Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions (Bezanilla and Pollard, 2000). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.     

The role of thioredoxin 1 in the mycophenolic acid-induced apoptosis of insulin-producing cells

Mycophenolic acid (MPA) is one of many effective immunosuppressive drugs. However, MPA can induce cellular toxicity and impair cellular function in β-cells. To explore the effects of MPA and the relation between MPA and Trx-1, we used various methods, including an Illumina microarray, to identify the genes regulated during pancreatic β-cell death following MPA treatment. INS-1E cells (a pancreatic β-cell line) and isolated rat islets were treated with MPA for 12, 24, or 36 h, and subsequent microarray analysis showed that (Trx1) gene expression was significantly reduced by MPA. Further, Trx1 overexpression increased the cell viability, decreased the activations of c-jun N-terminal kinase (JNK) and caspase-3 by MPA, and attenuated ROS upregulation by MPA. Furthermore, siRNA knockdown of Trx1 increased MPA-induced cell death and the activations of p-JNK and caspase-3, and MPA significantly provoked the apoptosis of insulin-secreting cells via Trx1 downregulation. Our findings suggest that the prevention of Trx1 downregulation in response to MPA is critical for successful islet transplantation.     

2D:4D ratios predict hand grip strength (but not hand grip endurance) in men (but not in women)

In humans, the ratio of the second digit to the fourth digit — the 2D:4D ratio — is a sexually dimorphic trait (men, on average, exhibit lower 2D:4D ratios than do women) that is influenced by prenatal testosterone exposure, but not by circulating testosterone levels in adulthood. Consequently, 2D:4D ratios are commonly used as indirect measures of prenatal testosterone exposure. Many studies have examined the associations of 2D:4D ratios with sexually dimorphic adaptations that are thought to be influenced by such exposure, including physical prowess. The existing literature, however, remains unclear as to (1) whether 2D:4D ratios are more closely linked to strength or to endurance; and (2) whether 2D:4D ratios are linked with physical prowess for both men and women. In 100 men and 122 women, the relationship of 2D:4D ratios with maximum voluntary contraction (MVC) scores (hand grip strength) and maximum endurance time (MET) scores (local muscular endurance) using a hand dynamometer was examined. Controlling for age, height, weight, and average digit length, we found that 2D:4D ratios significantly predicted MVC scores in men, but not in women. 2D:4D ratios did not significantly predict MET scores for either sex. These results suggest that prenatal testosterone exposure in this sample is significantly related to hand grip strength in men, but not in women (and to local muscular endurance in neither sex), and, therefore, that strength, rather than local muscular endurance, potentially drives the relationship between 2D:4D ratios and physical prowess.     

Functional properties of a new voltage-dependent calcium channel alpha(2)delta auxiliary subunit gene (CACNA2D2)

We have positionally cloned and characterized a new calcium channel auxiliary subunit, alpha(2)delta-2 (CACNA2D2), which shares 56% amino acid identity with the known alpha(2)delta-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of alpha(2)delta-2 expression is different from alpha(2)delta-1, and alpha(2)delta-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, alpha(2)delta-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with alpha(1B) and beta(3) subunits in Xenopus oocytes, alpha(2)delta-2 increased peak size of the N-type Ca(2+) currents 9-fold, and when co-expressed with alpha(1C) or alpha(1G) subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa alpha(2)delta-2 polypeptide in some but not all lung tumor cells. We conclude that the alpha(2)delta-2 gene encodes a functional auxiliary subunit of voltage-gated Ca(2+) channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca(2+) signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.     

Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21(CIPI/Waf1) and Bax proteins, as well as activation of p34(cdc2) kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27(Kip1) expression. Olomoucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-mediated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cdc2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: the role of protein translocation

Summary The voltage-dependent channel formed in planar lipid bilayers by colicin E1, or its channel-forming C-terminal fragments, is susceptible to destruction by the nonspecific protease pepsin under well-defined conditions. In particular, pepsin acts only from thecis side (the side to which colicin has been added) and only upon channels in the closed state. Channels in the open state are refractory to destruction bycis pepsin, and neither open nor closed channels are destroyed bytrans pepsin. Colicin E1 channels are normally turned on bycis positive voltages and turned off bycis negative voltages. For large (>80 mV) positive voltages, however, channels inactivate subsequent to opening. Associated with the inactivated state, some channels become capable of being turned on bycis negative voltages and turned off bycis positive voltages, as if the channel-forming region of the molecule has been translocated across the membrane. Consistent with this interpretation is the ability now oftrans pepsin to destroy these reversed channels when they are closed, but not when they are open, whereascis pepsin has no effect on them in either the open or closed state. Our results indicate that voltage gating of the E1 channel involves translocation of parts of the protein across the membrane, exposing different domains to thecis andtrans solutions in the different channel states.     

The essential role of yeast topoisomerase III in meiosis depends on recombination

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5' topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Delta mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Delta mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Delta mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.     

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a “partitioning center” represents yet another facet of the benign parasitism of the yeast plasmid.     

The homeostasis but not the differentiation of T cells is regulated by p27(Kip1)

The cyclin-dependent kinase inhibitor p27(Kip1) is a critical regulator of T cell proliferation. To further examine the relationship of T cell proliferation and differentiation, we examined the ability of T cells deficient in p27(Kip1) to differentiate into Th subsets. We observed increased Th2 differentiation in p27(Kip1)-deficient cultures. In addition to increases in CD4(+) and CD8(+) T cells, there is a similar increase in gamma delta T cells in p27(Kip1)-deficient mice compared with wild-type mice. The increase in Th2 differentiation is correlated to an increase of IL-4 secretion by CD4(+)DX5(+)TCR alpha beta(+)CD62L(low) T cells but not to increased expansion of differentiating Th2 cells. While STAT4- and STAT6-deficient T cells have diminished proliferative responses to IL-12 and IL-4, respectively, proliferative responses are increased in T cells doubly deficient in p27(Kip1) and STAT4 or STAT6. In contrast, the increased proliferation and differentiative capacity of p27(Kip1)-deficient T cells has no effect on the ability of STAT4/p27(Kip1)- or STAT6/p27(Kip1)-deficient CD4(+) cells to differentiate into Th1 or Th2 cells, respectively. Thus, while p27(Kip1) regulates the expansion and homeostasis of several T cell subsets, it does not affect the differentiation of Th subsets.     

Cytokinesis in yeast meiosis depends on the regulated removal of Ssp1p from the prospore membrane

Intracellular budding is a developmentally regulated type of cell division common to many fungi and protists. In Saccaromyces cerevisiae, intracellular budding requires the de novo assembly of membranes, the prospore membranes (PSMs) and occurs during spore formation in meiosis. Ssp1p is a sporulation-specific protein that has previously been shown to localize to secretory vesicles and to recruit the leading edge protein coat (LEP coat) proteins to the opening of the PSM. Here, we show that Ssp1p is a multidomain protein with distinct domains important for PI(4,5)P(2) binding, binding to secretory vesicles and inhibition of vesicle fusion, interaction with LEP coat components and that it is subject to sumoylation and degradation. We found non-essential roles for Ssp1p on the level of vesicle transport and an essential function of Ssp1p to regulate the opening of the PSM. Together, our results indicate that Ssp1p has a domain architecture that resembles to some extent the septin class of proteins, and that the regulated removal of Ssp1p from the PSM is the major step underlying cytokinesis in yeast sporulation.     

MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells

The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 microM), PD98059 (40 microM), or PD184352 (10 microM) with 10-100 microM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (DeltaPsi(m)), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and pro-caspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of DeltaPsi(m), implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.     

Cathepsin B but not cathepsins L or S contributes to the pathogenesis of Unverricht-Lundborg progressive myoclonus epilepsy (EPM1)

The inherited epilepsy Unverricht-Lundborg disease (EPM1) is caused by loss-of-function mutations in the cysteine protease inhibitor, cystatin B. Because cystatin B inhibits a class of lysosomal cysteine proteases called cathepsins, we hypothesized that increased proteolysis by one or more of these cathepsins is likely to be responsible for the seizure, ataxia, and neuronal apoptosis phenotypes characteristic of EPM1. To test this hypothesis and to identify which cysteine cathepsins contribute to EPM1, we have genetically removed three candidate cathepsins from cystatin B-deficient mice and tested for rescue of their EPM1 phenotypes. Whereas removal of cathepsins L or S from cystatin B-deficient mice did not ameliorate any aspect of the EPM1 phenotype, removal of cathepsin B resulted in a 36-89% reduction in the amount of cerebellar granule cell apoptosis depending on mouse age. The incidence of an incompletely penetrant eye phenotype was also reduced upon removal of cathepsin B. Because the apoptosis and eye phenotypes were not abolished completely and the ataxia and seizure phenotypes experienced by cystatin B-deficient animals were not diminished, this suggests that another molecule besides cathepsin B is also responsible for the pathogenesis, or that another molecule can partially compensate for cathepsin B function. These findings establish cathepsin B as a contributor to the apoptotic phenotype of cystatin B-deficient mice and humans with EPM1. They also suggest that the identification of cathepsin B substrates may further reveal the molecular basis for EPM1.     

The yeast Aac2 protein exists in physical association with the cytochrome bc1-COX supercomplex and the TIM23 machinery

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc(1))-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc(1)-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc(1)-COX supercomplex is perturbed, whereby a decrease in the III(2)-IV(2) assembly state relative to the III(2)-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc(1)-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc(1)-COX-TIM23 supercomplex are also discussed.     

Rap1p telomere association is not required for mitotic stability of a C(3)TA(2) telomere in yeast

Telomeric DNA usually consists of a repetitive sequence: C(1-3)A/TG(1-3) in yeast, and C(3)TA(2)/T(2)AG(3) in vertebrates. In yeast, the sequence-specific DNA- binding protein Rap1p is thought to be essential for telomere function. In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end. A tlc1h strain has short but stable telomeres and no growth defect. We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG(1-3) single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere. A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length. However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis. We conclude that Rap1p localization to a C(3)TA(2) telomere is not required for its essential mitotic functions.     

Activation of the yeast cell wall integrity MAPK pathway by zymolyase depends on protease and glucanase activities and requires the mucin-like protein Hkr1 but not Msb2

Yeast adaptation to conditions in which cell wall integrity is compromised mainly relies on the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway. Zymolyase, a mixture of cell wall-digesting enzymes, triggers a peculiar signaling mechanism in which activation of the CWI pathway is dependent on the high-osmolarity glycerol MAPK pathway. We have identified inhibitors of the principal enzyme activities present in zymolyase and tested their effect on the activation of the MAPK of the CWI pathway, Slt2/Mpk1. Eventually, only β-1,3-glucanase and protease activities were essential to elicit Slt2 activation and confer lytic power to zymolyase. Moreover, we show that the osmosensor Hkr1 is required for signaling, being the most upstream element identified to date.