In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.

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 cell cortex of Dictyostelium amebae contains an actin-rich cytoplasmic matrix. Changes in geometry of this matrix are believed to regulate protrusive activity and motility of the cell cortex . Two actin-binding proteins (120,000 and 95,000 daltons [120K and 950 are present in the cell cortex, and their properties, many of which are described here for the first time, suggest that they regulate growth and organization of cortical microfilaments . The 120K protein is a flexible dimer 35 nm in length with a native molecular mass of 241,000 . It nucleates the polymerization of actin and crosslinks the filaments to form branched networks like those seen in situ in the cell cortex . The production of a branched network of short crosslinked filaments results in a lattice that would theoretically generate the maximum rigidity with minimum amount of polymer . This sort of lattice would be very useful as a space-filling cytoskeleton capable of resisting deformation . The 120K protein inhibits the actin-stimulated Mg ATPase of myosin . Competition for actin binding between 120K and myosin, the impenetrability of the 120K-actin network to myosin, and the rigidity of actin filaments that are crosslinked by 120K could all contribute to the decrease in the actin-stimulated Mg ATPase of myosin . The properties of 120K are consistent with a role for this protein in regulating the site of actin filament growth and gelation in the cell but not the assembly of actin-containing structures that would participate in force generation by a sliding-filament mechanism involving myosin . The 95K protein is a rigid dimer 40 nm in length with a native molecular mass of between 190,000 and 210,000. Its physical and antigenic properties lead us to conclude that the 95K protein is Dictyostelium a-actinin . Unlike 120K, it crosslinks actin filaments into lateral arrays and increases the actin-stimulated Mg ATPase of myosin . Both activities are regulated by Cat+. The properties of 95K are consistent with a role in organizing actin filaments in the cell into lateral arrays that are capable of efficient interaction with myosin to produce force for cell motility .The cytoplasmic matrix is a pervasive filamentous network in the cytoplasm of many eukaryotic cells. This structure was first described by Wolosewick and Porter (42) as a microtrabecular lattice of heterogeneous morphology containing all three of the major filament classes (microfilaments, intermediate filaments, and microtubules) crosslinked to each other by narrow "trabeculae." Recently, workers in many labora-THE JOURNAL OF CELL BIOLOGY " VOLUME 99 NO . 1 PT . microfilaments and membranes, furtheremphasizing the view of the cytoplasm as a mechanically integrated network. These studies are largely summarized in the papers of this supplement.We have been studying the actin-rich zones of the cytomatrix in ameboid cells. In vegetative amebae of Dictyostelium discoideum this zone is found in the cell cortex . The cytoplasmic matrix in the cortex is heterogeneous, being composed o...

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.

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.

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PS, Ca, and Alb in NSCLC and Ca, Alb, and a history of chemotherapy in SCLC are useful for determining an indication of operation for spinal metastases from lung cancer.

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.