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. 2003 Jul 8;100(14):8241-6.
doi: 10.1073/pnas.1331629100. Epub 2003 Jun 25.

Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells

Lawrence Rajendran  1 , Madhan Masilamani, Samuel Solomon, Ritva Tikkanen, Claudia A O Stuermer, Helmut Plattner, Harald Illges
Affiliations

Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells

Lawrence Rajendran et al. Proc Natl Acad Sci U S A. .

Abstract

Hematopoietic cells have long been defined as round, nonpolar cells that show uniform distribution of cell surface-associated molecules. However, recent analyses of the immunological synapse and the importance of lipid microdomains in signaling have shed new light on the aspect of lymphocyte polarization during the activation processes, but none of the molecules implicated so far in either the activation process or the microdomain residency are known to have a preferential localization in nonactivated cells. Chemical crosslinking and fluorescence resonance energy transfer methods have allowed the visualization of certain glycosylphosphatidylinositol-anchored proteins in lipid rafts but so far no microdomain resident protein has been shown to exist as visible stable platforms in the membrane. We report here that two lipid microdomain resident proteins, flotillins/reggies, form preassembled platforms in hematopoietic cells. These platforms recruit signaling molecules upon activation through lipid rafts. The preassembled platforms significantly differ from the canonical cholesterol-dependent "lipid rafts," as they are resistant to cholesterol-disrupting agents. Most evidence for the functional relevance of microdomains in living cells remains indirect. Using laser scanning confocal microscopy, we show that these proteins exist as stable, microscopically patent domains localizing asymmetrically to one pole of the cell. We present evidence that the asymmetric concentration of these microdomain resident proteins is built up during cytokinesis.

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Figures

Fig. 1.
Fig. 1.
Exclusive asymmetric localization of flotillins in PAPs and their DRM partitioning in resting cells. (a) Expression of flotillins in different hematopoietic cells. Lanes 1–9: pro-B cell line KM3, pre-B cell Nalm6, Epstein–Barr virus-transformed mature B cell lines Dakiki, Raji, plasma B cell line HSSultan, peripheral blood B lymphocytes, T lymphocytes, T cell leukemic Jurkat cells, and the promonocytic cell line, U937, respectively. M, marker lane. (A) PCR amplification of flotillin-1. GAPDH amplification serves as control. (B and C) Detection of flotillin-1 and -2 by Western blotting. Both flotillins run at 48 kDa under reducing conditions and the Ponceau stainings serve as loading controls. (b) Nonactivated pre-B cell line Nalm6 (A), mature B cell lines Raji (B), Ramos (C), T leukemic Jurkat cells (D), and promonocytic cell line U937 (E) were stained with mouse monoclonal antiflotillin-1 (green) costained with affinity-purified rabbit polyclonal anti-reggie-1/flotillin-2 (red) antibodies. All of the cells exhibited the cap-like staining. (c) Reggie-1-EGFP is selectively targeted to the PAPs. Jurkat cells were electroporated with the plasmid and the cells were analyzed by laser scanning confocal microscopy. The overexpressed fusion protein was also targeted to the PAPs. (d) Jurkat cells were stained with antiflotillin-1 antibodies and costained with phalloidin green. Intense intracellular staining of flotillins shows the outlines of nuclei (arrowheads) and helps perceive the orientation of the PAPs with respect to the nucleus. Bright-field images (differential interference contrast microscopy, DIC) show the intact, round morphology of these cells. (e) Detergent insolubility of flotillins. Cells (A–E as in b) were lysed in cold 1% Triton X-100 and a sucrose gradient was overlaid onto the lysate as mentioned in Materials and Methods. The detergent-insoluble fractions 2–5 were identified by CTx-B-HRP and anti-lyn antibody staining. Note that the B cell receptor (IgM) under nonactivating conditions was excluded from the detergent-insoluble fractions. (f) The first three rows represent nonactivated Jurkat T cells stained with flotillin-2/reggie-1 antibodies (red) and anti-lck, anti-CD59, and anti-CD71 antibodies (green). The fourth row shows anti-ezrin, radixin, and moesin (ERM) (red) and antiflotillin-2 (green) costaining in U937 promonocytic cells. The bottom row represents the B cell line Raji stained with anti-CD21 (red) and antiflotillin-2 (green) antibodies. None of the molecules except flotillins showed a polarized localization. (Bars = 5 μm.)
Fig. 2.
Fig. 2.
Lipid raft clustering recruits raft-associated signaling molecules to the preassembled platforms and activates T cells. (a) CTx-B crosslinking (red) induces patching and recruits signaling molecules CD3 and Thy1 (green) to PAPs but not CD55. Noncrosslinked T cells (Left) show uniform distribution of GM1 (CTx-B, red), CD3, Thy1, and CD55 (green) but very polarized expressions of flotillins-1 and -2. (b) Some cells showed uniform distribution of GM1 in CTx-B crosslinked cells (red) and those cells also lacked flotillin (green) polarization. (c) Jurkat T cells were activated with PMA and CTx-B for the times indicated. The lysates were run on a reducing SDS/PAGE and blotted with antiphospho ERK-1/2 antibodies. The blots were stripped and blotted with antiflotillin-1 antibodies. Ponceau staining and flotillin levels showed loading of equal amounts of proteins. (d) Immunoprecipitation with antiflotillin-1 antibodies from CTx-B crosslinked and noncrosslinked Jurkat T cells. (Upper) Immunoprecipitates were blotted and stained with anti-phosphotyrosine (PY)-HRP antibodies. Arrows indicate flotillin-interacting phosphorylated protein bands enriched upon CTx-B crosslinking. (Lower) Loading control in the same immunoprecipitates by using antiflotillin-1 antibodies. (Bars = 5 μm.)
Fig. 3.
Fig. 3.
Flotillins form stable cholesterol-independent preassembled microdomains and platforms. (Upper) Confocal staining of untreated (-CDX) and CDX-treated (+CDX) (12.5 mM, 45 min, 37°C) Jurkat (A) and U937 (B) cells with antiflotillin-1 antibodies. The corresponding bright-field images (differential interference contrast microscopy, DIC) are shown. (Lower) Western blotting of sucrose gradient fractions of detergent-lysed (1% Triton X-100, 4°C) untreated (-CDX) and CDX-treated (+CDX) Jurkat (A) and U937 (B) cells. Insol represents the detergent-insoluble fractions and Sol represents the soluble fractions. The lysates were processed as described in Materials and Methods and blotted with antiflotillin-1, lck, and lyn antibodies. (Bars = 5 μm.)
Fig. 4.
Fig. 4.
Cytokinesis in correlation with polarized expression of flotillins. Jurkat T cells were treated with nocodazole for 12 h to synchronize cells in the G2/M transition phase, washed, and further cultured in fresh medium without nocodazole for another 1–2 h to allow the cells to proceed into mitosis. (A) The asynchronous culture showing the microtubule organizing centers stained with β-tubulin antibody (red) and flotillin-2 antibody (green). (B) A cell undergoing cytokinesis evidenced by the position of midbody in the division plane (arrowheads). Note the accumulation of flotillins in the division plane. (C) The cells after division. Most of the cells have flotillins polarized in PAPs. DIC (differential interference contrast microscopy) represents the bright-field images of the cells. (Bars = 5 μm.)
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