Effect of mutations of murine lens alphaB crystallin on transfected neural cell viability and cellular translocation in response to stress.
We examined the influence of over-expressed native and mutant murine lens alphaB crystallin on the response of a murine neural cell line to heat and ionic strength shock. Native and mutant (F27R and KK174/175LL) murine alphaB crystallin amplicons were subcloned into a Lac-Switch IPTG-inducible RSV promoter eukaryotic vector, and transfected into N1E-115 cells using lipofectin. Expression was induced maximally 8 h after addition of IPTG (optimal final concentration 1 mM) to the medium. Cells grew normally after transfection with native and mutant murine alphaB crystallin. We demonstrated expression of the protein using specific anti-alpha crystallin antibodies. Viability under severe heat and ionic strength stress increased when cells had been transfected with native alphaB crystallin, but not with mutants F27R or KK174/175LL. Heat shock caused translocation of both native and mutant alphaB crystallins into the central region of the cells. These results show that mutations in alphaB crystallin that effect its chaperone-like activity may also influence viability of N1E-115 neural cells under stress, while not influencing the distribution of the protein within the cell.
Wiesmann
K E
KE
Division of Cell and Molecular Biology, School of Animal and Microbial Sciences, The University of Reading, Berks, UK.
Coop
A
A
Goode
D
D
Hepburne-Scott
H W
HW
Crabbe
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D002470:Cell Survival; D003459:Crystallins / Q000235:genetics* / Q000378:metabolism; D018869:Heat-Shock Response; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D009994:Osmolar Concentration; D010641:Phenotype; D016415:Sequence Alignment; D014162:Transfection; D014407:Tumor Cells, Cultured
D003459:Crystallins