deficient (mad) mutants are the subclass of benomyl-sensitive mutants in which the completion of mitosis is not delayed in the presence of benomyl and that die as a consequence of their premature exit from mitosis. A number of properties of the mad mutants indicate that they are defective in the feedback control over the exit from mitosis: their killing by benomyl requires passage through mitosis; their benomyl sensitivity can be suppressed by an independent method for delaying the exit from mitosis; they have normal microtubules; and they have increased frequencies of chromosome loss. We cloned MAD2, which encodes a putative calcium-binding protein whose disruption is lethal. We discuss the role of feedback controls in coordinating events in the cell cycle.

Amino Acid Sequence↗

PubMed2004

The Hog1 MAP kinase pathway and the Mec1 DNA damage checkpoint pathway independently control the cellular responses to hydrogen peroxide.

The DNA damage checkpoint pathway and the MAP kinase pathway respond to various forms of environmental as well as endogenous stresses through signal transduction mechanisms involving protein kinases. Both pathways are intertwined in mammalian cells, but potential crosstalk between these two pathways in budding yeast has not been examined yet. We show that the Rad53 checkpoint kinase and the Hog1 MAP kinase of Saccharomyces cerevisiae become phosphorylated upon exposure to hydrogen peroxide, indicative of activation of the DNA damage checkpoint and MAP kinase pathways in response to oxidative stress. Rad53 kinase is equally activated in wild type and in hog1-Delta cells. Likewise, the activation of Hog1 MAP kinase is not dependent on Mec1 kinase, the central checkpoint kinase in budding yeast. Mutants in either pathway are sensitive to hydrogen peroxide and the double mutants exhibit a near perfectly additive phenotype. These data demonstrate that the DNA damage checkpoint pathway and the MAP kinase pathway respond to oxidative stress independently of each other and suggest that these two stress signaling pathways are activated by different types of insults induced by hydrogen peroxide.

Cell Cycle↗

PubMed2000

Cytokinesis without myosin II.

The ability of substrate-anchored Dictyostelium cells to divide without myosin II has opened the possibility of analysing the formation of cleavage furrows in the absence of a contractile ring made of filamentous myosin and actin. Similar possibilities exist in mutants of budding yeast and, less strictly, also in drug-treated mammalian cells. Myosin-II-independent activities in Dictyostelium include the microtubule-induced programming of the cell surface into ruffling areas and regions that are converted into a concave furrow, as well as the translocation of cortexillins and cross-linked membrane proteins towards the cleavage furrow. A centripetal flow of actin filaments followed by their disassembly in the cleavage furrow is proposed to underlie the translocation.

Actins↗

PubMed1998

A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II.

The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis.

Animals↗

PubMed2002

A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae.

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.

Amino Acid Sequence↗

PubMed2005

Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase.

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.

Anaphase↗

PubMed2001

Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin.

Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G(1) cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with ELONGATOR: Intriguingly, strains lacking non-Elongator HATs (gcn5, hat1, hpa3 and sas3) or non-Elongator transcription elongation factors TFIIS (dst1) and Spt4p (spt4) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin.

Acetyltransferases↗

PubMed2001

Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae.

Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

Alleles↗

PubMed1996

Molecular cloning and characterization of a cDNA clone that encodes a Cdc2 homolog from Nicotiana tabacum.

We have isolated a cDNA clone (cdc2Nt1) that encodes a homolog of p34(cdc2/CDC28) kinase from tobacco (Nicotiana tabacum). The cdc2Nt1 protein showed extensive similarity to other homologs of Cdc2 from plants. Complementation studies showed that the cdc2Nt1 gene was able to overcome cell cycle arrest at both the G1/S and the G2/M transitions of cdc28ts mutants of budding yeast, demonstrating that the cdc2Nt1 protein was able to replace the Cdc28 kinase at both the G1/S and the G2/M transitions. Analysis of gene expression demonstrated that the cdc2Nt1 gene was transcribed constitutively throughout the cell cycle but that it was preferentially expressed in activity dividing tobacco BY-2 cells.

Amino Acid Sequence↗

PubMed1992

A variant limb deformity transcript expressed in the embryonic mouse limb defines a novel formin.

The formins constitute a set of protein isoforms encoded by the alternatively spliced transcripts arising from the limb deformity (ld) locus of the mouse. Mutations in this locus disrupt formation of the anteroposterior axis of the embryonic limb. Although ld transcripts are widely expressed during embryogenesis, we have identified a novel transcript that is expressed in the mesenchyme and apical ectodermal ridge of the developing limb. This pattern of expression coincides with the earliest morphological defects observed in ld mutant limb buds. Moreover, the formin encoded by this transcript bears a highly acidic amino terminus, as distinguished from the basic amino terminus encoded by other ld transcripts suggesting that it may have a distinct biochemical function.

Amino Acid Sequence↗

PubMed2006

The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply.

Zinc ions are required to maintain the biological activity of numerous proteins. However, when mislocalized or accumulated in excess, Zn(2+) ions are toxic because of adventitious binding to proteins and displacement of other metal ions, among them Fe(2+), from their binding sites. Heterologous expression of a previously uncharacterized Arabidopsis thaliana metal tolerance protein, MTP3, in the zrc1 cot1 mutant of budding yeast restores tolerance to, and cellular accumulation of, zinc and cobalt. An MTP3-GFP fusion protein localizes to the vacuolar membrane when expressed in Arabidopsis. Ectopic over-expression of MTP3 increases Zn accumulation in both roots and rosette leaves of A. thaliana, and enhances Zn tolerance. Exposure of wild-type plants to high but non-toxic concentrations of Zn or Co, or Fe deficiency, strongly induce MTP3 expression specifically in epidermal and cortex cells of the root hair zone. Silencing of MTP3 by RNA interference causes Zn hypersensitivity and enhances Zn accumulation in above-ground organs of soil-grown plants and of seedlings exposed to excess Zn or to Fe deficiency. Our data indicate that, in wild-type A. thaliana, the AtMTP3 protein contributes to basic cellular Zn tolerance and controls Zn partitioning, particularly under conditions of high rates of Zn influx into the root symplasm.

Arabidopsis↗