Biochemical and Immunocytochemical Localization of Heat-Shock Proteins in <i>Drosophila</i> Cultured Cells

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Proceedings Chapter

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Biochemical and Immunocytochemical Localization of Heat-Shock Proteins in Drosophila Cultured Cells

TANGUAY, Robert M., et al.

TANGUAY, Robert M., et al . Biochemical and Immunocytochemical Localization of Heat-Shock Proteins in Drosophila Cultured Cells. In: Wang, E. & Fischman, D. Intermediate filaments . New York : New York Academy of Sciences, 1985. p. 712-714

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Biochemical and

Immunocyto£hemical Localization of Seat-Shock Prct2ins in DrasoA11hi!('.

Cultured Cells

^a

ROBERT M. TANGUAY, JEAN-LOUP DUBAND, FRANCINE LETIRE, JEAN-PAUL VALET,.

ANDRE PATRICK ARRIGO,

^b

AND LOUIS NICOLE

Molecular and Human Genetics

CHUL Research Center Departement Medecine

Universite Laval Ste Foy, P.Q. Canada Gl V 4G2

• Departement Biologie Moleculaire Universite Geneve Geneva. Switzerland

Treatment of living cells at supraoptimal temperatures or with various chemical or physical aggressors induces the synthesis of a group of proteins known as heat-shock proteins (HSP).'.i The function of these ubiquitous proteins is unclear although a role in cellular protection has been suggested.l In Drosophila cultured cells, biochemical fractionation of HS cells shows an enrichment of most HSPs (with the exception of ESP 82) in the nuclear pellet following

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[n the course of studies on the char- acterization oi a major intermediate filament-like cytoskeletal protein of 46,000. in these cells,^6·'we observed that the group of low molecular weight HSPs tended to

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purify with a Triton-high salt insoluble' cytoskeletal fraction (FIGURE 1). In order to investigate the significance of this finding and to elucidate the function of HSPs, we prepared polyclonal antibodies against HSP 82, 70, 68, and 23 and studied their intracellular distribution by immu.nofluorescence techniques following heat shock and during recovery. The antibodies were purified by affinity and their specificity checked by immunoblotting. The results are summarized in FIGURE 2.

HSP 82 shows an exclusive cytoplasmic distribution with a reversible concentration near the plasma membrane during HS. The HSP 68-70 complex, which is present in the cytoplasm and perinuclear regjon of unshocked cells, is found both in th~ nuclear and the cytoplasmic compartments following HS. In the nucleus, they are found in nucleoli as well as in the nucleoplasm. HSP 23 is only present in. stressed cells and concentrates in the nucleolus and perinuclear region upon heat s.hock. When stresses other th:!r~ :i: ::: ~re used, the HSPs do not migrate in the nuc1c:us. Thus immunoflu- orescent localization studies confirm and clarify previous cellular fractionation studies.

a Supported by the Medical Research Council of Canada.

712

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^TANGUAY^elal.: HEAT-SHOCK PROTEINS 713

The co-purification of the low M, HSPs with one of the cytoskeletal elements is probably significant since these proteins shuttle between the soluble and insoluble cellular fractions depending on the physiological state of the cells. While these could be cytoskdetal proteins, their behavior instead suggests rhat they can hind to such

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FIGURE 1. Co-purification of HSPs with cytoskcletal elements (l) Total cell extract, (2) Triton high salt insoluble cytoskeleton,' (3) Urea-solubilized cytoskeleton, (4) Fraction after reassociation by dialysis, and (5) M.W. standards. (Left) Stained gel. (Right) Fluo- rogram.

cytoskeletal elements directly or via interacting molecules such as RNA. Localization- function relationship should be cautiously interpreted since the localization differs depending on the nature of the stress.•

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714 ANNALS NEW YORK ACADEMY OF SCIENCES

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FIGURE 2. Summary of HSPs localization by immunoftuorescence.

4 .. ; · •• JU:FERENCES

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I. AsHBURNER, M. & J. J. BONNER. 1979. Cell 17: 241-254.

2. SCHLESINGER. M. J., M. ASHBURNER & A. TtSSJERES, Eds. 1982 .. Heat Shock: from Bacteria LO Man. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY.

3. TANGUAY, R. M. 1983. Can. J. Biochem. Cell Biol. 61: 387-394.

4. ARRIGO, A. P.,

s:

^FA.KAN^&A. TtsSIERES. 1980. Dev. Biol. 78: 86-103.

s.

^TANGUAY,^{R. M.}^&^M.^VINCENT.^1982.^{Can. J.}^Biochem.60: 306-315.

6. VtNCENT, M. & R. M. TANGUAY. 1982. J. MoL Biol. 162: :i65-378.

7. FALKNER, F. G .• H. SAUMWEBER & H. BlESSMAN. 1981. J. Cell Biol. 91: 175-183.

8. FRANKE, W. W., E. SCHMID, K. WEBER & M. OSBORN. 1979. Exp. Cell Res. 118: 95-109.

9. TANGUAY, R. M. 1985. Intracellular and possible functions of heat sh.ock proteins. In Changes in Gene E;i;pression in Response to Environmental Stress. B. Atkinson & D.

Walden, Eds.: 91-113. Academic Ptcss. New York.

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