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The directional tuning of the barber-pole illusion.

In order to study the integration of local motion signals in the human visual system, we measured directional tuning curves for the barber-pole illusion by varying two crucial aspects of the stimulus layout independently across a wide a range in the same experiment. These were the orientation of the grating presented behind the rectangular aperture and the aspect ratio of the aperture, which in combination determine the relative contributions of local motion signals perpendicular to the gratings and parallel to the aperture borders, respectively. The strength of the illusion, ie the tendency to perceive motion along the major axis of the aperture, obviously depends on the spatial layout of the aperture, but also on grating orientation. Subjects were asked which direction they perceived and how compelling their motion percept was, revealing different strategies of the visual system to deal with the barber-pole stimulus. Some individuals respond strongly to the unambiguous motion information at the boundaries, leading to multistable percepts and multimodal distributions of responses. Others tend to report intermediate directions, apparently being less influenced by the actual boundaries. The general pattern of deviations from the motion direction perpendicular to grating orientation--a decrease with aspect ratio approaching unity (ie square-shaped apertures) and with gratings approaching parallel orientation to the shorter aperture boundary--is discussed in the context of simple phenomenological models of motion integration. The best fit between model predictions and experimental data is found for an interaction between two stimulus parameters: (i) cycle ratio, which is the sine-wave gratings equivalent of the terminator ratio for line gratings, describing the effects from the aperture boundaries, and (ii) the grating orientation, responsible for perpendicular motion components, which describes the influence of motion signals from inside the aperture. This suggests that the most simple cycle (terminator) ratio explanation cannot fully account for the quantitative properties of the barber-pole illusion.

Adult↗

Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycle-regulated proteolysis.

The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. At the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell, CtrA is localized to the cell pole just before its degradation. Analysis of the requirements for CtrA polar localization and CtrA proteolysis revealed that both processes require a motif within amino acids 1-56 of the CtrA receiver domain, and neither process requires CtrA phosphorylation. These results strongly suggest that CtrA polar localization is coupled to its cell cycle-regulated proteolysis. The polarly localized DivK response regulator promotes CtrA localization and proteolysis, but it does not directly recruit CtrA to the cell pole. Mutations in the divJ and pleC histidine kinases perturb the characteristic asymmetry of CtrA localization and proteolysis in the predivisional cell. We propose that polar recruitment of CtrA evolved to ensure that CtrA is degraded only in the stalked half of the predivisional cell, perhaps by localizing a proteolytic adaptor protein to the stalked pole. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address.

Bacterial Proteins↗

The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

Cell Cycle↗

Temperature profile for glacial ice at the South Pole: implications for life in a nearby subglacial lake.

Airborne radar has detected approximately 100 lakes under the Antarctic ice cap, the largest of which is Lake Vostok. International planning is underway to search in Lake Vostok for microbial life that may have evolved in isolation from surface life for millions of years. It is thought, however, that the lakes may be hydraulically interconnected. If so, unsterile drilling would contaminate not just one but many of them. Here we report measurements of temperature vs. depth down to 2,345 m in ice at the South Pole, within 10 km from a subglacial lake seen by airborne radar profiling. We infer a temperature at the 2,810-m deep base of the South Pole ice and at the lake of -9 degrees C, which is 7 degrees C below the pressure-induced melting temperature of freshwater ice. To produce the strong radar signal, the frozen lake must consist of a mix of sediment and ice in a flat bed, formed before permanent Antarctic glaciation. It may, like Siberian and Antarctic permafrost, be rich in microbial life. Because of its hydraulic isolation, proximity to South Pole Station infrastructure, and analog to a Martian polar cap, it is an ideal place to test a sterile drill before risking contamination of Lake Vostok. From the semiempirical expression for strain rate vs. shear stress, we estimate shear vs. depth and show that the IceCube neutrino observatory will be able to map the three-dimensional ice-flow field within a larger volume (0.5 km(3)) and at lower temperatures (-20 degrees C to -35 degrees C) than has heretofore been possible.

Antarctic Regions↗

Clathrin-immunoreactive sites in the Golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells.

By analogy with receptor-mediated endocytosis, clathrin-coated membrane segments at locations other than at the plasma membrane have been implicated in intracellular membrane transport/sorting. The crucial role of the Golgi apparatus in these processes prompted us to study the distribution at this level of clathrin-immunoreactive sites in various polypeptide hormone-secreting cells. With a polyclonal antibody recognizing the polymerized form of clathrin (structural coats or cages), we found that the Golgi area had the highest degree of specific labeling of various cytoplasmic regions, including the subplasmalemmal web. Moreover, the Golgi labeling was not homogeneously distributed, most of the immunoreactive sites being associated with membrane segments at the trans pole. The labeled membranes comprised regions of trans cisternae with and without condensing secretory material, newly formed (maturing) secretory granules freshly released from trans cisternae, and typical coated vesicles. The cis Golgi cisternae, as well as the cytoplasmic region containing transfer (shuttle) vesicles and transitional cisternae of the rough endoplasmic reticulum, were only weakly labeled. Clathrin concentration, degree of assembly of triskelions into coats, duration of clathrin association with membranes, or presence of a coat protein different from clathrin could account for the low immunoreactivity observed at the cis pole as compared to the trans pole.

Animals↗

A maternal mRNA localized to the animal pole of Xenopus eggs encodes a subunit of mitochondrial ATPase.

We have previously isolated several cDNA clones of mRNAs that have the unusual property of being localized to either the animal pole or the vegetal pole of frog eggs. To gain insight into the function of these maternal mRNAs we have determined their DNA sequence and deduced the sequence of the proteins they encode. Here we report that An2, an mRNA localized to the animal pole of Xenopus oocytes and eggs, codes for the alpha chain of mitochondrial ATPase. Furthermore, we compare the intracellular localization of the An2 mRNA and mitochondria in oocytes and eggs and find that they do not have the same degree of localization. In the light of these results we discuss possible reasons for the maternal localization of the An2 mRNA.

Adenosine Triphosphatases↗

A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm.

The biological effects of endogenous bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta family, on embryonic development of Xenopus laevis were investigated by using a functionally defective mutant of the BMP-4 receptor (delta mTFR11), which blocks the BMP signaling pathway. Injection of delta mTFR11 RNA into either the animal pole area or ventral marginal cells at the two-cell stage induced a dorsal phenotype in the explant of ventral mesoderm with animal pole tissue from stage 10+ embryo, even though the normal fate of this explant is a "mesenchymal ball" containing blood cells. These explants with the dorsal phenotype contained muscle, neural tissue, eye capsule, and cement gland. Northern blot analysis showed an increase of cardiac alpha-actin mRNA and a decrease of T alpha-globin mRNA expression, providing further evidence of a conversion from ventral to dorsal phenotype. Although injection of delta mTFR11 RNA did not induce mesoderm in an animal cap culture, the same tissue injected with delta mTFR11 RNA can alter the differentiation fate of uninjected ventral mesodermal explant from ventral to dorsal type, suggesting specific interaction of animal pole tissue and prospective ventral mesoderm in vivo.

Animals↗

Three distinct distributions of F-actin occur during the divisions of polar surface caps to produce pole cells in Drosophila embryos.

The F-actin distribution was studied during pole cell formation in Drosophila embryos using the phalloidin derivative rhodaminyl-lysine-phallotoxin. Nuclei were also stained with 4'-6 diamidine-2-phenylindole dihydrochloride to correlate the pattern seen with the nuclear cycle. The precursors of the pole cells, the polar surface caps, were found to have an F-actin-rich cortex distinct from that of the rest of the embryo surface and an interior cytoplasm that was less intensely stained but brighter than the cytoplasm deeper in the embryo. They were found to divide once without forming true cells and then a second time when cells formed as a result of a meridional and a basal cleavage. Three distinct distributions of the cortical F-actin have been identified during these cleavages. It is concluded that the first division, which cleaves the polar caps but does not separate them from the embryo, involves very different processes from those that lead to the formation of the pole cells. A contractile-ring type of F-actin organization may not be present during the first cleavage but is suggested to occur during the second.

Actins↗

Quantitative subcellular study of apical pole membranes from chicken oxyntic cells in resting and HCl secretory state.

Vertebrate oxyntic cells, responsible for gastric HCl production, undergo a remarkable morphological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular membranes, the tubular system, occupies the luminal cytoplasm. Actin filaments frame a cortical network between the tubular system and the luminal plasma membrane. With the onset of HCl secretion, the tubular system becomes incorporated into the luminal plasma membrane. Villous processes containing microfilaments fill the secretory surface. This morphological reorganization of membranes and cytoskeletal matrix could regulate HCl secretion by translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface. In this paper, we describe the isolation of membranes that selectively belong to the tubular system or to the cytoplasmic processes of the secretory surface of chicken oxyntic cells. Chicken oxyntic cells are the main cellular component of the proventricular glands. A resting state was obtained after cimetidine treatment, whereas the HCl-secretory state was induced by histamine. We present a comparative analysis of resting and stimulated chicken gastric glands by quantitative subcellular fractionation. The HCl secretory state was related to specific modifications in membrane fractions derived from the secretory pole of oxyntic cells. Morphological and functional reorganization of oxyntic cells was closely correlated with changes in: the sedimentation pattern of the marker enzyme of the apical pole membrane (K-NPPase), the total activity of K-NPPase and nonmitochondrial Mg-ATPase, the valinomycin dependence of K-ATPase, and polypeptides that cosediment in purified membrane fractions. Changes in the distribution pattern of K-NPPase after fractionation of histamine-stimulated glands were consistent with the replacement of the small vesicles typical of resting glands by dense membrane profiles, analogous to the luminal processes of stimulated oxyntic cells. SDS-PAGE showed that, in purified membrane fractions of stimulated glands, the concentration of 28-, 43-, and 200-kD polypeptides increased while that of 95- and 250-kD polypeptides decreased. The present results define the tubular system of oxyntic cells as an organelle with properties different from those of endoplasmic reticulum, mitochondria, and plasma membrane. The biochemical and physico-chemical properties of this membraneous system changed when the organization of the membranes and the cytoskeletal matrix of the apical pole was modified by the onset of HCl secretion.

4-Nitrophenylphosphatase↗

MPS1 and MPS2: novel yeast genes defining distinct steps of spindle pole body duplication.

It is crucial to the eucaryotic cell cycle that the centrosome undergo precise duplication to generate the two poles of the mitotic spindle. In the budding yeast Saccharomyces cerevisiae, centrosomal functions are provided by the spindle pole body (SPB), which is duplicated at the time of bud emergence in G1 of the cell cycle. Genetic control of this process has previously been revealed by the characterization of mutants in CDC31 and KAR1, which prevent SPB duplication and lead to formation of a monopolar spindle. Newly isolated mutations described here (mps1 and mps2, for monopolar spindle) similarly cause monopolar mitosis but their underlying effects on SPB duplication are unique. The MPS1 gene is found by electron microscopy to be essential for proper formation of the site at which the new SPB normally arises adjacent to the existing one. By contrast, a mutation in MPS2 permits duplication to proceed, but the newly formed SPB is structurally defective and unable to serve as a functional spindle pole. Distinct temporal requirements for the CDC31, MPS1, and MPS2 gene functions during the SPB duplication cycle further demonstrate the individual roles of these genes in the morphogenetic pathway.

Cell Cycle↗

NDC1: a nuclear periphery component required for yeast spindle pole body duplication.

The spindle pole body (SPB) of Saccharomyces cerevisiae serves as the centrosome in this organism, undergoing duplication early in the cell cycle to generate the two poles of the mitotic spindle. The conditional lethal mutation ndc1-1 has previously been shown to cause asymmetric segregation, wherein all the chromosomes go to one pole of the mitotic spindle (Thomas, J. H., and D. Botstein. 1986. Cell. 44:65-76). Examination by electron microscopy of mutant cells subjected to the nonpermissive temperature reveals a defect in SPB duplication. Although duplication is seen to occur, the nascent SPB fails to undergo insertion into the nuclear envelope. The parental SPB remains functional, organizing a monopolar spindle to which all the chromosomes are presumably attached. Order-of-function experiments reveal that the NDC1 function is required in G1 after alpha-factor arrest but before the arrest caused by cdc34. Molecular analysis shows that the NDC1 gene is essential and that it encodes a 656 amino acid protein (74 kD) with six or seven putative transmembrane domains. This evidence for membrane association is further supported by immunofluorescent localization of the NDC1 product to the vicinity of the nuclear envelope. These findings suggest that the NDC1 protein acts within the nuclear envelope to mediate insertion of the nascent SPB.

Amino Acid Sequence↗

Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.

A highly enriched spindle pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional eleven gene products that had not previously been localized to the spindle pole. Immunoelectron microscopy localized eight of these components to different parts of the spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049-6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.

Amino Acid Sequence↗

ZYG-9, a Caenorhabditis elegans protein required for microtubule organization and function, is a component of meiotic and mitotic spindle poles.

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.

Adenosine Triphosphatases↗

Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein.

Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49-59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

Animals↗

Formation of spindle poles by dynein/dynactin-dependent transport of NuMA.

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.

Biological Transport↗

A kinesin-related protein, KRP(180), positions prometaphase spindle poles during early sea urchin embryonic cell division.

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.

Amino Acid Sequence↗

Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole.

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.

Animals↗

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Animals↗