Previous attempts to detect exogenous Hsc/Hsp70 binding to Aplysia bag cells used monoclonal antibodies to Hsc/Hsp70. Although some results suggestive of cell surface binding and patching of the bound protein were detected using this approach, it was not possible to be sure that only the added Hsc/Hsp70 was being detected and that the anti-Hsc/Hsp70 was not also detecting endogenous Hsc/Hsp70 produced by the bag cell. To avoid this problem, we labeled the Hsc/Hsp70 by reacting it with the succinimidyl proprionate ester form of fluorescein according to the recommendations of the manufacturer (Molecular Probes). The Hsc/Hsp70 was prepared from calf muscle by Boris A. Margulis (Russian Academy of Sciences, St. Petersburg, Russia) and consisted of about a 1:1 mixture of the constitutive and inducible forms. Ovalbulmin (Sigma A-2512) was similarly labeled to use as a control protein. Bag cells were incubated in 10 or 20 5g/ml fluorescein-labeled Hsc/Hsp70 (fl-Hsc/Hsp70) after one day in culture. The live cells were then examined immediately using the Zeiss confocal microscope or were first fixed in para-formaldehyde 30 - 45 min. after exposure to fl-Hsc/Hsp70 and mounted on slides. Weak fluoresecence was detected in some cells, but because of native autofluorescence of the bag cells, it was not clear if this was above background. There was no detectable difference in uptake between ovalbumin and Hsc/Hsp70. Similar tests of uptake were done using cultures of skate retinal neurons (prepared with the help of Leena Patel and R. Paul Malchow, University of Illinois at Chicago) because these cells exhibited much less autofluorescence. The retinal neurons were examined by confocal microscopy while exposed to fl-Hsc/Hsp70 and low levels of fluorescent material were seen to appear inside the neurons over a period of 30 min or more. To test the effect of Hsc/Hsp70 on free radical damage to bag cells, the cells were plated directed into medium containing either 10 5g/ml Hsc/Hsp70 or bovine serum albumin (BSA, cell culture grade, Calbiochem, cat. #126579). Surprisingly, the cells cultured in the presence of BSA did very poorly, being weakly attached and extending no neurites. Those in Hsc/Hsp70 faired better, being well attached and showing short processes, but still did not do as well as cells cells cultured in regular ASW. On day 2 in culture, the cells were challenged with 10 5l hydrogen peroxide/3 ml culture medium. There was no clear difference in the blebbing and neurite retraction responses of the Hsc/Hsp70 treated cells and those in plain ASW, but the BSA treated cells showed greater signs of damage (somal blebbing and detachment from the coverslip) than either of the other two groups. This difference probably was a function of the poorer condition of the BSA treated cells at the start of the experiment.
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