Selective blocking of clathrin-mediated ... - Semantic Scholar

2 downloads 0 Views 333KB Size Report
amphiphysin and EPS15 have been subjected to dominant negative overex- pression, however mutant proteins have their limitations as well (13–15). Therefore ...
Research Reports

Selective blocking of clathrin-mediated endocytosis by RNA interference: epsin as target protein Davy Vanden Broeck and Marc J.S. De Wolf BioTechniques 41:475-484 (October 2006) doi 10.2144/000112265

Epsin is an essential accessory protein exclusively implicated in clathrin-mediated endocytosis and therefore an ideal target to study involvement of this entry route in the uptake of bioligands. The technique of RNA interference (RNAi) was exploited to generate a cell line constitutively silencing epsin expression in a sequence-specific manner. In these Caco-2eps- cells, quantitative reverse transcription PCR (RT-PCR) revealed a severe depletion of the epsin messenger RNA (mRNA) level in cells, reaching a factor >106. The reduction at the mRNA level in the Caco-2eps- cells was paralleled by a decrease of 75% at the protein level. In order to evaluate transfection effects at the functional level, uptake of transferrin and epidermal growth factor (EGF) in transfected Caco-2eps- and control cells was evaluated. In control cells, respectively, approximately 72% of transferrin and approximately 66% of EGF were internalized, whereas in Caco-2eps- cells only approximately 25% of transferrin and approximately 34% of EGF was taken up, confirming that in the transfected cells, endocytosis via coated pits was prominently compromised. The reduced uptake was not the result of an inhibition of transferrin recycling. The effects of direct treatment with chlorpromazine on Caco-2 cells, also monitored from the degree of transferrin internalization, were compared with those elicited by RNAi.

INTRODUCTION Endocytosis, a hallmark of all eukaryotic cells, results in the intracellular uptake of membrane proteins, lipids, and a variety of extracellular ligands. Endocytosis is involved in cellular processes such as nutrient uptake, morphogenesis, synaptic molecule recycling, and regulation of the cell surface expression of signaling receptors, transporters, and channels (1). The first endocytic event, the internalization step, has been extensively studied in both lower and higher eukaryotic cells. Studies in mammalian cells have revealed the existence of multiple endocytic pathways, including macro-pinocytosis and clathrinmediated endocytosis, as well as internalization via caveolae, caveolae-like structures, and lipid rafts (2–4). Multiple studies have focused on uptake through clathrin-coated pits because about 50% of all ligands are internalized by this route. Therefore clathrin-mediated

endocytosis is often considered as a first tentative internalization pathway for a given molecule (2). One major obstacle in identifying a particular endocytic pathway as unique for the uptake of a ligand resides in developing an experimental strategy exclusively suppressing that specific internalization route without affecting other internalization pathways. For example, cholesterol extracting or sequestrating drugs such as cyclodextrins, filipin, or nystatin disrupt not only caveolae/lipid rafts, but destabilize clathrin-coated pits as well (5,6). This lack of specific pharmacological inhibitors frequently hampers the study of internalization processes. Other experimental approaches such as dominant negative overexpression, gene targeting, selective inactivation, and RNA interference (RNAi) have been exploited in the study of clathrinmediated endocytosis (7–12). A frequently used tool to disrupt internalization via coated pits is dominant

negative overexpression of a K44A mutant of dynamin; next to dynamin, amphiphysin and EPS15 have been subjected to dominant negative overexpression, however mutant proteins have their limitations as well (13–15). Therefore alternative approaches like gene targeting, selective inactivation, and RNAi have been exploited. Here, a method was developed to suppress clathrin-dependent uptake very specifically. To this end, the RNAi technique was used to silence expression of a core component of the clathrin-mediated endocytosis in a sequence-specific manner. Introduction of small interfering RNA (siRNA) in mammalian cells induces strong and specific suppression of the gene of interest. However, this effect is transient due to the short lifespan of synthetic RNAs and the absence of RNA-dependent RNA polymerase in mammalian cells, thus limiting its applications (16–18). To overcome these limitations, a vector-based production

University of Antwerp, Antwerp, Belgium Vol. 41 ı No. 4 ı 2006

www.biotechniques.com ı BioTechniques ı 475

Research Reports

of short hairpin RNA (shRNA) was used, offering perspectives to construct a cell line that permanently restrains translation of messenger RNA (mRNA) encoding for the target protein. Based on recent insights at the molecular level of clathrin-mediated endocytosis, we have chosen epsin as the most appropriate factor to target shRNA. According to Ford et al. (19) epsin is an essential accessory protein implicated in the clathrin-mediated uptake. It is widely expressed in many mammalian cell types (20–25) and a homologous protein has also been found in Drosophila melanogaster (24). Epsin is composed of different domains including an epsin N-terminal homology (ENTH) domain, clathrin binding motifs, ubiquitin interacting consensus motifs, and C-terminal Eps15 homology (EH) domain binding sequences (25). The ENTH domain has been proposed to represent a separately folded protein module (20) displaying a super helix of seven α-helices with a supplementary phosphatidylinositide

(PIP2)-induced α0 helix misaligned with the super helical axis (26–28). Insertion of the α0 helix in the inner plasma membrane leaflet is crucial for induction of membrane curvature and therefore one of the first events in coat formation (27). The ENTH domain is highly conserved (29,30) and unique for clathrin-mediated endocytosis at the plasma membrane (20,31–36), rendering it suited to select stretches of 20 nucleotides to target shRNA (22,31,37,38). All these features make epsin an ideal target protein to suppress in order to study involvement of clathrin-mediated endocytosis in the uptake of bioligands. MATERIALS AND METHODS Cell Culture Human intestinal colon carcinoma cells, Caco-2wt (passages 5 to 34) and Caco-2eps- cells (passages 1 to 12) were cultured in modified Eagle’s medium

(MEM) supplemented with 10% heat-inactivated fetal bovine serum, nonessential amino acids, 1 mg/L amphotericin B, 50 mg/L gentamicin, 50 mg/L penicillin/streptomycin, and 55 mg/L sodium pyruvate (all from Gibco®; Invitrogen, Carlsbad, CA, USA). Cell medium was changed twice a week, and cells were passed weekly. After transfection, cell medium was supplemented with 2 μg/mL selection antibiotic Zeocin® (Invitrogen). RNAi Selection Based on sequences from National Center for Biotechnology Information (NCBI) GenBank® with accession nos. AF073727 (epsin), BC070036 (epsin2), AF062085 (epsin2b), NM_017957 (epsin3), and AF434813 (epsin4), a multiple alignment and homology search was performed using ClustalW (version 1.8) from the Baylor College of Medicine Human Genome Sequencing Center (HGSC; searchlauncher.bcm. tmc.edu/multi-align/multi-align.

Table 1. Oligonucleotides, Primers, and Probes Target

Sequence Oligonucleotides Used to Generate shRNA

psiRNA epsin forward psiRNA epsin reverse

5′-ACCTCGAAGAACATCGTGCACAACTATCAAGAGTAGTTGTGCACGATGTTCTTCTT-3′ 5′-CAAAAAGAACATCGTGCACAACTACTCTTGATAGTTGTGCACGATGTTCTTCG-3′ Synthetic siRNA Sequence (Control within the Same Region)

siRNA epsin forward

5′-GAAUGACCAUGGCAAGAACUTT-3′

siRNA epsin reverse

5′-AGUUCUUGCCAUGGUCAUUCTT-3′

psiRNA scrambled forward

5′-GCATATGTGCGTACCTAGCAT-3′

Functional, Nontargeting Control psiRNA scrambled reverse

5′-CGTATACACGCATGGATCGTA-3′ Primers and TaqMan Probe for Quantification of Epsin Expression

Epsin forward

5′-GATCTGGAAGCGGCTCAATG-3′

Epsin reverse

5′-GAGGTACTCCATCAGCGTCATG-3′

Epsin TaqMan probe

5′-FAM-CTTGTAAACGTGACGCCAGTTCTTCCAT-3′ TAMRA Primers and TaqMan Probe for Quantification of GAPDH Expression

GAPDH forward

5′-CCACATCGCTCAGACACCAT-3′

GAPDH reverse

5′-GTGACCAGGCGCCCAAT-3′

GAPDH TaqMan probe

5′-FAM-CGACCAAATCCGTTGACTCCGACCTT-3′ TAMRA Primers and TaqMan Probe for Quantification of HGPRT Expression

HGPRT forward

5′-GGCAGTATAATCCAAAGATGGTCAA-3′

HGPRT reverse

5′-TGGCTTATATCCAACACTTCGT-3′

HGPRT TaqMan probe

5′-FAM-GGCAGTATAATCCAAAGATGGTCAA-3′ TAMRA

siRNA, small interfering RNA; shRNA , short hairpin RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HGPRT, hypoxanthine guanine phosphoribosyl transferase.

476 ı BioTechniques ı www.biotechniques.com

Vol. 41 ı No. 4 ı 2006

Research Reports

html). A stretch of 21 nucleotides was selected from the conserved region located 106 (Figure 1), emphasizing the effective silencing by the epsin targeting shRNA. A second synthetic siRNA (Table 1) with an alternative consensus sequence reduced the epsin expression by >90%, without affecting expression profiles of the housekeeping genes. In negative controls (Caco-2ss), expression of epsin was not affected at all, an indication that the transfection procedure did not elicit any nonspecific effects (Figure 1). In order to exclude secondary transfection effects, expression profiles of two genes encoding for the housekeeping enzymes GAPDH and HGPRT were compared in both transfected and control cells. Real-time PCR curves of all three Caco-2wt, Caco-2ss, and Caco-2eps- nearly coincided, resulting in almost identical CT values. These data reveal that neither the carbohydrate nor nucleotide metabolism were compromised by the transfection procedure and the epsin suppression, an essential requisite for

40

20

0 0

10

20

30

40

50

Time (min)

Figure 3. Effect of reduced epsin expression on the internalization of 125I-EGF. Caco-2wt (°) and Caco-2eps- cells (n) in suspension were cooled on ice, and 125I-EGF [106 counts per minute (cpm)] was allowed to bind for 60 min. Cells were washed three times with ice-cold phosphate-buffered saline (PBS), further incubated at 37°C for the indicated times, and subsequently placed on ice to block endocytosis. Surface bound 125I-EGF was removed using 0.1 M HAc, pH 2.5, and samples were assayed for radioactivity. EGF, epidermal growth factor.

and NaOH solutions were counted in a Model LS6500 scintillation counter (Beckman Coulter, Fullerton, CA, USA). It was shown that acid washing removed >95% of cell-surface bound 125 I-transferrin (data not shown). Transferrin recycling was assessed according to Moskowitz et al. (11). Briefly, after binding of 125I-transferrin, cells were incubated for 1 h at 37°C to allow internalization, subsequently cells were cooled to 4°C, and surface bound transferrin was acid striped. Afterwards, cells were incubated with 3 μg/mL unlabeled transferrin for the indicated times at 37°C, cooled on ice, and the medium was collected and further assayed for radioactivity. Treatment of Cells with Cationic Amphiphilic Drugs To confirm the requirement for clathrin-coated pits as the vehicle for internalization of transferrin, experiments have been performed with chlorpromazine (CPZ), a drug reported to prevent coated pit assembly at the cell surface. Prior to the administration of 125Itransferrin, cells were starved for 30 min in serum-free medium and then preincubated at 37°C for 60 min in MEM supplemented with 25 mM 480 ı BioTechniques ı www.biotechniques.com

HEPES, 1% BSA, and increasing amounts of CPZ. Thereafter, 125I transferrin was added, and cells were kept on ice allowing the protein to bind. Endocytosis of 125I-transferrin was initiated by increasing the temperature up to 37°C, and incubation continued for the indicated intervals. The extent of endocytosis was measured as described in the previous section. RESULTS Cell Integrity No differences in cell morphology could be observed between control and transfected cells by light microscopy. Viability of Caco-2eps- cells as deter-

Table 2. Assessment of Viability and Cytotoxicity Treatment

Viability (% vs

Caco-2wt

Caco-2wt)

Cytotoxicity (MTT) (% vs Caco-2wt)

100

100

Caco-2eps-

96.43 ± 2.76

95.36 ± 3.57

Caco-2ss

96.03 ± 3.69

96.87 ± 3.41

94.17 ± 4.77

95.01 ± 3.12

92.71 ± 6.03

92.21 ± 5.10

93.30 ± 3.65

94.22 ± 2.39

Caco-2wt

+ CPZ (100 μM)

Caco-2eps- + CPZ (100 μM) Caco-2ss +

CPZ (100 μM)

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Vol. 41 ı No. 4 ı 2006

Research Reports

cells, amounting to 45.1% and 43.0%, respectively.

Internalized 125I-Transferrin Released in the Medium (% of total)

60 Caco-2 Caco-2 eps-

50

DISCUSSION 40 30 20 10 0 0

5

10

15

20

25

30

35

Time (min) Figure 4. Effect of reduced epsin expression on the recycling of 125I-transferrin. Caco-2wt (°) and Caco-2eps- cells (n) in suspension were cooled on ice, and 125I-transferrin [106 counts per minute (cpm)] was allowed to bind for 60 min. Cells were washed three times with ice-cold phosphate-buffered saline (PBS), further incubated at 37°C for 1 h, and subsequently placed on ice to block endocytosis. Surface bound 125I-transferrin was stripped using 0.1 M HAc, pH 2.5. Cells were further incubated in the presence of 3 μg/mL unlabeled transferrin at 37°C for the indicated times. Radioactivity in the medium was determined at each time point.

application of siRNA-induced silencing of specific target genes (Figure 1). Western Blot Analysis In order to evaluate expression of epsin at the protein level, Western blot analysis was applied. Densitometric scans revealed a strong reduction of cellular epsin content amounting to approximately 75% in transfected Caco-2eps- cells versus controls (Figure 1E). The reduction of mRNA in the Caco-2eps- cells was thus paralleled by a concurring decrease of protein, although much less pronounced. Internalization of Transferrin/EGF In order to evaluate the transfection effects at the functional level, the uptake of transferrin and EGF in transfected Caco-2eps- and control cells was monitored. Transferrin/EGF and their receptors are known to be exclusively internalized via coated pits and can be considered as markers for this endocytic pathway. As depicted in Figure 2, in Caco-2wt cells, 72.5% of surface bound transferrin was internalized, whereas in Caco-2eps- cells, only 24.6% was taken up, confirming that in these cells endocytosis via coated pits was Vol. 41 ı No. 4 ı 2006

prominently impeded. Equal results were obtained when monitoring EGF internalization. Figure 3 shows 65.9% internalization of surface bound EGF in Caco-2wt, while in Caco-2eps- only 33.9% was taken up. To confirm the observed requirement for clathrin-coated pits as the vehicle for internalization of transferrin in an independent way, experiments have been performed with CPZ, a drug reported to prevent coated pit assembly at the cell surface. Pretreatment of cells with 100 μM CPZ did not reduce the transferrin internalization to the same extent as observed in the Caco-2epscells with only 34.6% being taken up. Also, administration of 100 μM CPZ to Caco-2eps- cells did not result in any significantly additive effect, as only 23.5% of surface bound 125I-transferrin were internalized (Figure 2). Recycling of Transferrin Since a reduction in internalization of transferrin may be influenced by an inhibition of transferrin recycling, we looked whether in Caco-2eps- cells the rate of recycling was perturbed. As shown in Figure 4, recycling of transferrin displayed only minor differences between Caco-2wt and Caco-2eps-

In order to facilitate the study of internalization pathways, we have constructed a Caco-2eps- cell line, constitutively and sequence specifically suppressing (Table 1) epsin expression, which is an essential accessory component of clathrin-mediated endocytosis. To confirm the specificity of the epsin shRNA, we transfected Caco-2 cells with a second RNA duplex (Table 1) targeting a different sequence within the same highly conserved open reading frame. This alternative siRNA silenced expression of epsin to a similar extent. We chose epsin as the target for shRNA-mediated silencing, because application of this technique on other proteins such as clathrin heavy chain (CHC) and adaptor protein 2 (AP-2) suffers from cross-reaction phenomena with other endocytic pathways (44). CHC, although inherently specific for clathrin-mediated endocytosis, is not unique for internalization at the plasma membrane, since it is also involved in vesicular transport at the trans-Golgi network (TGN) (45). Whereas AP-2 has an important function in the recognition of certain cargo motifs, it is not recognized by all cargo molecules internalized via coated pits. In addition, several lines of evidence suggest that coated pits can form independently of AP-2 interaction with cargo (46–48). For epsin, to our knowledge, no restrictions in specificity for clathrin-mediated endocytosis at the plasma membrane have been reported. Highly specific reduction of epsin in Caco-2eps- markedly reduced the uptake of transferrin and EGF, marker proteins of clathrin-mediated endocytosis. Other studies (7,9,44,48,49) have shown that uptake of molecules entering the cytoplasm via clathrin-coated vesicles is also depressed in cells depleted in either CHC, amphiphysin, eps15, or AP-2 down to 20%, 18%, 20%, and 15%, respectively. In our experiments, epsin expression at the protein level was reduced by approximately 75%, resulting in an equal inhibition www.biotechniques.com ı BioTechniques ı 481

Research Reports

of clathrin-mediated endocytosis. Reduction at the protein level was much less pronounced as compared with inhibition at the mRNA expression level. This discrepancy might be partially explained by a more appreciable contribution of the background, when quantifying weak protein bands by densitometry. An alternative explanation could be the very efficient translation of the residual mRNA encoding for epsin, a protein with a very stable basal expression. Finally, the half-life time of epsin might be long enough to ensure a high steady state level of the protein. As outlined above, an approximately 75% reduction was sufficient to suppress clathrin-mediated endocytosis significantly and interfere with normal function of this endocytic pathway. The partial blocking of clathrinmediated uptake has the advantage that cell viability is not significantly reduced, moreover complete inhibition would undoubtedly lead to induction of apoptosis. In this respect, Hinrichsen and coworkers have shown that a reduction of clathrin performance to 20% is the absolute limit to ensure cell viability (44). In addition, Iversen et al. have reported that transferrin receptor uptake can never be abolished completely in living cells (49). Moreover, according to these authors, inhibition of transferrin uptake was already initiated, when cellular clathrin concentration was reduced by no more than 10%. As an alternative approach to specifically inhibit clathrin-mediated endocytosis, dominant negative overexpression of mutant dynamin has been favored. Dynamin is a GTPase with an essential role in clathrinmediated endocytosis; it is assumed to be specifically involved in the fission of clathrin-coated vesicles. A K44A point mutation of dynamin converts the protein into a dominant negative inhibitor of clathrin-mediated endocytosis (13,14). Although dominant negative overexpression of mutant dynamin is generally used to inhibit clathrin-dependent endocytosis, according to Yao et al. (15) this experimental strategy does not allow the distinction between caveolae- and clathrin-mediated uptake. Furthermore, contrary to RNAi, overexpression 482 ı BioTechniques ı www.biotechniques.com

of a protein always results in very high intracellular amounts of mutant protein, with the risk of unpredictable secondary side effects. Finally, considering the translation efficiency (a typical mRNA yields approximately 5000 protein copies), RNAi targeting mRNA rather than the protein itself is potentially a much more efficient approach to block specific protein functions (50). Apart of genetic engineering approaches, endocytic processes have also been studied by using a number of drugs including cholesterol sequestrating and extracting drugs such as filipin and methyl β cyclodextrin (MβCD). These types of drugs have been used to specifically perturb internalization processes via lipid rafts and caveolae (9) but have also been reported to interfere with clathrinmediated uptake. Cationic amphiphilic drugs (CADs) such as CPZ are known to cause clathrin lattices to assemble on endosomal membranes and at the same time prevent coated pit assembly at the cell surface, by reversing an on/ off switch that controls AP-2 binding to membranes (51). Therefore, we have compared the effects elicited by RNAi to direct treatment of the cells with CADs on clathrin-mediated endocytosis in Caco-2 cells as evident from the degree of transferrin internalization. From our results, it is clear that in Caco-2eps- cells transferrin uptake is more compromised than in CAD-treated cells, although no significant differences were observed. Furthermore, exposing Caco-2eps- cells to CPZ only slightly increased the inhibition of transferrin uptake. Since high concentrations of CPZ have been reported to elicit numerous side effects (46), the RNAi approach is thought to be much more specific. In conclusion, by using the powerful technique of RNAi, we succeeded in developing an experimental cell line model to study clathrin-mediated internalization processes. Indeed, in this Caco-2eps- cell line, uptake of transferrin and EGF was strongly impaired, whereas only minor effects on the recycling of transferrin were noticed.

ACKNOWLEDGMENTS

The authors are indebted to Rony Goossens and Karen Verstraeten for skillful technical assistance. They thank Lieve Naessens for care in the layout. They are also grateful to Prof. Dr. Em. Albert Lagrou for reading the manuscript and helpful discussions. This work was partially financially supported by a RAFO grant, no. RAFO/1 DEWOM KP02. COMPETING INTERESTS STATEMENT

The authors declare no competing interests. REFERENCES 1. Dupré, S., D. Urban-Grimal, and R. Hagenuenauer-Tsapis. 2004. Ubiquitin and endocytic internalization in yeast and animal cells. Biochim. Biophys. Acta 1695:89-111. 2. Johannes, L. and C. Lamaze. 2002. Clatrindependant or not: is it still the question? Traffic 3:443-451. 3. Pelkmans, L. and A. Helenius. 2002. Endocytosis via caveolae. Traffic 3:311-320. 4. Nichols, B. 2003. Caveosomes and endocytosis of lipid rafts. J. Cell Sci. 116:4707-4714. 5. Rodal, S.K., G. Skretting, O. Garred, F. Vilhardt, B. Van Deurs, and K. Sandvig. 1999. Extraction of cholesterol with methylbeta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol. Biol. Cell 10:961-974. 6. Subtil, A., I. Gaidarov, K. Kobylarz, M.A. Lampson, J.H. Keen, and T.E. McGraw. 1999. Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc. Natl. Acad. Sci. USA 96:6775-6780. 7. Benmerah, A., C. Lamaze, B. Bègue, S.L. Schmid, A. Dautry-Varsat, and N. CerfBensussan. 1998. AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J. Cell Biol. 140:1055-1062. 8. Bennett, E.M., S.X. Lin, M.C. Towler, F.R. Maxfield, and F.M. Brodsky. 2001. Clathrin hub expression affects early endosome distribution with minimal impact on receptor sorting and recycling. Mol. Biol. Cell 12:2790-2799. 9. Wigge, P., Y. Vanllis, and H.T. McMahon. 1997. Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain. Curr. Biol. 7:554-560. 10. Wettey, F.R., S.F.C. Hawkins, A. Stewart, J.P. Luzio, J.C. Howard, and A.P. Jackson. 2002. Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes. Science 297:1521-1525. 11. Moskowitz, H.S., J. Heuser, T.E. McGraw, and T.A. Ryan. 2003. Targeted chemical

Vol. 41 ı No. 4 ı 2006

Research Reports

disruption of clathrin function in living cells. Mol. Biol. Cell 14:4437-4447. 12. Motley, A., N.A. Bright, M.N.J. Seaman, and M.S. Robinson. 2003. Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162:909-918. 13. Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127:915-934. 14. Sever, S., H. Damke, and S.L. Schmid. 2000. Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated cytosis. J. Cell Biol. 150:1137-1148. 15. Yao, Q., J. Chen, H. Cao, J.D. Orth, J.M. McCaffery, R.V. Stan, and M.A. McNiven. 2005. Caveolin-1 interacts directly with dynamin-2. J. Mol. Biol. 348:491-501. 16. Sijen, T., J. Fleenor, F. Simmer, K.L. Thijssen, S. Parrish, L. Timmons, R.H. Plasterk, and A. Fire. 2001. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476. 17. Paddison, P.J. and G.J. Hannon. 2002. RNA interference: the new somatic cell genetics? Cancer Cell 2:17-23. 18. Saksela, K. 2003. Human viruses under attack by small inhibitory RNA. Trends Microbiol. 11:345-347. 19. Ford, M.G., I.G. Mils, B.J. Peter, Y. Vallis, G.J. Praefcke, P.R. Evans, and H.T. McMahon. 2002. Curvature of clathrin-coated pits driven by epsin. Nature 419:361-366. 20. Chen, H., S. Fre, V.I. Slepnev, M.R. Capua, K. Takei, M.H. Butler, P.P. Di Fiore, and P. de Camilli. 1998. Epsin is an EH-domainbinding protein implicated in clathrin-mediated endocytosis. Nature 394:793-797. 21. Rosenthal, J.A., H. Chen, V.I. Slepnev, L. Pellegrini, A.E. Salcini, P.P. di Fiore, and P. de Camilli. 1999. The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module. J. Biol. Chem. 27:33959-33965. 22. Spradling, K.D., T.J. Burke, J. Lohi, and B.K. Pilcher. 2000. Cloning and initial characterization of human epsin 3, a novel matrixinduced keratinocyte specific transcript. J. Invest. Dermatol. 115:332. 23. De Camilli, P., H. Chen, J. Hyman, E. Panepucci, A. Bateman, and A.T. Brunger. 2002. The ENTH domain. FEBS Lett. 513:1118. 24. Cadavid, A.L., A. Ginzel, and J.A. Fischer. 2000. The function of the Drosophila fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Development 127:1727-1736. 25. Legendre-Guillemin, V., S. Wasiak, N.K. Hussain, A. Angers, and P.S. McPherson. 2004. ENTH/ANTH proteins and clathrinmediated membrane budding. J. Cell Sci. 117:9-18. 26. Hyman, J., H. Chen, P.P. Di Fiore, P. De Camilli, and A.T. Brunger. 2000. Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukaemia Zn(2)+ finger protein (PLZF). J. Cell Biol. 149:537-546. Vol. 41 ı No. 4 ı 2006

27. Ford, M.G., B.M. Pearse, M.K. Higgins, Y. Vallis, D.J. Owen, A. Gibson, C.R. Hopkins, P.R. Evans, and H.T. McMahon. 2001. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291:10511055. 28. Itoh, T., S. Koshiba, T. Kigawa, A. Kikuchi, S. Yokoyama, and T. Takenawa. 2001. Role of the ENTH domain in phosphatidylinositol-4,5-biphosphate binding and endocytosis. Science 291:1047-1051. 29. Stahelin, R.V., F. Long, B.J. Peter, D. Murray, P.P. De Camilli, H.T. McMahon, and W. Cho. 2003. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains. J. Biol. Chem. 278:28993-28999. 30. Nossal, R. and J. Zimmerberg. 2002. Endocytosis: curvature to the ENTH degree. Curr. Biol. 12:770-772. 31. Rosenthal, J.A., H. Chen, V.I. Slepnev, L. Pellegrini, A.E. Salcini, P.P. Di Fiore, and P. De Camilli. 1999. The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein molecule. J. Biol. Chem. 274:33959-33965. 32. Tebar, F., S.K. Bohlander, and A. Sorkin. 1999. Clathrin assembly lymphoid myeloid leukaemia (CALM) protein: localization in endocytic coated pits, interactions with clathrin; and the impact of overexpression on clathrin-mediated traffic. Mol. Biol. Cell 10:2687-2702. 33. Wendland, B., K.E. Steece, and S.D. Emr. 1999. Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis. EMBO J. 18:43834393. 34. Morgan, J.R., K. Prasad, W. Hao, G.J. Augustine, and E.M. Lafer. 2000. A conserved clathrin assembly motif essential for synaptic vesicle endocytosis. J. Neurosci. 20:8667-8676. 35. Engqvist-Goldstein, A.E., R.A. Warren, M.M. Kessels, J.H. Keen, J. Heuser, and D.G. Drubin. 2001. The actin-binding proteinHip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro. J. Cell Biol. 154:12091223. 36. Metzler, M., V. Legendre-Guillemin, L. Gan, V. Chopra, A. Kwok, P.S. McPherson, and M.R. Hayden. 2001. HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J. Biol. Chem. 276:39271-39276. 37. Morinaka, K., S. Koyama, S. Nakashima, T. Hinoi, K. Okawa, A. Iwamatsu, and A. Kikuchi. 1999. Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis. Oncogene 18:5915-5922. 38. Strausberg, R.L., E.A. Feingold, L.H. Grouse, J.G. Derge, R.D. Klausner, F.S. Collins, L. Wagner, C.M. Shenmen, et al. 2002. Generation and initial analysis of more than 15000 full-length human and mouse cDNA sequences. Proc. Natl. Acad. Sci. USA. USA 99:16899-16903. 39. Tuschl, T., P.D. Zamore, R. Lehmann, D.P. Bartel, and P.A. Sharp. 1999. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13:3191-3197.

40. Reynolds, A., D. Leake, Q. Boese, S. Scaringe, W.S. Marchall, and A. Khvorova. 2004. Rational siRNA design for RNA interference. Nat. Biotechnol. 22:326-330. 41. Pfaffl, M.W. 2000. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29:2006-2007. 42. Carmichael, J., W.G. DeGraff, A.F. Gazdar, J.D. Minna, and J.B. Mitchell. 1987. Evaluation of a tetrazolium-based semi-automated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47:936942. 43. Fraker, P.J. and J.C. Speck, Jr. 1978. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80:849-857. 44. Hinrichsen, L., J. Harborth, L. Andrees, K. Weber, and E.J. Ungewickell. 2003. Effect of clathrin heavy chain- and α-adaptin-specific small inhibitory RNAs on endocytic accessory proteins end receptor trafficking in HeLa cells. J. Biol. Chem. 278:45160-45170. 45. Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2002. Intracellular vesicular traffic. Mol. Biol. Cell. 4:711-766 46. Nesterov, A., R.E. Carter, T. Sorina, G.N. Gill, and A. Sorkin. 1999. Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis. EMBO J. 18:2489-2499. 47. Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo. Curr. Biol. 11:665-676. 48. Rappoport, J.Z., S.M. Simon, and A. Benmerah. 2004. Understanding living clathrin-coated pits. Traffic 5:327-337. 49. Iversen, T.G., G. Skretting, B. Van Deurs, and K. Sandvig. 2003. Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA. Proc. Natl. Acad. Sci. USA 100:5175-5180. 50. Nordenberg, J., E. Fenig, M. Landau, R. Weizman, and A. Weizman. 1999. Effects of psychotropic drugs on cell proliferation and differentiation. Biochem. Pharmacol. 58:12291236. 51. Wang, L.H., K.G. Rothberg, and R.G.W. Anderson. 1993. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123:1107-1117.

Received 16 March 2006; accepted 4 August 2006. Address correspondence to Marc J.S. De Wolf, UA-Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B2020 Antwerp, Belgium. e-mail: [email protected]

To purchase reprints of this article, contact: [email protected] www.biotechniques.com ı BioTechniques ı 483