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Cell Biology: Corrections Proc 3214 Cell Biology: Corrections Proc. Natl. Acad. Sci. USA 86 (1989) Correction. In the article "Animal cell lysosomes rapidly Correction. In the article "The role of lipids in Plasmodium exchange membrane protein" by Yuping Deng and Brian falciparum invasion oferythrocytes: A coordinated biochem- Storrie, which appeared in number 11, June 1988, of Proc. ical and microscopic analysis" by Ross B. Mikkelsen, Natl. Acad. Sci. USA (85, 3860-3864), the authors request Markus Kamber, Kalyan S. Wadwa, Peck-Sun Lin, and that the following correction be noted in the acknowledgment Rupert Schmidt-Ullrich, which appeared in number 16, Au- (p. 3864). Anti-LIMP I antibody (clone 14E12) was prepared gust 1988, of Proc. Natl. Acad. Sci. USA (85, 5956-5960), the in the laboratory of Dr. Ignacio V. Sandoval (National authors request the following change. The first sentence in the Institutes of Health) and was a gift of Dr. Sandoval. Dr. section describing the purification of human monoclonal Sandoval's present address is Centro de Biologia Molecular, antibodies should read: Two Epstein-Barr virus-transformed Campus de Cantoblanco, Universidad Autonoma de Madrid, human B-lymphocyte lines, SS2F4 and SS2D2 (developed and 28049 Madrid, Spain. provided by H. C. Whittle and M. Blackman, Medical Re- search Council Laboratories, Fajara, The Gambia; ref. 22), secreting monoclonal antibodies against Pf195 were fused (23) Correction. In the article "Phosphorylation and disassembly with the HAT (hypoxanthine/aminopterin/thymidine)- of intermediate filaments in mitotic cells" by Ying-Hao sensitive, ouabain-resistant lymphoblastoid cell line KR-4 (24) Chou, Ellen Rosevear, and Robert D. Goldman, which for increased stability (M.K., unpublished results). appeared in number 6, March 1989, ofProc. Natl. Acad. Sci. USA (86, 1885-1889), the authors request that the following change be noted. On p. 1885, lines 9-12 of the Introduction should read as follows: ".... However, a detailed correla- tion of the state of IF reorganization with the level of phosphorylation within the same cell has not been carried out. .. ." Downloaded by guest on October 1, 2021 Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3860-3864, June 1988 Cell Biology Animal cell lysosomes rapidly exchange membrane proteins (cell fusion/vesicular stomatitis virus/immunofluorescence/sucrosomes/monoclonal antibody) YUPING DENG AND BRIAN STORRIE Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Communicated by James Bonner, February 9, 1988 (receivedfor review October 15, 1987) ABSTRACT The lysosome has been chosen as a model to lysosomes from different animal species. The distribution of study the exchange of native membrane proteins within an species-specific membrane protein antigens between lyso- organelle population. Heterologous lysosomes were brought somes was then detected by immunofluorescence using together by vesicular stomatitis virus-mediated cell fusion. The monoclonal antibodies. After interspecies cell fusion, animal distribution of lysosomal membrane protein was visualized by cell lysosomes were found to rapidly exchange native mem- indirect immunofluorescence using species-specific monoclonal brane proteins and contents. antibody. LAMP-2, a mouse lysosomal membrane protein, and HLAMP-B, a human lysosomal membrane protein, were found MATERIALS AND METHODS to transfer to Chinese hamster ovary cell sucrosomes (sucrose- swollen lysosomes). This transfer occurred in the presence of Cell Culture. CHO-K1 (hamster) cells were cultured in cycloheximide. The exchange of LAMP-2 and LIMP I, a rat Ham's F12 medium. 3T3 (mouse), HeLa (human), and lysosomal membrane protein, was observed between native normal rat kidney (NRK) cells were cultured in Dulbecco's lysosomes in a mouse (3T3)-rat (normal rat kidney) cell fusion. modified Eagle's medium. Both media were supplemented Extensive transfer/exchange was observed within 1.5-2 hr with 10% heat-inactivated fetal bovine serum. All cells were postfusion, which is consistent with the kinetics of endocytic routinely grown in 60-mm plastic tissue culture dishes at content exchange between lysosomes. Both membrane protein 37°C. Cells were harvested by 0.05% trypsin treatment. For and content transfer between lysosomes were inhibited by cell fusion experiments, 2 x 105 cells were plated on 22-mm2 nocodazole, a disrupter of microtubules, as was endocytic glass coverslips. For viral infection, the serum supplement delivery to sucrose-swollen lysosomes. In the presence of was lowered to 2%, and penicillin (50 units/ml) and strepto- nocodazole, tubular lysosomes disappeared. Both tubular ly- mycin (50 ,ug/ml) were added to the medium. sosomes and microtubules may be important for the transfer/ Viral Stocks. Concentrated VSV stocks were prepared as exchange. The interspecies cell fusion/monoclonal antibody described (4). Dilutions ofthe VSV stocks for cell fusion were approach developed here should be readily applicable to determined empirically. If the VSV concentration was too determining if membrane protein exchange is a property of high, the syncytia detached from the coverslips. If the other organelles such as Golgi apparatus and mitochondria. concentration was too low, the extent ofcell fusion was low. Different dilutions of VSV were used for each pair of cell No cellular organelle exists independently inside a cell. Like fusions. parts of a machine, organelles reciprocate and cooperate (for Lysosomal Content Labeling Conditions. All the content a general discussion, see ref. 1). Golgi apparatus, endoplas- markers were incubated with cells in complete culture media mic reticulum, and lysosomes are interrelated. Proteins syn- for 18-20 hr. The concentrations used were sucrose, 0.03 M; thesized in rough endoplasmic reticulum are transported to yeast invertase (grade 3; Sigma), 1 mg/ml; fluorescein iso- Golgi and then to lysosomes. Likewise, transport of newly thiocyanate (FITC) dextran (molecular mass = 72 kDa; synthesized proteins from the rough endoplasmic reticulum by Sigma), 1 mg/ml. way of Golgi to the plasma membrane has been shown. Cell Fusion. Eighteen to 20 hr after plating, cells grown on Organelle resident proteins may also be transported between 22-mm2 coverslips were infected with diluted VSV stock at organelles. Mature LEP 100, a membrane protein normally 37°C. Coverslips were rocked gently every 10 min. One hour resident in lysosomes, cycles between lysosomes, endosomes, after infection, free virus was removed by rinsing the cells and the plasma membrane (2). twice with medium, fresh medium was added, and the cells Little is known about native protein transport/exchange were cultured at 37°C for 4 hr to give maximum VSV G within an organelle population. Exchange of newly synthe- protein expression at the cell surface. Donor cells (2 x 105) sized vesicular stomatitis virus (VSV) G protein, a foreign were added to the recipient coverslips and cultured in molecule, from one stack of Golgi cisternae to another has marker-free medium for 2 hr at 37°C in order to chase markers been observed in cell fusion experiments (3). Recently, from endosomes into lysosomes. At 4 hr postinfection, experiments done in this laboratory (4) have indicated rapid growth medium was removed, and cells were rinsed once and and extensive exchange of endocytized contents between then incubated with pH 5 fusion medium (3) for 10 min at lysosomes in intraspecies cell fusions of Chinese hamster 37°C. Cells were then rapidly rinsed with growth medium and ovary (CHO) cells with CHO cells. These exchanges of incubated at 37°C. foreign molecules within an organelle population raise the Immunofluorescence. Cells were rinsed three times in possibility that exchange ofnative organelle resident proteins Dulbecco's phosphate-buffered saline (PBS) at 24°C and may occur. fixed in 3.7% paraformaldehyde in PBS containing 0.03 M We have used the lysosome as a model to study the sucrose for 10 min at 24°C. Cells were rinsed three times with exchange ofnative molecules within an organelle population. PBS and then incubated with the appropriate monoclonal By cell fusion, we brought together, in the same cytoplasm, antibody in 0.1% saponin for 15 min at 37°C. Coverslips were The publication costs ofthis article were defrayed in part by page charge Abbreviations: VSV, vesicular stomatitis virus; CHO, Chinese payment. This article must therefore be hereby marked "advertisement" hamster ovary; NRK, normal rat kidney; FITC, fluorescein isothio- in accordance with 18 U.S.C. §1734 solely to indicate this fact. cyanate. 3860 Cell Biology: Deng and Storrie Proc. Natl. Acad. Sci. USA 85 (1988) 3861 rinsed three times with PBS and then incubated with FITC- 8), and HLAMP-B, an =120-kDa human glycoprotein (T. conjugated goat anti-mouse IgG or goat anti-rat IgG (Cappel, August, personal communication). The distribution of each Organon Teknika, West Chester, PA) second antibody for 30 of these proteins was established by immunofluorescence min. In double-label experiments, FITC-conjugated goat-anti using species-specific monoclonal antibodies. No reaction of rat IgG (no cross-reactivity with mouse IgG, Organon the monoclonal antibodies with CHO cells was observed in Teknika) and Texas red-conjugated goat anti-mouse IgG (no control experiments. Immediately after fusion of 3T3 cells cross-reactivity with rat IgG, Jackson ImmunoResearch, with CHO cells, sucrosomes were negative for LAMP-2 West Grove, PA) second antibodies were used. All antibodies
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