Search

Polo-like kinase 4 (Plk4) is the master-regulator of centriole assembly, and cell cycle-dependent regulation of its activity maintains proper centrosome number. During most of the cell cycle, Plk4 levels are nearly undetectable due to its ability to autophosphorylate and trigger its own ubiquitin-mediated degradation. However, during mitotic exit, Plk4 forms a single aggregate on the centriole surface to stimulate centriole duplication. Whereas most Polo-like kinase family members are monomeric, Plk4 is unique because it forms homodimers. Notably, Plk4 trans -autophosphorylates a degron near its kinase domain, a critical step in autodestruction. While it is thought that the purpose of homodimerization is to promote trans -autophosphorylation, this has not been tested. Here, we generated separation-of-function Plk4 mutants that fail to dimerize and show that homodimerization creates a binding site for the Plk4 activator, Asterless. Surprisingly, however, Plk4 dimer mutants are catalytically active in cells, promote centriole assembly, and can trans -autophosphorylate through concentration-dependent condensate formation. Moreover, we mapped and then deleted the weak-interacting regions within Plk4 that mediate condensation and conclude that dimerization and condensation are not required for centriole assembly. Our findings suggest that Plk4 dimerization and condensation function simply to down-regulate Plk4 and suppress centriole overduplication.

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole "targeting-factor." In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4's kinase domain. Thus, Asl-A's phosphorylation state determines which of Asl-A's two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication., (© 2018 McLamarrah et al.)

Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO2 . UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function., (© 2016 The Protein Society.)

Recent studies have indicated the potential for dietborne metals as an important exposure pathway for metal toxicity in freshwater organisms. We conducted a study in which freshwater cladocerans (Ceriodaphnia dubia) were fed green algae (either Pseudokirchneriella subcapitata or Chlorella vulgaris) that were grown in Ag- or Cu-contaminated media. In one series of toxicity tests patterned after the U.S. Environmental Protection Agency's three-brood C. dubia chronic toxicity test, we exposed C. dubia to waterborne Ag or Cu while feeding them normal amounts of uncontaminated yeast-Cerophyll-trout chow (YCT) slurry and either algae grown in standard media or algae grown in standard media supplemented with Ag or Cu (added as AgNO3 or CuSO4 x 5H2O). These parallel tests demonstrated that dietborne metal did not contribute to survival or reproduction effects beyond the effects caused by waterborne metal alone. We also conducted dietborne-only toxicity tests patterned after two other recently published experimental designs in which (1) C. dubia were fed only metal-contaminated algae for 4 h, transferred to fresh water, and fed uncontaminated algae and YCT slurry for the duration of the three-brood test or (2) C. dubia were fed standard amounts of metal-contaminated algae and uncontaminated YCT slurry for the entire three-brood test. In contrast to previous studies, we did not find consistent dietborne metal toxicity or standard concentration-response relationships in those two experiments. Instead, among-experiment variation in intracellular partitioning of metals in the algae fed to the C. dubia, among-laboratory differences in experimental procedures, selective feeding by C. dubia to avoid metal-contaminated algae, an interaction between reproductive status of the C. dubia and dietborne metal concentration, or a combination of these might help explain the apparently inconsistent results.

The biotic ligand model (BLM) for the acute toxicity of cationic metals to aquatic organisms incorporates the toxicity-modifying effects of dissolved organic matter (DOM), but the default parameterization (i.e., assuming 10% of DOM is humic acid) does not differentiate DOM from different sources. We exposed a cladoceran (Ceriodaphnia dubia) to Ag in the presence of DOM from filtered YCT (standard yeast-Cerophyll(R)-trout chow food recommended by the U.S. Environmental Protection Agency [EPA] for cladocerans), from the Suwannee River (GA, USA; relatively little anthropogenic input), and from the Desjardins Canal in Hamilton (ON, Canada; receives treated municipal wastewater effluent). In all three treatments, the dissolved organic carbon (DOC) concentration was 2 mg/L (the concentration following addition of YCT slurry at the U.S. EPA-recommended volume ratio). The average 48-h median effects concentration (EC50) ratios for dissolved Ag in the presence and absence of DOM [i.e., (EC50 with DOM)/(EC50 without DOM)] were as follows: Suwannee River, 1.6; Desjardins Canal, 2.2; and YCT filtrate, 26.8. Therefore, YCT filtrate provided much more protection against Ag toxicity than that provided by DOM from the surface waters. The major spectral characteristic that differentiated YCT filtrate from the other two types of DOM was a strong tryptophan peak in the excitation- emission matrix for YCT. These results have important implications for interpreting Ag toxicity tests in which organisms are fed YCT, and they suggest BLM-calculated toxicity predictions might be improved by incorporating specific chemical constituents or surrogate indices of DOM. Another component of the protective effect against Ag toxicity, however, might be that the dissolved fraction of YCT served as an energy and/or nutrient source for C. dubia.

Concentrations of cationic metals (e.g., Ag, Cd, Cu, Ni, Pb, Zn) and other water quality parameters (e.g., pH, alkalinity, hardness, dissolved organic carbon (DOC) concentration) often cycle daily in surface waters, and the toxicity of the metals to aquatic organisms is altered by variations in those water quality parameters. Consequently, a method is needed to predict the LC50s (median lethal concentrations) of dissolved metals in temporally varying water quality. In this study, we combined the biotic ligand model (BLM), which predicts toxicity of cationic metals across a wide range of water quality conditions, with a one-compartment uptake-depuration (OCUD) model, which predicts toxicity of a chemical at any exposure time in either continuous or time-variable exposures, to test whether we could accurately predict pulse-exposure toxicity of Cu to fathead minnow (FHM; Pimephales promelas) larvae. First, we conducted continuous-exposure toxicity tests to calculate 1- to 96-h Cu LC50s for the FHM larvae. Then we re-parameterized the default Cu BLM for FHM until the corresponding predicted Cu LA50s (medial lethal accumulations at the biotic ligand) collapsed together into a narrow band and also fit the generalized pattern of an OCUD model [i.e., a steeply sloping plot of ln(LA50) versus ln(time) at short exposure times, followed by a gradual approach to an incipient lethal level at longer exposure times]. Next, in 72-h tests, we exposed FHM larvae to 2- or 8-h square-wave pulses of elevated Cu concentration followed by recovery in uncontaminated water for the remaining 22 or 16 h in each of three consecutive 24-h pulse-and-recovery cycles, at pH 6 or 7 in water containing either 0.5 or 2 mEq/L hardness and 0 or 20 mg DOC/L. Using the combined BLM-OCUD model developed from continuous-exposure data, we then predicted the Cu LA50s in the pulse-exposure tests and compared those LA50s to the observed pulse-exposure Cu LA50s. Although predicted pulse-exposure LA50s were within approximately 4x of the observed pulse-exposure LA50s, delayed deaths during the recovery phases of the exposures precluded more accurate predictions of pulse-exposure Cu LA50s and, as a consequence, of pulse-exposure dissolved Cu LC50s. We conclude that one global OCUD equation linked to a re-parameterized Cu BLM for FHM can be used to predict the acute toxicity of continuous and pulse exposures of Cu to FHM larvae across a range of water quality conditions; but to improve the accuracy of those predictions, a mechanism must be developed to account for delayed deaths.

We conducted laboratory toxicity tests in support of the development of a biotic ligand model (BLM) to predict acute toxicity of zinc (Zn) to fathead minnows (Pimephales promelas). To test the effect of dissolved organic matter (DOM) on Zn toxicity, we exposed larval fathead minnows to Zn in water containing elevated concentrations of dissolved organic carbon (DOC) in 96-h static-renewal toxicity tests. We tested DOM isolated from four surface waters: Cypress Swamp, Delaware; Edisto River, South Carolina; Suwannee River, Georgia; and Wilmington, Delaware, wastewater treatment effluent. The DOM isolates from the Edisto River and Wilmington wastewater treatment effluent contained elevated concentrations of NaCl (20-110x control NaCl) due to the use of a Na+-exchange resin to remove Ca2+ and Mg2+ during the DOM isolation process. Therefore, we also performed Zn toxicity tests in which we added up to 20 mM NaCl to exposure solutions containing Cypress Swamp and Suwannee River DOM. A threshold concentration of 11 mg DOC/L was needed to decrease Zn toxicity, after which the 96 h Zn LC50 was positively correlated with DOC concentration. Elevated NaCl concentrations did not alter Zn toxicity in the presence of DOM. In conjunction with data from other studies with fish and invertebrates, results of this study were used to calibrate Version 2.1.1 of the Zn BLM. BLM-predicted LC50s for our exposure waters containing elevated DOM concentrations were within the range of acceptable deviation relative to the observed LC50s (i.e., 0.5-2x observed LC50s); however, BLM-predicted LC50s for our exposure waters containing < 1 mg DOC/L were 2-3x lower than the observed LC50s (i.e., the BLM over-predicted the toxicity). Therefore, the current composite-species BLM for Zn could be improved for fathead minnows if that species were modeled separately from the other species used to calibrate Version 2.1.1.

Food is added to exposure solutions in cladoceran chronic toxicity tests and sometimes in acute toxicity tests, but its effects on the bioavailability of toxicants have not been studied extensively. We compared the toxicity of waterborne Ag and Cu to Ceriodaphnia dubia in the presence or absence of food (a mixture of a yeast--Cerophyll--trout chow slurry and a green alga) in two series of acute toxicity tests. In the first series, we added food to Ag or Cu exposure solutions 0, 30, 60, or 120 min before transferring C. dubia into the solutions. In the second series, we exposed C. dubia to waterborne Ag or Cu for 30, 60, and 120 min in the absence of food. Adding food before transferring C. dubia into the exposure solutions greatly decreased the toxicity of Ag, but had less effect on the toxicity of Cu. In contrast, adding food after transferring C. dubia into the exposure solutions did not alter the toxicity of Ag considerably and did not alter the toxicity of Cu as much as the reverse sequence. Median effects concentrations (EC50s) for 30-, 60-, and 120-min unfed Ag tests were within the range of EC50s for 48-h unfed Ag tests, suggesting most uptake that contributes to acute Ag toxicity to C. dubia occurs within the first 30 to 60 min. However, uptake that contributes to acute Cu toxicity to C. dubia appears to occur over more than 2 h. Therefore, standard food decreases the toxicity of waterborne Ag and Cu to C. dubia, and the timing of adding food to exposure solutions is especially important in Ag tests.

Mechanistic models predicting copper (Cu) toxicity to aquatic biota in natural waters require organic and inorganic water chemistry, and quantified values for Cu binding by sensitive biological receptors. In bioaccumulation experiments using larval fathead minnows (FHM; Pimephales promelas), we investigated time to asymptotic accumulation of Cu and quantified the conditional stability constants (binding affinity; log K(Cu-FHM)) and binding-site densities of Cu-FHM complexation. Cu bioaccumulation increased rapidly, approaching an asymptote in exposures longer than 12 h, indicating that Cu loading at 24 h is an appropriate exposure duration for modeling Cu complexation by larval FHM. Results of Langmuir and Scatchard analyses of other bioaccumulation experiments produced log K(Cu-FHM) values of 6.52, and binding-site densities of 0.39 micromol g(-1)dry weight. These whole-body log K(Cu-FHM) values are approximately an order of magnitude lower than those reported for adult FHM gills. However, binding-site densities for larval and adult FHM are similar. Under similar test conditions, comparable concentrations of aqueous Cu cause 50% mortality in adult and larval FHM suggesting that binding site densities determine comparable metal-tissue loadings and have greater influence on Cu bioavailability than binding affinity.

We tested the hypothesis that whole-body accumulation of Cu at 50% mortality (i.e. the median lethal accumulation, LA50 value) in a freshwater oligochaete (Lumbriculus variegatus) is constant across a wide range of water quality, whereas the LC50 values of Cu(total) and the cupric ion (Cu(2+)) in solution are not. We exposed the worms in intermittent-flow, water-only chambers to a series of Cu concentrations at a variety of combinations of pH and water hardness (pH 6.5, 7.5 and 8.5 crossed with hardnesses of 0.5, 2, 4, 6 and 15 mEql(-1)) at 17-20 degrees C. In addition to monitoring mortality at 48 h, we determined whole-body Cu uptake in half of the replicate chambers at 6 h. LC50 values of Cu(total) and Cu(2+) increased as water hardness increased, as expected from traditional LC50 vs. hardness regressions. Moreover, LC50 values of Cu(total) remained approximately constant and LC50 values of Cu(2+) decreased considerably as pH increased, as expected from principles of cation competition and binding by inorganic ligands. However, LA50 values of Cu(body) remained approximately constant (0.17-0.34 micromol Cug(-1) dry wt.) in all pH x hardness combinations. Thus, consistent with the biotic-ligand model, Cu accumulation might be a constant predictor of acute mortality to L. variegatus whereas aqueous Cu concentrations are not.