Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Apr 18.
Published in final edited form as: Chem Biol. 2013 Apr 18;20(4):614–618. doi: 10.1016/j.chembiol.2013.03.016

Fluorogenic Probe for Constitutive Cellular Endocytosis

Michael N Levine 1, Trish T Hoang 1, Ronald T Raines 1,2,*
PMCID: PMC3671891  NIHMSID: NIHMS463396  PMID: 23601650

SUMMARY

Endocytosis is a fundamental process of eukaryotic cells that is critical for nutrient uptake, signal transduction, and growth. We have developed a molecular probe to quantify endocytosis. The probe is a lipid conjugated to a fluorophore that is masked with an enzyme-activatable moiety known as the trimethyl lock. The probe is not fluorescent when incorporated into the plasma membrane of human cells but becomes fluorescent upon internalization into endosomes, where cellular esterases activate the trimethyl lock. Using this probe, we found that human breast cancer cells undergo constitutive endocytosis more rapidly than do matched noncancerous cells. These data reveal a possible phenotypic distinction of cancer cells that could be the basis for chemotherapeutic intervention.

INTRODUCTION

Endocytosis is the key regulator of macromolecular internalization into eukaryotic cells (Conner and Schmid, 2003). In this intricate process, proteins mediate the invagination of the plasma membrane and then its fusion to pinch off a lipid bilayer-encased vesicle within a cell (Doherty and McMahon, 2009). Many endocytic pathways operate in parallel. The most studied pathway, clathrin-mediated endocytosis, occurs constitutively in all cell types and generally involves the binding of a ligand to a receptor prior to internalization (McMahon and Boucrot, 2011). Another pathway, caveolae-mediated endocytosis, is characterized by vesicles enriched in glycosphingolipids, cholesterol, and the integral membrane protein, caveolin (Hansen and Nichols, 2009). These endocytic pathways are the portals for delivery of essential nutrients, such as iron via transferrin and cholesterol via lipoprotein particles. Deleteriously, these pathways can facilitate the transit of pathogens (Gruenberg and van der Goot, 2006; Kerr and Teasdale, 2009).

Endocytosis regulates the concentration of cell-surface receptors by transporting them to and from the plasma membrane (Mosesson et al., 2008). Accordingly, endocytosis has a direct influence on signal transduction pathways that can malfunction in cancer patients (Hanahan and Weinberg, 2011). Conversely, differences in endocytosis between cancerous and noncancerous cells could lead to new treatment options. For example, pancreatic-type ribonucleases (ptRNases) have emerged as putative cancer chemotherapeutic agents (Lee and Raines, 2008; Arnold, 2008; Fang and Ng, 2011; Lomax et al., 2012). These cationic enzymes are internalized by receptor-independent endocytosis, and then escape from endosomes to the cytosol where they catalyze the degradation of cellular RNA. The basis for their cancer cell-specific toxicity is not clear, but could entail differential rates of endocytosis.

Two types of assays have been used to monitor constitutive endocytosis (Ivanov, 2007). In one, endocytosis has been quantified by assaying the uptake of soluble enzymes, such as horseradish peroxidase (Steinman and Cohn, 1972). Data are acquired by fixing cells, and then staining them with a colorimetric substrate. This assay is discontinuous and vulnerable to the artifacts that can accompany the use of fixed cells (Richard et al., 2003). Alternatively, the fate of a fluorescent lipid has been monitored continuously by microscopy (Kok et al., 1990; Cochilla et al., 1999; Maier et al., 2002; Rea et al., 2004; Bissig et al., 2012). These assays require extensive washing to remove unincorporated lipid and are not amenable to automated cell counting and sorting techniques.

We sought to develop a facile means to assess endocytosis continuously in live cells. An ideal molecular probe would have no background fluorescence, and would be able to distinguish the lumen of endosomes from the plasma membrane. We reasoned that a lipid with a headgroup that is responsive to an endosomal enzyme could serve as the basis as such a probe, as fluorescence generated over time would reflect the rate of endocytosis. Here we report on the efficacy of our strategy.

RESULTS AND DISCUSSION

To test our strategy, we designed lipid 1. Lipid 1 contains a “trimethyl lock” moiety in which an acetyl ester acts as a molecular trigger (Levine and Raines, 2012). This ester linkage is known to be stable to hydrolysis at physiological pH (Chandran et al., 2005; Lavis et al., 2006a; Lavis et al., 2006b; Levine et al., 2008). Although the ester linkage in lipid 1 is insulated from the fluorophore, its hydrolysis is coupled to the cleavage of its otherwise recalcitrant amide bond. Analogous probes have been used to quantify the endocytosis of soluble molecules (Johnson et al., 2007; Mangold et al., 2008; Turcotte et al., 2009; Chao et al., 2010; Chao and Raines, 2011), but not membrane-associated ones. Our expectation here was that upon endocytosis, the headgroup of lipid 1 would encounter endosomal esterases (Tian et al., 2012). The ensuing hydrolysis of the acetyl ester would unmask the rhodamine moiety and label the lumen with fluorescence (Figure 1). The modularity of lipid 1 facilitated its synthesis by a route ending with the conjugate addition of 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanol to a trimethyl lock–rhodamine–maleimide fragment.

Figure 1. Structure and function of lipid 1.

Figure 1

Open in a new tab

Fluorescence is unmasked by an esterase encountered upon endocytosis.

The phosphatidylglycerol moiety of lipid 1 is endogenous to humans and incorporates spontaneously into cellular membranes (Christie and Han, 2010). Most importantly for us, incorporated lipid 1 paints HeLa cells incubated at 37 °C with a punctate staining pattern, as shown in Figure 2A. This pattern is indicative of vesicular localization, and demonstrates that lipid 1 does indeed report on constitutive endocytosis. Notably, no fluorescence was observed in cells incubated at 4 °C (Figure 2B), a temperature that does not allow for endocytosis (Tomoda et al., 1989).

Figure 2. Lipid 1 reports on endocytosis.

Figure 2

Open in a new tab

HeLa cells in serum-free Dulbecco’s modified Eagle’s medium (DMEM) were labeled for 3 h at 4 °C with lipid 1 (20 μM), washed with serum-free medium, and then incubated for 3 h at (A) 37 °C, or (B) 4 °C. Left panels, confocal images; right panels, overlay of confocal and bright-field images; blue dye, Hoechst 33342; scale bars, 20 μm.

Endocytic pathways are complex (Bissig et al., 2012). What then is the fate of lipid 1 after endocytosis? As shown in Figure 3A, we found that lipid 1 does not colocalize with fluorescently labeled transferrin, which is a marker of recycling endosomes (Ghosh et al., 1994). Consistent with this finding, lipid 1 that had been unmasked by a cellular esterase does not reappear on the plasma membrane (Figure 3B). These data indicate that lipid 1 does not recycle to the plasma membrane, but instead enters endosomes and traffics to other destinations. This attribute is desirable because fluorescence from lipid 1 reports only on new endocytic events (and not repetitious entry). As shown in Figure 3C, we found that lipid 1 does colocalize partially with LysoTracker® Red, a marker of late endosomes or lysosomes (Zhang et al., 1994), evincing its joining the canonical endosome-to-lysosome pathway along with trafficking to other subcellular compartments.

Figure 3. Lipid 1 does not recycle to the cell surface.

Figure 3

Open in a new tab

HeLa cells in serum-free DMEM were labeled for 3 h at 4 °C with lipid 1 (20 μM), washed with serum-free medium, and then incubated for 3 h at 37 °C. (A) Image after an Alexa Fluor® 594–transferrin conjugate (1 μM) was added for the final 1 h of a 3-h incubation. (B) Image after a 24-h incubation. (C) Image after LysoTracker® Red (50 nM) was added for the final 20 min of a 3-h incubation. Blue, Hoechst 33342; scale bars, 20 μm.

Lipid 1 can report on the rate of endocytosis. HeLa cells were labeled at 4 °C with lipid 1 and then incubated for various times at 37 °C. Fluorescence was quantified by flow cytometry. As shown in Figure 4, the mean fluorescence per cell increases over time until ~2 h. This time course is consistent with morphological observations of mouse fibroblasts, which were seen to engulf their cell surface every 125 min (Steinman et al., 1976).

Figure 4. Time course of endocytosis by HeLa cells.

Figure 4

Open in a new tab

HeLa cells in serum-free DMEM were labeled for 3 h at 4 °C with lipid 1 (20 μM), washed with serum-free medium, and incubated for various times at 37 °C. Fluorescence was quantified by flow cytometry. Values in arbitrary units (AU) are the mean ± SD from assays of 10,000 cells.

Finally, lipid 1 can reveal differences in endocytic rates between similar cells. HTB-125 and HTB-126 are noncancerous and cancerous breast cell lines that were derived from the same patient (An et al., 2008). We used lipid 1 to assess endocytosis by cells in these matched lines, quantifying the results by flow cytometry. As shown in Figure 5, the increase in fluorescence over 3 h of incubation at 37 °C was 2.5-fold for HT B-125 cells. This increase was less than half that for HTB-126, which was 6.0-fold. Thus, in these cell lines, endocytosis is significantly more rapid in the cancerous than in the noncancerous cells. These differential rates could reflect more rapid turnover of cell-surface receptors in cancer cells (Mayor and Pagano, 2007), promoting a cancerous phenotype (Mosesson et al., 2008). We note that such an intrinsic difference in endocytic rate could provide an opportunity for therapeutic intervention by increasing the relative uptake of ptRNases or other macromolecular drugs (Sahay et al., 2010; Stephan and Irvine, 2011; Hymel and Peterson, 2012).

Figure 5. Rate of endocytosis is greater in cancerous cells.

Figure 5

Open in a new tab

Matched breast cell lines HTB-125 (noncancerous) and HTB-126 (cancerous) in serum-free DMEM were labeled for 3 h at 4 °C with lipid 1 (20 μM), washed with serum-free medium, and incubated for 0 or 3 h at 37 °C. Fluorescence was quantified by flow cytometry. Values are the mean ± SD from triplicate assays of 2000 cells. HTB-125 cells: 3.7 ± 0.7 and 9.4 ± 0.7 AU; HTB-126: 3.1 ± 0.6 and 18.6 ± 2.2 AU.

SIGNIFICANCE

We have designed and synthesized a headgroup-modified lipid that can be incorporated into the plasma membrane, and that becomes fluorescent upon initiation of endocytosis. The lipid is trafficked to late endosomes and lysosomes and does not enter the recycling endosomal pathway. The increase in fluorescence can be monitored over time by flow cytometry and is utilized to reveal that cells from a breast cancer line undergo endocytosis more rapidly than do matched noncancerous cells. These data enable and encourage the use of lipid 1 in the exploration of endocytosis.

EXPERIMENTAL PROCEDURES

General

All reagents, unless noted, were from Aldrich Chemical (Milwaukee, WI) or Fisher Scientific (Hanover Park, IL), and were used without further purification. Thin-layer chromatography was performed by using aluminum-backed plates coated with silica gel containing F254 phosphor, and was visualized by UV illumination or developed with ceric ammonium molybdate stain. Flash chromatography was performed on open columns with silica gel-60 (230–400 mesh).

NMR spectra were obtained with a Bruker DMX-400 Avance spectrometer at the National Magnetic Resonance Facility at Madison (NMRFAM). Mass spectrometry was performed with an Applied Biosystems MDS SCIEX 4800 matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometer at the Mass Spectrometry Facility in the Biotechnology Center, University of Wisconsin–Madison.

The term “concentrated under reduced pressure” refers to the removal of solvents and other volatile materials using a rotary evaporator at water aspirator pressure (<20 torr) while maintaining the water-bath temperature below 50 °C. The term “high vacuum” refers to vacuum (<0.1 torr) achieved by a mechanical belt-drive oil pump.

Synthesis of Lipid 1

Maleimidourea–Rh110 trimethyl lock (20 mg, 0.026 mmol) was synthesized as described previously (Lavis et al., 2006a), and dissolved in anhydrous chloroform (5 mL). The resulting solution was added to a flame-dried 10-mL round-bottom flask that had been flushed with Ar(g). Anhydrous triethylamine (20 μL, 0.14 mmol) was added, followed by 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanol, sodium salt (Avanti Polar Lipids, Alabaster, AL; 20 mg, 0.027 mmol). The flask was covered in foil, and the reaction mixture was stirred for 3 h under Ar(g). Reaction progress was monitored by thin-layer chromatography (10% v/v methanol in DCM). Once the reaction was complete, the solvent was evaporated under reduced pressure and the residue was placed under high vacuum overnight. The crude product was purified by silica gel chromatography (10–15% v/v methanol in DCM) to yield 1 as a white powder (26 mg, 66%). 1H NMR (400 MHz, CDCl3) δ: 8.52 (bs, 1H), 7.94 (d, J = 6.3 Hz, 1H), 7.79 (s, 1H), 7.64–7.50 (m, 2H), 7.39 (s, 2H), 7.05 (d, J = 6.9 Hz, 1H), 6.97 (bs, 1H), 6.76 (s, 1H), 6.64–6.56 (m, 2H), 6.51 (t, J = 7.2 Hz, 2H), 6.13 (s, 1H), 5.20 (s, 1H), 4.35 (d, J = 10.8 Hz, 1H), 4.15–3.86 (m, 6H), 3.52–3.40 (m, 2H), 3.24–2.92 (m, 7H), 2.87–2.75 (m, 1H), 2.63–2.57 (m, 2H), 2.41 (s, 3H), 2.34 (s, 3H), 2.28–2.15 (m, 7H), 1.64 (s, 6H), 1.56–1.46 (m, 4H), 1.33–1.14 (m, 48H), 0.87 (t, J = 6.2 Hz, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ: 178.5, 175.4, 174.0, 173.7, 172.1, 170.4, 170.0, 156.0, 153.0, 151.8, 151.7, 150.1, 142.5, 140.1, 139.0, 137.3, 135.4, 133.2, 133.1, 129.9, 128.3, 126.5, 125.0, 124.2, 123.5, 115.4, 114.7, 114.0, 111.6, 107.6, 106.0, 83.6, 70.6, 65.3, 63.9, 62.9, 50.9, 40.3, 39.6, 37.0, 36.5, 34.4, 34.2, 32.1, 30.2–29.2, 27.1, 26.7, 25.7, 25.1, 25.0, 22.8, 22.0, 20.3, 14.3 ppm. 31P NMR (162 MHz, CDCl3) δ: −1.7 ppm. MS (MALDI): m/z 1487.75 [M+Na]+ ([C80H113O17N4NaPS]+ = 1487.75).

Mammalian Cell Culture

HeLa, HTB-125, and HTB-126 cells were from the American Type Culture Collection (ATCC) (Manassas, VA). HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing fetal bovine serum (FBS; 10% v/v), penicillin (100 units/mL), and streptomycin (100 μg/mL). HTB-125 cells were grown in Hybri-Care Medium supplemented with sodium bicarbonate (1.5 g/L), mouse epidermal growth factor (30 ng/mL), FBS (10% v/v), penicillin (100 units/mL), and streptomycin (100 μg/mL). HTB-126 cells were cultured in DMEM supplemented with bovine insulin (10 μg/mL), FBS (10% v/v), penicillin (100 units/mL), and streptomycin (100 μg/mL). Media and supplements were from Invitrogen (Carlsbad, CA), Sigma–Aldrich (Milwaukee, WI), or ATCC. Cells were cultured at 37 °C in a humidified incubator containing CO2(g) (5% v/v).

Microscopy

Imaging was performed with a Eclipse TE2000-U laser scanning confocal microscope from Nikon (Tokyo, Japan) equipped with a AxioCam digital camera from Carl Zeiss (Oberkochen, Germany). A blue-diode laser was used to provide excitation at 408 nm, and emission light was passed through a 35-nm band-pass filtered centered at 450 nm. An argon-ion laser was used to provide excitation at 488 nm, and emission light was passed through a 40-nm band-pass filter centered at 515 nm. A HeNe laser was used to provide excitation at 543 nm, and emission light was passed through a 75-nm band-pass filter centered at 605 nm.

HeLa cells were plated 24 h prior to experiments at a density of 1 × 105 cells in 1-cm diameter glass-bottom dishes from Electron Microscopy Sciences (Hatfield, PA) in 1 mL of medium. On the day of an experiment, all cells, media, and pipette tips were pre-cooled to 4 °C for 2 h. Next, cells were washed with serum-free DMEM (3 × 1 mL). Stock solutions of lipid 1 (50 mM in DMSO) were diluted to 10 mM with absolute ethanol. From this stock, 1 μL was added to 500 μL of serum-free DMEM, which was then vortexed vigorously, and applied to the HeLa cells. Vehicle-treated cells were treated with 500 μL of serum-free DMEM containing 1 μL of ethanol. The labeling reaction was allowed to proceed for 3 h at 4 °C, after which, the cells were washed with serum-free DMEM (3 × 1 mL). Cells were incubated for the given amount of time at 37 °C. LysoTracker® Red (50 nM) from Invitrogen was used to stain acidic vesicles for the final 20 min of incubation at 37 °C. Endocytic marker Alexa Fluor® 594–transferrin (1 μM) from Invitrogen was used to stain recycling endosomes for the final 1 h of incubation at 37 °C. Nuclear counterstaining was performed with Hoechst 33342 (2 μg/mL) from Invitrogen for the final 5 min at 37 °C. Cells were washed with serum- free DMEM prior to imaging.

Flow Cytometry

Flow cytometry was performed at the University of Wisconsin Carbone Cancer Center with a FACSCalibur instrument equipped with a 488 argon-ion air-cooled laser from Becton Dickinson (Franklin Lakes, NJ). Fluorescence emission light was passed through a 30-nm band pass filter centered at 530 nm. Cell lines were plated 24 h prior to experiments at a density of 3 × 105 HeLa cells and 3.7 × 104 cells HTB-125 or HTB-126 cells in 6 mL of medium (vide supra) in T-25 tissue culture flasks from BD Biosciences (San Jose, CA). On the day of an experiment, all cells, media, and pipette tips were pre-cooled to 4 °C for 2 h. Next, the cells were washed with serum-free DMEM (3 × 1 mL). Stock solutions of lipid 1 (50 mM in DMSO) were diluted with absolute ethanol to 10 mM. This stock solution in ethanol was added to serum-free DMEM to a final concentration of 20 μM, and was mixed vigorously by vortexing. The medium was removed from the cells and was replaced with 1 mL of the labeling solution. Vehicle treated cells were treated with 1 mL of serum-free DMEM containing 2 μL of ethanol. The labeling reaction was allowed to proceed for 3 h at 4 °C, after which, the cells were washed with serum-free DMEM (3 × 1 mL). Cells were incubated for a known time at 37 °C. Cells were then washed with DPBS (1 mL; Invitrogen) and treated with trypsin/EDTA (0.25% w/v; 750 μL) for 5 min at 37 °C. The trypsin was neutralized with DMEM containing FBS (10% v/v; 750 μL), and the cells were collected by centrifugation (5 min at 400g). The supernatant was decanted, and the cell pellet was resuspended in 1 mL of DPBS. The cells were collected again by centrifugation (5 min at 400g). The supernatant was decanted, and the pellet was resuspended and fixed with 100 μL of aqueous formaldehyde (2% v/v) for 30 min in a vial covered with aluminum foil. This solution was diluted by adding DPBS to 1 mL, and the cells were collected by centrifugation (5 min at 400g). The supernatant was decanted, and the cell pellet was resuspended in 1 mL of DPBS. The suspension was strained through a 35-μm filter into a polystyrene flow cytometry test tube from BD Biosciences. The fixed cells were stored on ice until analyzed (~1–4 h). The mean fluorescence per cell was determined in triplicate for 10,000 HeLa cells, 2000 HTB-125 cells, and 2000 HTB-126 cells, and the data were analyzed with FlowJo software from Tree Star (Ashland, OR).

Supplementary Material

01

Acknowledgments

We are grateful to M. T. Walker for assistance in assay optimization and T. F. J. Martin for the use of his confocal microscope and contributive discussions. T.T.H. was supported by a Graduate Research Scholars Advance Opportunity Fellowship and Molecular Biosciences Training Grant T32 GM07215 (NIH). This work was supported by Grants R01 CA073808 and R01 GM044783 (NIH), and made use of the National Magnetic Resonance Facility at Madison, which is supported by Grants P41 RR002301 and P41 GM066326 (NIH), and the Mass Spectrometry Facility, which is supported by Grants P50 GM064598 and R33 DK007297 (NIH), and Grants DBI-0520825 and DBI-9977525 (NSF).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information depicts 1H, 13C, and 31P NMR spectrum of lipid 1 and can be found with this article online at http://dx.doi.org/10.1016/j.chembiol.2013.xx.xxx.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. An S, Kumar R, Sheets ED, Benkovic SJ. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science. 2008;320:103–106. doi: 10.1126/science.1152241. [DOI] [PubMed] [Google Scholar]
  2. Arnold U. Aspects of the cytotoxic action of ribonucleases. Curr Pharm Biotechnol. 2008;9:161–168. doi: 10.2174/138920108784567263. [DOI] [PubMed] [Google Scholar]
  3. Bissig C, Johnson S, Gruenberg J. Studying lipids involved in the endosomal pathway. Methods Cell Biol. 2012;108:19–46. doi: 10.1016/B978-0-12-386487-1.00002-X. [DOI] [PubMed] [Google Scholar]
  4. Chandran SS, Dickson KA, Raines RT. Latent fluorophore based on the trimethyl lock. J Am Chem Soc. 2005;127:1652–1653. doi: 10.1021/ja043736v. [DOI] [PubMed] [Google Scholar]
  5. Chao TY, Lavis LD, Raines RT. Cellular uptake of ribonuclease A relies on anionic glycans. Biochemistry. 2010;49:10666–10673. doi: 10.1021/bi1013485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chao TY, Raines RT. Mechanism of ribonuclease A endocytosis: Analogies to cell-penetrating peptides. Biochemistry. 2011;50:8374–8382. doi: 10.1021/bi2009079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Christie WW, Han X. Lipid Analysis: Isolation, Separation, Identification and Lipidomic Analysis. 4. Cambridge, UK: Woodhead Publishing Limited; 2010. [Google Scholar]
  8. Cochilla AJ, Angleson JK, Betz WJ. Monitoring secretory membrane with FM1-43 fluorescence. Annu Rev Neurosci. 1999;22:1–10. doi: 10.1146/annurev.neuro.22.1.1. [DOI] [PubMed] [Google Scholar]
  9. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422:37–44. doi: 10.1038/nature01451. [DOI] [PubMed] [Google Scholar]
  10. Doherty GJ, McMahon HT. Mechanisms of endocytosis. Annu Rev Biochem. 2009;78:857–902. doi: 10.1146/annurev.biochem.78.081307.110540. [DOI] [PubMed] [Google Scholar]
  11. Fang EF, Ng TB. Biochim. Biophys Acta. 2011;1815:65–74. doi: 10.1016/j.bbcan.2010.09.001. [DOI] [PubMed] [Google Scholar]
  12. Ghosh RN, Gelman DL, Maxfield FR. J Cell Sci. 1994;107:2177–2189. doi: 10.1242/jcs.107.8.2177. [DOI] [PubMed] [Google Scholar]
  13. Gruenberg J, van der Goot FG. Mechanisms of pathogen entry through the endosomal compartments. Nat Rev Mol Cell Biol. 2006;7:495–504. doi: 10.1038/nrm1959. [DOI] [PubMed] [Google Scholar]
  14. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]
  15. Hansen CG, Nichols BJ. Molecular mechanisms of clathrin-independent endocytosis. J Cell Sci. 2009;122:1713–1721. doi: 10.1242/jcs.033951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hymel D, Peterson BR. Synthetic cell surface receptors for delivery of therapeutics and probes. Adv Drug Deliv Rev. 2012;64:797–810. doi: 10.1016/j.addr.2012.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ivanov AI, editor. Exocytosis and Endocytosis. Totowa, NJ: Humana Press; 2007. [Google Scholar]
  18. Johnson RJ, Chao TY, Lavis LD, Raines RT. Cytotoxic ribonucleases: The dichotomy of Coulombic forces. Biochemistry. 2007;46:10308–10316. doi: 10.1021/bi700857u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kerr MC, Teasdale RD. Defining macropinocytosis. Traffic. 2009;10:364–371. doi: 10.1111/j.1600-0854.2009.00878.x. [DOI] [PubMed] [Google Scholar]
  20. Kok JW, Beest M, Scherphof G, Hoekstra D. A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway. Eur J Cell Biol. 1990;53:173–184. [PubMed] [Google Scholar]
  21. Lavis LD, Chao TY, Raines RT. Fluorogenic label for biomolecular imaging. ACS Chem Biol. 2006a;1:252–260. doi: 10.1021/cb600132m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lavis LD, Chao TY, Raines RT. Latent blue and red fluorophores based on the trimethyl lock. Chem Bio Chem. 2006b;7:1151–1154. doi: 10.1002/cbic.200500559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee JE, Raines RT. Ribonucleases as novel chemotherapeutics: The ranpirnase example. Bio Drugs. 2008;22:53–58. doi: 10.2165/00063030-200822010-00006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Levine MN, Lavis LD, Raines RT. Trimethyl lock: A stable chromogenic substrate for esterases. Molecules. 2008;13:204–211. doi: 10.3390/molecules13020204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levine MN, Raines RT. Trimethyl lock: A trigger for molecular release in chemistry, biology, and pharmacology. Chem Sci. 2012;3:2412–2420. doi: 10.1039/C2SC20536J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lomax JE, Eller CH, Raines RT. Rational design and evalution of mammalian ribonuclease cytotoxins. Methods Enzymol. 2012;502:273–290. doi: 10.1016/B978-0-12-416039-2.00014-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maier O, Oberle V, Hoekstra D. Fluorescent lipid probes: Some properties and applications. Chem Phys Lipids. 2002;116:3–18. doi: 10.1016/s0009-3084(02)00017-8. [DOI] [PubMed] [Google Scholar]
  28. Mangold SL, Carpenter RT, Kiessling LL. Synthesis of fluorogenic polymers for visualizing cellular internalization. Org Lett. 2008;10:2997–3000. doi: 10.1021/ol800932w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mayor S, Pagano RE. Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol. 2007;8:603–612. doi: 10.1038/nrm2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2011;12:517–533. doi: 10.1038/nrm3151. [DOI] [PubMed] [Google Scholar]
  31. Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: An emerging feature of cancer. Nat Rev Cancer. 2008;8:835–850. doi: 10.1038/nrc2521. [DOI] [PubMed] [Google Scholar]
  32. Rea R, Li J, Dharia A, Levitan ES, Sterling P, Kramer RH. Streamlined synaptic vesicle cycle in cone photoreceptor terminals. Neuron. 2004;41:755–766. doi: 10.1016/s0896-6273(04)00088-1. [DOI] [PubMed] [Google Scholar]
  33. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B. Cell-penetrating peptides—a reevaluation of the mechanism of cellular uptake. J Biol Chem. 2003;278:585–590. doi: 10.1074/jbc.M209548200. [DOI] [PubMed] [Google Scholar]
  34. Sahay G, Alakhova DY, Kabanov VA. Endocytosis of nanomedicines. J Control Rel. 2010;145:182–195. doi: 10.1016/j.jconrel.2010.01.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Steinman RM, Brodie SE, Cohn ZA. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976;68:665–687. doi: 10.1083/jcb.68.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Steinman RM, Cohn ZA. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro. J Cell Biol. 1972;55:186–204. doi: 10.1083/jcb.55.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stephan MT, Irvine DJ. Enhancing cell therapies form the outside in: Cell surface engineering using synthetic nanomaterials. Nano Today. 2011;6:309–325. doi: 10.1016/j.nantod.2011.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tian L, Yang Y, Wysocki LW, Arnold AC, Hu A, Ravichandran B, Sternson SM, Looger LL, Lavis LD. Selective esterase–ester pair for targeting small molecules with cellular specificity. Proc Natl Acad Sci USA. 2012;109:4756–4761. doi: 10.1073/pnas.1111943109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tomoda H, Kishimoto Y, Lee YC. Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages. J Biol Chem. 1989;264:15445–15450. [PubMed] [Google Scholar]
  40. Turcotte RF, Lavis LD, Raines RT. Onconase cytotoxicity relies on the distribution of its positive charge. FEBS J. 2009;276:4270–4281. doi: 10.1111/j.1742-4658.2009.07098.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhang YZ, Diwu Z, Mao F, Leung W, Haugland RP. Mol. Biol Cell. 1994;5 (Suppl):113a. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS463396-supplement-01.pdf (1.9MB, pdf)

RESOURCES