Distribution of proteins between nucleus and cytoplasm of amoeba proteus

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but—because the cytoplasm is 50 times larger than the nucleus—about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained. Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm. New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic “contamination” of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments. We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm’s large volume ensures that CP will be abundant there. Extending Bonner’s idea of “quasi-functional nuclear binding sites” for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.     

Effects of pH and ATP on the cytoplasm of Amoeba proteus: relation between cytoplasm motility and actin polymerization

The effect of pH and ATP was studied on isolated cytoplasm of Amoeba proteus. These two parameters were shown to influence both the motility and the organization of actin filaments in the isolated cytoplasm. Furthermore, our results demonstrate that there is a relationship between the motility and the polymeric state of actin. When the isolated cytoplasm is non-motile, actin is highly polymerized into long filaments arranged parallel in bundles. When this cytoplasm is motile, however, actin can either be weakly polymerized, i.e. observed as few short filaments, or can be polymerized in long branched filaments forming a loose network.     

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.     

The role of nucleus and cytoplasm in the inheritance of multiplication rates in Amoeba proteus cultured at different temperatures.

In Amoeba proteus the inheritance of the multiplication rate is controlled exclusively by the nucleus in successive cell generations. This result has been obtained with four different types of nucleo-cytoplasmic ‘hybrids’ at three different temperatures, 10 °, 17 °, and 25 °C. No contribution of the cytoplasm towards the inheritance of this character has been found.     

Observation of filaments of 10nm in the demembraned cytoplasm of Amoeba proteus

We report here the first observation of 10 nm filaments in a protozoan, Amoeba proteus. These intermediate sized filaments were observed in spread cytoplasmic preparations of amoeba as stable cytoplasmic components over a wide range of pH (5.0-9.0). Although their morphology is grossly similar to the vertebrate intermediate filaments by negative staining, the filaments of amoeba show a characteristic helical structure with a 25 nm axial periodicity and do not display fibrillar projection along their length or at their extremity.     

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm

Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif.     

Effect of Rho family GTP-binding proteins on Amoeba proteus

Summary.  While there is a number of studies on the effects of Rho GTPases on the actin-based cytoskeleton in higher eukaryotes, studies in protozoans are rather limited. The problem seems to be intriguing since the structure of protozoan cytoskeletons is distinct from most vertebrate cells. By blocking endogenous Rho family proteins of highly motile Amoeba proteus with C3 transferase and antibodies against human RhoA and Rac1, we tried to assess the in vivo role of these proteins in amoebae. In migrating amoebae, both proteins are concentrated in the cortical layer and seem to colocalize with filamentous actin. Endogenous Rac1, but not RhoA, is accumulated in the perinuclear cytoskeleton. Blocking Rac- or Rho-like proteins caused distinct and irreversible changes in the locomotive shape of the examined amoebae and significant inhibition of their migration. Amoebae microinjected with anti-Rac1 antibodies were contracted, shortened, and developed only few wide pseudopodia. More pronounced changes were observed in cells treated with anti-RhoA antibodies. They exhibited an atypical locomotion not leading to their effective displacement. After treatment with 50 μg of C3 transferase per ml, cells rapidly contracted and almost completely rounded up, became refractile with the granules beaten into a dense mass, detached from the surface and died. Ten times lower concentration of the enzyme caused similar changes as the inhibition of endogenous RhoA-like protein. These results indicate that Rho family-based regulation plays a key role in amoebic migration. Received May 2, 2002; accepted August 2, 2002; published online November 29, 2002     

Functional characterization of contractile vacuole isolated from Amoeba proteus

Contractile vacuoles (CVs) released from cells of Amoeba proteus were used to analyze its function in vitro. When CV was transferred to a hypertonic medium, its volume decreased within 10 sec. When it was subsequently returned to its original medium, it quickly started swelling. However, it ruptured before recovering its initial volume. These results suggested that the CV membrane is semi-permeable and that the fluid is collected by the osmotic gradient in vivo. The water permeability of membrane of isolated CV was calculated from the rate of osmotic volume change to be 0.94 microm/sec . OsM. This high value suggested that CV membrane is equipped with water channel. CV contracted (or burst) quickly upon addition of 1 mM ATP. Contraction was induced by ATP, but not by other nucleotides, GTP, ITP, ADP, or the analogues of ATP, AMP-PNP and ATPgammaS. It was suggested that the contraction of isolated CV was caused by increase in the tension of its membrane by ATP.     

Isolation and partial characterization of two plasmid deoxyribonucleic acids from endosymbiotic bacteria of Amoeba proteus.

Obligatory endosymbiotic bacteria in a strain of Amoeba proteus were found to harbor two distinct species of plasmid, pHJ11 and pHJ12. Their molecular weights were 39 x 10(6) and 14 x 10(6), respectively.