Satoshi Ogihara

Changing Patterns of Gene Expression in Dictyostelium Prestalk Cell Subtypes Recognized by In Situ Hybridization with Genes from Microarray Analyses

ABSTRACT We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

This study underscores the emerging role of NHE6 as a novel regulatory protein in the apical surface development of human hepatoma HepG2 cells. A limited range of endosomal pH facilitated by NHE6.1 is suggested to be important for securing the polarized distribution of membrane lipids and proteins and maintenance of apical bile canaliculi.

Green algal microtubule-associated protein with a molecular weight of 90 kDa which bundles microtubules

SummaryCytoplasmic streaming in the freshwater, coencytic green alga,Dichotomosiphon tuberosus, is regulated by light. Conspicuous changes are observed in the number of microtubules cross-linked together in bundles when the cytoplasmic streaming is modulated by light. In an attempt to identify the cross-linker, we stainedD. tuberosus cells with antibodies specific for several different microtubules-associated proteins (MAPs) from vertebrates. Antibodies raised against bovine adrenal 190 kDa MAP stained the algal cells, and the pattern of staining was quite similar to that obtained with tubulin-specific antibodies. Examination by immunoelectron microscopy revealed that the antibodies specific for the 190 kDa microtubule-associated protein (MAP) were located along the microtubules. Western blotting demonstrated that the antibodies crossreacted with a peptide fromD. tuberosus with a molecular weight of about 90 kDa. This peptide was heat-stable, a property shared by the bovine 190 kDa MAP. Moreover, this 90 kDa peptide, crossreacted with antibodies raised against a synthetic peptide, identical to the tubulin-binding domain found in the 190 kDa MAP and in a tau protein. Partially purified 90 kDa protein fromD. tuberosus has the ability to bundle microtubules when mixed with a tubulin fraction fromD. tuberosus, in the presence of taxol. These results suggest that the 90 kDa protein fromD. tuberosus is a MAP that bundles microtubules.

Calcium and ATP regulation of the oscillatory torsional movement in a Triton model of Physarum plasmodial strands

Abstract A segment of the plasmodial strand of Physarum polycephalum , when hung vertically, shows oscillatory torsional movements that accompany shortening and elongation, i.e., contraction and relaxation in the vertical direction. Reactivation of such movements was achieved in a fresh, demembranated model system through the use of Triton X-100 and chemically defined physiological solutions. Oscillatory rotations were reactivated in solutions containing 0.5 mM Mg-ATP and 10 −6 –10 −4 of free Ca 2+ . Lowering the Ca 2+ concentration to 10 −8 M brought about unidirectional rotations with concomitant vertical elongation of the strands, i.e., relaxation. Such unidirectional rotations were also observed either when Mg-ATP concentrations was raised to 5 mM, or when Mg-ATP was replaced with Mg-pyrophosphate. Higher concentrations of free Ca 2+ than 10 −4 M similarly caused unidirectional rotations, but there was no concomitant vertical elongation. These data mean that the oscillatory contraction and relaxation require a factor which is activated at Ca 2+ concentration above 10 −6 M and inactivated at 10 −8 M. The findings that Mg-pyrophosphate or high concentrations of Mg-ATP-induced relaxation suggest that the actomyosin is dissociated in the relaxation phase. In the light of the present study, it is not likely that the periodic movement in the plasmodia is caused by any oscillatory activities in either the ATP-producing system or the plasma membrane.

Identification of a birefringent structure which appears and disappears in accordance with the shuttle streaming inPhysarum plasmodia

SummaryR-HMM (rhodamine-heavy meromyosin) stained the birefringent fibrous structure which appears and disappears cyclically in parallel with the periodic shuttle streaming in the plasmodium ofPhysarum polycephalum. In addition, 0.6 M KI readily made the birefringent fibrils fade away. These results clearly show that the birefringent fibrils are composed of actin filaments and prove the possibility of actin filaments to alter in the aggregation state during the cyclic production of the motive force responsible for the cytoplasmic streaming.

Identification and characterization of novel calcium‐binding proteins of Dictyostelium and their spatial expression patterns during development

Five putative Ca2+‐binding proteins, CBP5, 6, 7, 8 and 9, all having EF‐hand motifs, were found by searching the Dictyostelium cDNA database (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html). 45Ca2+‐overlay experiments revealed that four of these (excluding CBP9) are real Ca2+‐binding proteins. Northern blot analysis revealed that the genes encoding CBP5, 6, 7 and 8 are all developmentally regulated. In situ hybridization analyses revealed that spatial expression of these genes was regulated in several different ways. CBP1, 2, 3, 5, 6 and 7 are expressed in prespore cells in the slug stage. Transcripts of the genes for CBP1 and 5 are enriched in prestalk subtype PstO cells. In contrast, CBP4 is expressed predominantly in PstO cells. CBP8 is evenly expressed at a very low level throughout the whole slug. Such distinct spatial expression patterns suggest that the CBP might be involved in morphogenesis and might have their own roles either in prespore or in prestalk cell differentiation of Dictyostelium.

Helicobacter pylori Disrupts Host Cell Membranes, Initiating a Repair Response and Cell Proliferation

Helicobacter pylori (H. pylori), the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA) have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+), single mutant (ΔvacA or ΔcagA) or double mutant (ΔvacA/ΔcagA) strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca2+ influx. Ca2+-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis.

Microtubule-dependent migration of the cell nucleus toward a future leading edge in amoebae ofPhysarum polycephalum

SummaryIn several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell locomotion. The centrosome is positioned between the leading edge pseudopod and the nucleus. This suggests that the polarized distribution of organelles in the cytoplasm is coupled spatially with structural and functional polarity in the cell cortex. To study cellular polarization with special interest in the roles of microtubules, we have analyzed the effects of microtubule-disrupting reagents and local laser irradiation on behaviors of both the nucleus and the centrosome in living amoebae ofPhysarum polycephalum. Physarum cells often have 2–3 pseudopods. One of the pseudopods keeps extending to become a stable leading edge while the rest retracts, a crucial step that reorients cells during locomotion. The nucleus, together with the centrosome, moves specifically toward the pseudopod that will become the leading edge. Disruption of microtubules with nocodazole randomizes positions of the nucleus, indicating the involvement of microtubules in the directional migration of the nucleus toward a specific pseudopod. The migration direction of the nucleus is reversed immediately after the UV laser is irradiated at regions between the nucleus and the future leading pseudopod. In contrast, irradiation at regions between the future tail and the nucleus does not affect nuclear migration. By immunofluorescence, we confirmed fragmentation of microtubules specifically in the irradiated region. These results suggest that the nucleus is pulled together with the centrosome toward the future leading-edge pseudopod in a microtubule-dependent manner. Microtubules seem to exert the pulling force generated in the cell cortex on the centrosome. They may serve as a mediator of shape changes initiated in the cell cortex to the organelle geometry in the endoplasm.