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Nuclear p26, a small heat shock/-crystallin protein, and its relationship to stress resistance in Artemia franciscana embryos

Julia K. Willsie and James S. Clegg^*

Section of Molecular and Cellular Biology, and Bodega Marine Laboratory, University of California (Davis), Bodega Bay, CA 94923, USA

View larger version (40K): [in a new window]   Fig. 1. Effects of heat shock on subcellular localization of p26 and final hatching levels. (A) Embryos were subjected to increasing temperatures (from 22°C to 50°C at 0.7°Cmin-1) and sampled at 22, 42, 46 and 50°C. Supernatant (S) and pellet (P) fractions were prepared for SDS–PAGE as described in Materials and methods. The arrow indicates p26 location on this Coomassie-Blue-stained gel. The positions of marker proteins (kDa) are shown. (B) Embryos from the experiment shown in A were also used in hatching assays or analyzed by western immunoblotting. The percentage of total p26 present in the pellet (filled circles) was determined by densitometry of western blots. Open circles indicate the percentage of larvae that hatched. Data points represent the means of three replicate experiments ± S.D.; error bars that are not visible are contained within the symbols.

View larger version (29K): [in a new window]   Fig. 2. Effect of heat shock on nuclear localization of p26 and transcription. (A) Coomassie-Blue-stained gel of proteins from nuclei isolated from control (C) and heat-shocked (HS) embryos (22–50°C) used in 15min mock run-on assays at pH8.0. Samples of nuclei were prepared for SDS–PAGE at both initiation (C0, HS0) and termination (C15, HS15) of the mock assay. Loading was equalized at 1x106 nuclei per lane, and the arrow indicates p26. (B) Western immunoblotting of the samples in A treated with antibody to detect p26. (C) Transcription rates of nuclei from control and heat-shocked embryos. Run-on assays were carried out as described in Materials and methods, at the pH indicated. Error bars are +1 S.D. based on three separate experiments.

View larger version (20K): [in a new window]   Fig. 3. Effect of heat shock and recovery on rates of transcription and subcellular localization of p26. Nuclear run-on assays measured transcription rates in nuclei isolated from embryos subjected to heat shock. Conditions included 22°C (control), 22–42°C, 22–46°C, 22–50°C and also 22–50°C followed by 2 or 4h of recovery at 25°C. Nuclei were then isolated and assayed as described in Materials and methods. Transcription data were normalized to control nuclei, and all assays were performed at pH8.0. Levels of p26 were obtained by immunoblotting and densitometry, and represent nuclear p26 as a percentage of the total. Error bars are +1 S.D., based on three separate experiments.

View larger version (25K): [in a new window]   Fig. 4. [14C]CO2 incorporation in control embryos and in embryos recovering from heat shock. Control (C) and heat-shocked (HS) (22–50°C) embryos were incubated at 25°C for 2 or 4h in [14C]NaHCO3. The protein (P), nucleic acid (NA) and precursor pool (Pre) fractions were obtained as described in Materials and methods, and 14C incorporation was determined by scintillation counting. The inset shows the results for protein and nucleic acid radioactivity on an expanded scale.

View larger version (92K): [in a new window]   Fig. 5. Autoradiogram of a thin-layer chromatogram of precursor fractions from control and heat-shocked embryos. Cold-TCA-soluble (precursor) fractions were obtained from control (C) and heat-shocked (HS, 22–50°C) embryos after incubation at 25°C in the presence of [14C]NaHCO3 for 2 or 4h.

View larger version (16K): [in a new window]   Fig. 6. Transcription rates of nuclei isolated from control and previously anoxic embryos. Embryos were subjected to 2 weeks of anoxia as described in Materials and methods. Nuclei isolated from these embryos were used in nuclear run-on assays at the pH values shown. Error bars represents 1 S.D. for three separate experiments.

View larger version (27K): [in a new window]   Fig. 7. Effects of pH on subcellular localization of p26 and transcription rates. Homogenates of control embryos were incubated in buffer K at pH6.5 to move p26 into nuclei, or at pH8.0 to keep p26 extra-nuclear. Nuclei were then isolated from both homogenates for further study. (A) Nuclei isolated from embryos homogenized at either pH8.0 (C, control) or pH6.5 (L, p26-loaded) were used in a 15min mock run-on assay. Samples of nuclei were prepared for SDS–PAGE at both initiation (C0, L0) and termination (C15, L15) of the assay. Equal amounts (1x106 nuclei) were loaded per lane, and the arrow indicates p26. (B) Detection of p26 in the samples from part A by western immunoblotting. (C) Transcription rates of nuclei isolated from homogenates incubated at pH8.0 and pH6.5. Nuclear run-on assays were carried out as described in Materials and methods, at the indicated pH. Error bars are +1 S.D. based on three separate experiments.

View larger version (32K): [in a new window]   Fig. 8. Confocal microscopy confirms that p26 translocates into nuclei in response to heat shock, anoxia and low pH. Nuclei were double stained using Alexa-Fluor 488 (Ai–Di), and DNA was detected using using Bopro-3 (Aii–Dii). Nuclei were isolated from control embryos (A), embryos heat-shocked from 22 to 50°C (B), embryos after 2 weeks of anoxia (C) and embryos homogenized and incubated at pH6.5 (D). The scale bar in D applies to all parts of the figure.