Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in <Emphasis Type="Italic">Dictyostelium</Emphasis>

The calmodulin-binding protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar protein. Prior to this study, the dynamics of specific nucleolar proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar protein and the only nucleolar calmodulin-binding protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.     

Rad53 homologue forkhead-associated kinase A (FhkA) and Ca<Superscript>2+</Superscript>-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Background

During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca²⁺-binding protein 4a (CBP4a), which is a binding partner of NumA1.

Methods

Polyclonal antibodies were produced in-house. Cells were fixed and probed with either anti-FhkA or anti-CBP4a in order to determine cellular localization during interphase and throughout the stages of mitosis. Colocalization with DAPI nuclear stain allowed us to determine the location of the nucleus and nucleolus while colocalization with anti-α-tubulin allowed us to determine the cell cycle stage.

Results

Here we verify two novel nucleolar proteins, Rad53 homologue FhkA which localized around the edge of the nucleolus and CBP4a which was detected throughout the entire nucleolus. Treatment with the Ca²⁺ chelator BAPTA (5?mM) showed that the nucleolar localization of CBP4a is Ca²⁺-dependent. In response to actinomycin D (0.05?mg/mL) CBP4a disappeared from the nucleolus while FhkA protruded from the nucleus, eventually pinching off as cytoplasmic circles. FhkA and CBP4a redistributed differently during mitosis. FhkA redistributed throughout the entire cell and at the nuclear envelope region from prometaphase through telophase. In contrast, during prometaphase CBP4a relocated to many large, discrete “CBP4a islands” throughout the nucleoplasm. Two larger “CBP4a islands” were also detected specifically at the metaphase plate region.

Conclusions

FhkA and CBP4a represent the sixth and seventh nucleolar proteins that have been verified to date in Dictyostelium and the third and fourth studied during mitosis. The protein-specific distributions of all of these nucleolar proteins during interphase and mitosis provide unique insight into nucleolar protein dynamics in this model organism setting the stage for future work.

     

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.     

Purinergic-mediated Ca2+ influx in Dictyostelium discoideum

The presence of five P2X-like genes (p2xA–E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca²⁺ influx in response to extracellular ATP and ADP (EC₅₀ = 7.5 μM and 6.1 μM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91 ± 0.04 s, required extracellular Ca²⁺, were inhibited by Gd³⁺, modified by extracellular pH and were not affected by deletion of either the single Gβ or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu²⁺ and Zn²⁺ inhibited purine-evoked Ca²⁺ influx with IC₅₀ values of 0.9 and 6.3 μM, respectively. 300 μM Zn²⁺ completely abolished the initial large rapid rise in intracellular Ca²⁺ revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.     

Plant Ca<Superscript>2+</Superscript>-ATPases

Plant calcium pumps, similarly to animal Ca²⁺ pumps, belong to the superfamily of P-type ATPase comprising also the plasma membrane H⁺-ATPase of fungi and plants, Na⁺/K⁺ ATPase of animals and H⁺/K⁺ ATPase of mammalian gastric mucosa. According to their sensitivity to calmodulin the plant Ca²⁺-ATPases have been divided into two subgroups: type IIA (homologues of animal SERCA) and type IIB (homologues of animal PMCA). Regardless of the similarities in a protein sequence, the plant Ca²⁺ pumps differ from those in animals in their cellular localization, structure and sensitivity to inhibitors. Genomic investigations revealed multiplicity of plant Ca²⁺-ATPases; they are present not only in the plasma membranes and ER but also in membranes of most of the cell compartments, such as vacuole, plastids, nucleus or Golgi apparatus. Studies using yeast mutants made possible the functional and biochemical characterization of individual plant Ca²⁺-ATMPases. Plant calcium pumps play an essential role in signal transduction pathways, they are responsible for the regulation of [Ca²⁺] in both cytoplasm and endomembrane compartments. These Ca²⁺-ATPases appear to be involved in plant adaptation to stress conditions, like salinity, chilling or anoxia.     

Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants

Summary A cal1-1 mutant of the yeast Saccharomyces cerevisiae showing Ca²⁺-dependent growth was isolated. Its growth continued exponentially in Ca²⁺-rich medium, but stopped in Ca²⁺-poor medium at 37°C. Mg²⁺ ions could not replace Ca²⁺ ions. In Ca²⁺-poor medium, the mutant cells stopped growing homogeneously at the stage of cell division cycle with a tiny bud. The nucleus in these arrested cells was in the G2 stage, judging from observation after nuclear staining and determination of the DNA content. Trifluoperazine-dependent pseudorevertants, which could grow in the presence of 20 M to 80 M trifluoperazine in Ca²⁺-poor medium at 37°C, were obtained from this cal1-1 mutant. The suppressor mutation, tfrl, itself conferred trifluoperazine resistance. Other calmodulin inhibitors structurally unrelated to trifluoperazine had similar effects to trifluoperazine on these pseudorevertants. These results suggest that Ca²⁺ ions and a calmodulin play important roles in the yeast cell division cycle at the stage of bud growth and nuclear division.Abbreviations Tfp trifluoperazine - DAP1 46-diamidino-2-phenylindole - EMS ethyl methanesulfonate - PD parental ditype - NPD nonparental ditype - T tetratype     

The role of asymmetric cell division in pteridophyte cell differentiation. I. Localized metal accumulation and differentiation inVittaria gemmae andOnoclea prothallia

Summary In gemmae ofVittaria graminifolia and prothallia ofOnoclea sensibilis, cell differentiation is initiated by nuclear migration and geometrically asymmetric cell division. The small daughter cells inVittaria develop into antheridia in the presence of gibberellic acid or into rhizoids or new prothallia in its absence. Antheridial differentiation from asymmetric division is induced inOnoclea byPteridium antheridiogen, whereas rhizoid or vegetative cell formation occurs in its absence. Although asymmetric cytokinesis initiates differentiation, it does not in itself determine the developmental fate of the smaller cell. Several histochemical techniques demonstrate that prior to nuclear migration and cell division, Ca²⁺ accumulates in the cytoplasm and wall of the cell at the site where asymmetric division will occur, regardless of the developmental fate of the small cell. The cytoplasmic localization of Ca²⁺ appears to reflect a mobilization of Ca²⁺ from within the cell that eventually moves into the cell wall. We propose that this internal accumulation of Ca²⁺ leads to a localized decrease in cytosolic [Ca²⁺] which in turn may regulate developmental events such as nuclear migration.Publishing prior to 1984 as Alix R. Bassel.     

Effects of Calcium-BAPTA Buffers and the Calmodulin Antagonist W-7 on Mouse Egg Activation

Results of numerous experiments indicate that the transient rise in intracellular Ca²⁺following sperm–egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca²⁺than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca²⁺, we manipulated buffered intracellular Ca²⁺concentrations by microinjecting Ca²⁺-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca²⁺concentrations between 0.5 and 1.0 μM,recruitment of maternal mRNAs is only partially stimulated at injected free Ca²⁺concentrations of 2.5 μM,and no evidence for cell cycle resumption was observed (up to 2.5 μMCa²⁺). Although the Ca²⁺- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca²⁺that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.     

The spinach plasma membrane Ca2+ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region

The spinach (Spinacia oleracea L.) leaf plasma membrane Ca²⁺-ATPase is regulated by calmodulin (3-fold stimulation) and limited proteolysis (trypsin; 4-fold stimulation). The plasma membrane Ca²⁺-ATPase was identified as a 120-kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120-kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109-kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109-kDa polypeptide represents a Ca²⁺-ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by ¹²⁵I-calmodulin overlay studies where calmodulin labeled the 120-kDa band in the presence of Ca²⁺, while the 109-kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP-dependent accumulation of ⁴⁵Ca²⁺ in isolated inside-out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca²⁺ and ATP. Calmodulin increased the V_max. for Ca²⁺ pumping 3-fold, and reduced K_m for Ca²⁺ from 1.6 to 0.9 µM. The K_m for ATP (11 µM) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca²⁺ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the V_max. for Ca²⁺ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

The Ca2+‐binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer

Plasma membrane‐associated Ca²⁺‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca²⁺/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca²⁺. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca²⁺/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca²⁺. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2^G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca²⁺ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.     

Cell Cycle-associated Changes of Guanylate Cyclase Activity in Synchronized Tetrahymena: a Possible Involvement of Calmodulin in its Regulation1

Guanylate cyclase activity decreased during the division phase of heat-shock synchronized Tetrahymena pyriformis, strain GL. However, when Ca²⁺ was removed by EGTA to negate the effects of the Ca²⁺-binding protein (calmodulin), which is required for the full activity of guanylate cyclase in this organism, no significant change in the enzymatic activity was observed throughout the cell cycle. On the other hand, the reduced guanylate cyclase activity at division phase was associated with a decreased level of calmodulin content. These results suggest that fluctuations in guanylate cyclase activity during the cell cycle would be dependent on the concentration of calmodulin.     

Binding of Ca<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> to factor IX/X-binding protein from venom of <Emphasis Type="Italic">Agkistrodon halys</Emphasis> Pallas: stabilization of the structure during GdnHCl-induced and thermally induced denaturation

Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca²⁺- and Zn²⁺-binding protein. The binding properties of Ca²⁺ and Zn²⁺ ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca²⁺ with lower affinity for Zn²⁺ and one specific for Zn²⁺ with lower affinity for Ca²⁺. The bindings of Ca²⁺ and Zn²⁺ in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn²⁺ is 1 order of magnitude higher than for Ca²⁺ for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn²⁺ in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca²⁺–Ca²⁺-AHP IX/X-bp, Zn²⁺–Zn²⁺-AHP IX/X-bp, and Ca²⁺–Zn²⁺-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca²⁺/Zn²⁺-induced tight packing of the protein. Ca²⁺ and Zn²⁺ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca²⁺ and Zn²⁺ have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn²⁺-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca²⁺ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn²⁺ plays both structural and active roles in the anticoagulation. In blood, Ca²⁺ binds to AHP IX/X-bp and stabilizes its structure, whereas Zn²⁺ cannot bind to AHP IX/X-bp owing to the low Zn²⁺ concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X.     

Rate and extent of protein localization is controlled by peptide‐binding domain association kinetics and morphology

Protein localization is an important regulatory mechanism in many cell signaling pathways such as cytoskeletal organization and genetic regulation. The specific mechanism of protein localization determines the kinetics and morphological constraints of protein translocation, and thus affects the rate and extent of localization. To investigate the affect of localization kinetics and morphology on protein localization, we designed a protein localization system based on Ca²⁺‐calmodulin and Src homology 3 domain binding peptides that can translocate between specific localizations in response to a Ca²⁺ signal. We used a stochastic biomolecular simulator to predict that such a protein localization system will exhibit slower and less complete translocations when the association kinetics of a binding domain and peptide are reduced. As well, we predicted that increasing the diffusion resistance by manipulating the morphology of the system would similarly impair translocation speed and completeness. We then constructed a network of synthetic fusion proteins and showed that these predictions could be qualitatively confirmed in vitro. This work provides a basis for explaining the different characteristics (rate and extent) of protein transport and localization in cells as a consequence of the kinetics and morphology of the transport mechanism.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

The calmodulin hypothesis of neurotransmission

Ca²⁺ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca²⁺ binding protein in brain, mediates the effects of Ca²⁺ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca²⁺ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca²⁺-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca²⁺ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca²⁺ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca²⁺-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calcium's actions at the synapse, and that the effects of Ca²⁺ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca²⁺ signal into a motor force in the process of neurotransmission.