RESUMEN
The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity. We have characterized two related kinesins in fission yeast, klp5+ and klp6+,, that are amino-terminal motors of the KIP3 subfamily. Analysis of null mutants demonstrates that neither klp5+ nor klp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior. klp5Delta and klp6Delta are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell. This phenotype is greatly enhanced in the cell cycle mutant cdc25-22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest. Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent. During the meiotic phase of the life cycle, both of these kinesins are essential. Spore viability is low in homozygous crosses of either null mutant. Heterozygous crosses of klp5Delta with klp6Delta have an intermediate viability, suggesting cooperation between these proteins in meiosis.
Asunto(s)
Cinesinas/metabolismo , Meiosis , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Tamaño de la Célula , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Intrones/genética , Cinesinas/química , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Familia de Multigenes , Mutación/genética , Unión Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismo , Factores de Tiempo , ras-GRF1/química , ras-GRF1/genética , ras-GRF1/metabolismoRESUMEN
We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Cinesinas/fisiología , Meiosis/fisiología , Proteínas Asociadas a Microtúbulos/genética , Mitosis/fisiología , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Alelos , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Dineínas/genética , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Cinesinas/clasificación , Cinesinas/genética , Cinesinas/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fosfoproteínas/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiología , Homología de Secuencia de Aminoácido , Huso Acromático , Temperatura , Tiabendazol/farmacologíaRESUMEN
The results of studies of Micrasterias rotata (Grev.) Ralfs, M. thomasiana Archer (biradiate and uniradiate forms) and Closterium sp. using one- and two-dimensional vibrating probes show that transcellular ionic currents are detectable only around cells undergoing expansion of the primary cell wall (half-cell); current enters local regions of expansion and exits over both the rigid surface of the secondary wall and regions of the primary wall where hardening of the wall prevents further expansion. Current densities remain at steady levels until expansion stops with maturation of the primary wall, whereupon currents are no longer detectable. The temporal and spatial correlation between the currents and regions of wall expansion is particularly evident because morphogenesis of the half-cell is a determinate process. Measurements of inward currents ranged from 0.1 to 5.4 µA · cm(-2), and outward currents ranged from-0.05 to -1.5 µA · cm(-2) measured at 18 µ from the cell surface. The results of ion substitution and channel-blocker studies indicate that the currents may be carried at least in part by Ca(2+), Cl(-), H(+) and K(+) ions. The possible role of a Ca(2+) influx during tip growth in desmids is discussed.