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doi:10.1369/jhc.5B6681.2005
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Volume 53 (8): 1037-1040, 2005
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
Elongation of Peroxisomes as an Indicator for Efficient Dynamin-like Protein 1 Knock Down in Mammalian Cells
Department of Cell Biology and Cell Pathology, University of Marburg, Marburg, Germany
Correspondence to: Michael Schrader, Department of Cell Biology and Cell Pathology, University of Marburg, Robert-Koch Str. 6, D-35037 Marburg, Germany. E-mail: schrader{at}mailer.uni-marburg.de
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Summary |
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 Top Summary Literature Cited |
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RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.
(J Histochem Cytochem 53:1037–1040, 2005)
Key Words: peroxisomes • dynamin-like protein 1 • siRNA • electroporation • oligofectamine
RNA INTERFERENCE (RNAi) HAS BECOME a powerful tool to investigate gene function through the specific suppression of a particular mRNA and thus knock down phenotypes for a specific protein. Gene silencing in mammalian cultured cells can be achieved by DNA-directed RNAi, in which a DNA vector is used to express short hairpin RNAs, which are cleaved by DICER, an RNase III like-enzyme, and incorporated into the RNA-inducing silencing complex (RISC)-mediated RNAi pathway, or by directly introducing small, interfering RNA duplexes (siRNA) into target cells. After the selection of suitable siRNA sequences, their suppressive effect on target gene expression has to be confirmed experimentally. Furthermore, optimal conditions for transfection and silencing have to be determined, which are dependent on the transfection reagent, cell type, cell density, and the amount of siRNA/DNA used.
Recently, RNAi has been proven to be a valuable tool to identify and investigate proteins that are involved in the biogenesis of peroxisomes (Petriv et al. 2002
; Koch et al. 2003; Fang et al. 2004). Although peroxisomes are indispensable for human health and normal development, as exemplified by the severity and lethality of numerous peroxisomal disorders, little is known about the proteins required for their growth and division.
The knock down of proteins involved in organelle formation and maintenance of morphology is often accompanied by particular morphological changes of the organelle, which allow insight into protein function. A thorough biochemical, ultrastructural, and quantitative analysis of the effects requires efficient silencing in a large number of cells that can easily be measured and monitored. We have recently demonstrated that the large guanosine triphosphatase DLP1/DRP1, which has been implicated in the fission of mitochondria, is also required for the division of mammalian peroxisomes (Koch et al. 2003, 2004). The knock down of DLP1 causes peroxisome elongation. We then took advantage of the particular morphological changes of peroxisomes observed after silencing of DLP1 to compare current transfection methods and to develop an efficient, fast, easy, and convenient protocol for cell transfection and gene silencing.
We have chosen chemical synthesis of siRNA duplexes, which are easier to transfect than are DNA-based vectors that express siRNA. The transfection of RNA silencing vectors is often followed by a selection of knock down cells; for example, with hygromycin. However, the selection medium can alter the delicate organelle morphology. To knock down the expression of DLP1 by RNA interference, 21-nucleotide RNAs were obtained from Dharmacon (Lafayette, CO). The siRNA sequence efficiently targeting human DLP1 (access no. NM012062) corresponded to the coding region 783–803. The DLP1 siRNA turned out to be effective in COS-7 (African green monkey) and HepG2 cells (human hepatoblastoma), but also in cells of rodent origin. The effectiveness of our selected DLP1 target sequence has recently been confirmed by another group using HeLa cells (Lee et al. 2004).
To establish a very efficient, fast, and convenient protocol for cell transfection and gene silencing, we decided to use the well-defined DLP1 siRNA to knock down DLP1 expression in COS-7 cells stably expressing a green fluorescent protein (GFP) construct bearing the C-terminal peroxisomal targeting signal 1 (GFP-PTS1). COS-7 cells were cultured under standard conditions in DMEM supplemented with 10% FCS (PAA Laboratories GmbH; Cölbe, Germany). siRNA was transfected into the cells using either oligofectamine (Invitrogen GmbH; Karlsruhe, Germany) or electroporation. As a control, cells were transfected with siRNA duplexes targeting luciferase (Dharmacon). The indicated transfection conditions concerning cell number, concentration of siRNA, and amount of transfection reagent have been determined in preceding optimization experiments. Conditions were selected to achieve a high level of siRNA transfection and knock down efficiency while maintaining a high level of cell viability. For transfection with oligofectamine, cells were plated on six-well plates (2 x 105 cells/well) and transfected 24 hr after plating (day 1) by adding 200 µl of transfection mix to a well containing 1 ml of serum- and antibiotic-free medium. The transfection mix was prepared as follows: 4 µl of oligofectamine were added to 11 µl of serum-free medium (solution A), and 4 µl of siRNA duplex (20 µM) was mixed with 181 µl of serum-free medium (solution B). Both solutions were incubated for 5 min at room temperature. Solution A was then added dropwise to solution B and incubated for 20 min at room temperature. The transfection medium was replaced after 5 hr by medium containing 10% FCS. After transfection, cells were either kept in culture for another 96 hr (until day 4) or were retransfected with DLP1 siRNA after 24 hr (day 2), or after 24 and 48 hr (days 2 and 3). For transfection by electroporation, cells grown to 90% confluency on an area of 75 cm2 (1 x 107 cells) were harvested by trypsination, washed in 10 ml of HBS solution (21 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose), resuspended in 0.5 ml HBS, and transferred to a 0.4-cm-gap sterile electroporation cuvette containing 20 µl of siRNA duplex (20 µM). Electroporation was performed in an Easyject Plus electroporator (Peqlab; Erlangen, Germany) at 230 V, 1500 µF, 25–30 msec duration. After electroporation, cells were immediately resuspended in DMEM/10% FCS and plated on six-well plates (2 x 105 cells/well) or cultured in 75 cm2 flasks for retransfection. Cells were either transfected with siRNA duplexes once (day 1) or were retransfected after 48 hr (day 3) using electroporation. In another set of experiments, cells were first transfected with siRNA duplexes by electroporation (day 1) and afterwards retransfected by oligofectamine to prolong the period of DLP1 depletion. Cells were usually assayed for silencing and peroxisome morphology on day 4 (96 hr after the first transfection) by immunofluorescence and immunoblotting (shorter incubation times are possible). For immunofluorescence, cells grown on glass cover slips were fixed with 4% paraformaldehyde in PBS, pH 7.4, and permeabilized with 0.2% Triton X-100. For visualization of endogenous DLP1, cells stably expressing GFP-PTS1 were incubated with rabbit anti-DLP1 antibodies (Yoon et al. 1998), and subsequently with goat anti-rabbit IgG conjugated to tetramethyl rhodamine isothiocyanate. Samples were examined using an Olympus BX-61 microscope (Olympus Optical Co. GmbH; Hamburg, Germany) equipped with a x100 objective (Olympus Plan-Neofluar; numerical aperture 1.35) and a F-view II CCD camera (Soft Imaging System GmbH; Münster, Germany). For gel electrophoresis and immunoblotting, cells were solubilized in lysis buffer (25 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.5% deoxycholate, 0.5% Triton X-100 supplemented with a protease inhibitor mix) for 30 min at 4C. Proteins were precipitated with 10% (w/v) trichloroacetic acid, and protein concentrations were determined using the BC assay (Interchim; Montlucon, France). Protein samples were separated by SDS-PAGE on 12.5% acrylamide gels, transferred to nitrocellulose using a semidry apparatus, and analyzed by immunoblotting using antibodies to DLP1, dynamin 2 (MC63) (Yoon et al. 1998), and 
-tubulin (DM1) (Sigma-Aldrich; Taufkirchen, Germany). Immunoblots were processed using HRP-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Corporation; Arlington Heights, IL).
Oligofectamine transfection of DLP1 siRNA has recently been shown to induce an elongation of the peroxisomal compartment in COS-7 cells (Koch et al. 2004). This notion was confirmed by transfection of COS-7 cells with DLP1 siRNA using electroporation (Figure 1A)
. Endogenous DLP1 was barely detectable in silenced cells by immunofluorescence (Figure 1A, asterisks). Adjacent cells, which were not or insufficiently transfected with the DLP1 siRNA (Figure 1), or controls transfected with luciferase siRNA (Figure 1B) showed high protein levels and a fine, punctate cytoplasmic staining pattern for DLP1. The GFP-labeled peroxisomes in controls or non-transfected cells had a spherical or rod-shaped appearance, whereas peroxisomes in DLP1-silenced cells displayed a highly elongated, tubular morphology (up to 15 µm in length). The special morphology is due to a complete block of peroxisomal fission resulting in the accumulation of peroxisomes in an elongated and constricted state (Koch et al. 2004). No differences in the length or number of elongated peroxisomes per cell were observed when either oligofectamine or electroporation was used for transfection. The special morphological changes of peroxisomes induced by DLP1 silencing allowed a thorough quantitative evaluation of the transfection efficiency and DLP1 knock down. For quantitative evaluation of peroxisome morphology, 200–300 cells per cover slip were examined and categorized as cells with elongated, tubular (>2 µm in length), or spherical peroxisomes (0.3–1 µm). Usually, three to five cover slips per preparation were analyzed, and three to five independent experiments were performed.
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A single transfection of COS-7 cells with DLP1 siRNA using oligofectamine was able to induce elongated, segmented peroxisomes in 32.5 ± 8.5% of the cell population (Figure 1C). Efficient silencing of endogenous DLP1 in these cells was confirmed by costaining with a DLP1-specific antibody (Figure 1A). A higher percentage of cells exhibiting elongated peroxisomes was obtained after a second (65 ± 7%) and a third round of transfection (75 ± 5%) using oligofectamine (Figure 1C). Peroxisome morphology or staining of endogenous DLP1 was not changed in control cells transfected with luciferase siRNA. Using electroporation,
75% of the cell population showed elongated peroxisomes (Figure 1D). The percentage of cells with elongated peroxisomes was not significantly increased after retransfection with oligofectamine or after a second electroporation step. The morphological observations were confirmed by immunoblotting (Figure 1E). Immunoblots of cell lysates demonstrated that the DLP1 protein level was most efficiently reduced after electroporation of DLP1 siRNA to 15–25% of the control level, whereas the protein levels of -tubulin and dynamin 2 remained unchanged. Because the transfection efficiency is usually 70–80%, the DLP1 protein level in those cells that took up the DLP1 siRNA is actually lower than indicated by the immunoblots performed with total cell lysate. Retransfection of electroporated cells did not result in a significant further reduction of the DLP1 protein level, whereas treatment with oligofectamine required retransfection for efficient silencing (to 20–35% of the control level). A significant reduction of DLP1 protein level was not observed in controls. These data demonstrate that peroxisome elongation serves as an excellent indicator for efficient DLP1 knock down. Although mitochondrial morphology is affected by DLP1 siRNA, it exhibits a greater heterogeneity and is more difficult to quantitate. We show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. The transfection protocol provided represents a very efficient, fast, easy, and convenient method for cell transfection and gene silencing in a large number of cells, which can be used for quantitative and biochemical studies. Cells are usually assayed on day 4 after transfection, but shorter incubation times are possible. Finally, the method allows cotransfection of DNA-vectors by chemical transfection reagents such as cationic liposomal and polyamine based agents (usually 48 hr after electroporation).
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Footnotes |
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Received for publication March 7, 2005; accepted March 23, 2005
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Literature Cited |
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TopSummaryLiterature Cited |
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Fang Y, Morrell JC, Jones JM, Gould SJ (2004) PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins. J Cell Biol 164:863–875
Koch A, Schneider G, Luers GH, Schrader M (2004) Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1. J Cell Sci 117:3995–4006
Koch A, Thiemann M, Grabenbauer M, Yoon Y, McNiven MA, Schrader M (2003) Dynamin-like protein 1 is involved in peroxisomal fission. J Biol Chem 278:8597–8605
Lee YJ, Jeong SY, Karbowski M, Smith CL, Youle RJ (2004) Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. Mol Biol Cell 15:5001–5011
Petriv OI, Pilgrim DB, Rachubinski RA, Titorenko VI (2002) RNA interference of peroxisome-related genes in C. elegans: a new model for human peroxisomal disorders. Physiol Genomics 10:79–91
Yoon Y, Pitts KR, Dahan S, McNiven MA (1998) A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells. J Cell Biol 140:779–793
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