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M E Porter

Publications and source records attributed to M E Porter.

17 recordsLinked to original sources
PubMed1992

Two distinct isoforms of sea urchin egg dynein.

Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-2 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.

Adenosine Triphosphatases↗
PubMed1992

Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas.

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.

Animals↗
PubMed1992

Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms.

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.

Animals↗
PubMed1990

Cytoplasmic dynein is localized to kinetochores during mitosis.

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.

Adenosine Triphosphatases↗
PubMed1988

Dynein isoforms in sea urchin eggs.

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.

Adenosine Triphosphatases↗
PubMed1988

Drosophila kinesin: characterization of microtubule motility and ATPase.

Preparations of kinesin, a microtubule-based force-producing protein, have been isolated from Drosophila melanogaster embryos by incubation of microtubules with a nonhydrolyzable ATP analogue and gel filtration of proteins released from the microtubules by ATP. These preparations induced MgATP-dependent microtubule gliding in vitro with a Km for MgATP of 44 microM and a Vmax for gliding of 0.9 micron/sec. Samples of Drosophila proteins that were active in motility assays possessed an average ATPase activity in solution of 17 nmol/min per mg that increased to an average of 106 nmol/min per mg in the presence of microtubules. The major polypeptides that copurified with these activities showed relative molecular masses of 115 kDa and 58 kDa. An antiserum raised against the 115-kDa polypeptide also recognized the 110-kDa component of squid kinesin preparations and the 130-kDa component of sea urchin kinesin preparations.

Animals↗
PubMed1988

Verbal deficits in Klinefelter (XXY) adults living in the community.

Forty-two adult males--10 with Klinefelter's syndrome (karyotype XXY), 17 with normal sex chromosome karyotype (XY) but with physical signs of hypogonadism, and 15 presumed to be normal both chromosomally and hormonally--completed tests of Verbal and Nonverbal ability. It was predicted: a) that the XXY group would perform more poorly than the other two groups on tests of Verbal ability, and b) that the XY, hypogonadal group would perform more poorly than the others on tests of Non-verbal ability. The first hypothesis was supported. However, in spite of measurable verbal deficits, the XXY men (whose parents were more highly educated than those of the other two groups) did not appear to be underachievers. Rather, they were, in general, as well educated, as likely to be employed, and of socioeconomic status equivalent to that of the other men in the study. These findings are discussed briefly in terms of implications for early childhood education and genetic counselling. The second hypothesis was not supported: this is attributed to presumed heterogeneity of etiology of hypogonadism in the XY, hypogonadal group.

Adult↗
PubMed1987

Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans.

C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, "plus" ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.

Adenosine Triphosphatases↗
PubMed1987

Characterization of the microtubule movement produced by sea urchin egg kinesin.

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.

Adenosine Triphosphate↗
PubMed1978

Characterization of a second myosin from Acanthamoeba castellanii.

We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii. The sequence of ion exchange chromatography, actomyosin precipitation, actin extraction, and gel permeation chromatography yields per 100 g of cells about 11 mg of myosin-II which is 90 to 96% pure. ATPase activity is highest in the presence of Ca2+, but the enzyme is also active in EDTA provided high concentrations of K+ are present. The molecule consists of two 175,000 molecular weight heavy chains, one or two 17,500 molecular weight light chains, and two 16,500 molecular weight light chains. Myosin-II is rich in acidic residues and contains about 32 residues of cysteine/mol. The sedimentation coefficient is 5.9 S. Intrinsic viscosity is 126 cc/g. By equilibrium ultracentrifugation, the molecular weight averages depended upon the initial loading concentration in a way that suggested a 400,000 molecular weight species is in equilibrium with a 200,000 molecular weight species. By electron microscopy the molecule was seen to have two globular heads at one end of a tail 90 nm long. In KCl solutions of less than 0.25 M, the myosin-II tails self-associate to form the backbone of very small (6.6 x 205 nm) bipolar filaments with central bare zones 97 nm long. Myosin-II binds to actin filaments, forming periodic arrowhead-shaped complexes, but its Mg2+ ATPase activity is activated only 50% or less by actin. When radioactive myosin-II is incubated up to 90 min in unlabeled Acanthamoeba homogenates, it is not degraded into smaller fragments, such as the 190,000 molecular weight myosin-I. Our observations and the detailed enzymatic data presented by Maruta and Korn ((1977) J. Biol. Chem. 252, 6501-6509) argue that the smaller Acanthamoeba myosin-I (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem, 248, 4682-2690) does not arise by fragmentation of myosin-II in the homogenate or extract.

Actins↗
PubMed1978

Alpha-actinin localization in the cleavage furrow during cytokinesis.

We used antibodies against alpha-actinin and myosin labeled directly with contrasting fluorochromes to localize these contractile proteins simultaneously in dividing chick embryo cells. During mitosis anti-alpha-actinin stains diffusely the entire cytoplasm including the mitotic spindle, while in the same cells intense antimyosin staining delineates the spindle. During cytokinesis both antibodies stain the cleavage furrow intensely, and until the midbody forms the two staining patterns in the same cell are identical at the resolution of the light microscope. Thereafter the anti-alpha-actinin staining of the furrow remains strong, but the antimyosin staining diminishes. These observations suggest that alpha-actinin participates along with actin and myosin in the membrane movements associated with cytokinesis.

Actinin↗
PubMedDate not supplied

Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle.

To understand the molecular basis of microtubule-associated motility during mitosis, the mechanochemical factors that generate the relevant motile force must be identified. Myosin, the ATPase that interacts with actin to produce the force for muscle contraction and other forms of cell motility, is believed to be involved in cytokinesis but not in mitosis. Dynein, the mechanochemical enzyme that drives microtubule sliding in eukaryotic cilia and flagella, has been identified in the cytoplasm of sea urchin eggs, but the evidence that it is involved in cytoplasmic microtubule-based motility (rather than serving as a precursor for embryonic cilia) is equivocal. Microtubule-associated ATPases have been prepared from other tissues, but their role in cytoplasmic motility is also unknown. Recent work on axoplasmic transport, however, has led to the identification of a novel mechanochemical protein called kinesin, which is thought to generate the force for moving vesicles along axonal microtubules. These results suggest that kinesin may also be a mechanochemical factor for non-axoplasmic forms of microtubule-based motility, such as mitosis. We describe here the identification and isolation of a kinesin-like protein from the cytoplasm of sea urchin eggs. We present evidence that this protein is localized in the mitotic spindle, and propose that it may be a mechanochemical factor for some form of motility associated with the mitotic spindle.

Adenosine Triphosphatases↗
PubMedDate not supplied

Reappraisal of the genetic map of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, we have found that linkage groups XII and XIII define only a single linkage group and that linkage groups XVI and XVII also define a single linkage group. The interdigitation of the genetic maps of linkage groups XII and XIII and of XVI and XVII is presented. At present, 17 linkage groups that display Mendelian segregation have been identified in C. reinhardtii.

Chlamydomonas↗