Tat Peptide Is Capable of Importing Large Nanoparticles Across Nuclear Membrane in Digitonin Permeabilized Cells
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Understanding the capabilities and limitations of nuclear import is crucial to efficient delivery of macromolecules and nanoparticles for diagnosis and targeted therapy of diseases. Here we report the Tat peptide-mediated import of different cargos into cell nucleus, including dye-labeled streptavidin protein, 43 and 90 nm fluorescent beads, as well as ~20 nm quantum dots for kinetic measurements. Our results revealed significant differences between Tat- and NLS-mediated nuclear import: unlike delivery with the NLS, Tat peptide-based delivery is not inhibited by WGA blockage nor does it require ATP. Surprisingly, Tat peptide was able to import 90 nm beads into the nuclei of digitonin-permeabilized cells, suggesting that its interaction with the nuclear envelope follows a mechanism different from that of NLS. The import kinetics was quantified using Tat peptide-conjugated QDs, yielding a kinetic constant of 0.0085 s−1. Taken together, our results suggest that, compared with NLS, Tat peptide-mediated nuclear import is faster, follows a different pathway, and is capable of importing large nanoparticles. These results have significant implications for the development of new approaches for delivery of cargo into the nuclei of living cells.
KeywordsNuclear delivery Nanoparticles Nuclear localizing sequence Tat
The ability of eukaryotic cells to transport large macromolecules across the nuclear membrane is essential for many cellular functions, and the mechanism of such a process has received extensive study.1,2,7,10,16,17,21,23 The nuclear membrane contains specialized protein complexes called nuclear pore complexes (NPC), which control the transport of macromolecules larger than 30–40 kDa from the cytoplasm to the nucleus.1 The transport of such macromolecules typically requires a nuclear localization signal (NLS) domain either in the primary sequence of a protein or attached to the macromolecule exogenously. The transport process involves two steps: (1) bringing a macromolecule to the NPC, and (2) translocating the macromolecule through the pore. The first step is energy-independent, whereas the second requires energy.5 It has been shown that conventional nuclear transport requires the presence of various cytosolic factors such as importins, karyopherin and ATP, and can be inhibited by blockage of the NPC with wheat germ agglutinin (WGA), cold incubation, or depletion of ATP and importins.2,5,10,23
The efficient delivery of molecular imaging probes and therapeutic agents into the nuclei of living cells is crucial for development of novel disease diagnosis and treatment strategies. In addition to the use of the NLS peptide for nuclear transport, it has been shown that the Tat peptide, an 11-amino-acid peptide of the HIV-1 Tat protein, can carry cargo across both the plasma membrane and the nuclear membrane.9,19 To reveal the mechanism responsible for Tat-based cargo delivery, extensive studies have been carried out using different constructs, including the Tat peptide alone, the Tat peptide complexed with a fluorescent protein or other reporters, the HIV-1 Tat protein, and the Tat peptide conjugated to nanoparticles or liposomes.3,6,9,11,13,18,20,25 Although most of these studies aimed to understand the interaction between the Tat peptide with the plasma membrane, and the mechanisms of its membrane translocation,6,8,11, 12, 13,20 very limited effort has been made to understand the function of Tat peptide for nuclear import.9,25 Since Tat peptide and its small molecular weight conjugates (e.g. with fluorescent dyes, short oligonucleotides) can diffuse through the NPC, studies using these constructs are not able to provide a better understanding of the import mechanism associated with the NPC.
Characteristics of cargos for nuclear import studies
Fluorescence reporter and wavelength
Alexa-647 conjugated to streptavidin, peak excitation = 647 nm, peak emission = 660 nm
Polymer nanoparticles coated with streptavidin
Formulation proprietary, peak excitation = 488 nm, peak emission = 515 nm
Polymer nanoparticles coated with streptavidin
Alexa-647 conjugated to streptavidin, peak excitation = 647 nm, peak emission = 660 nm
Streptavidin-coated quantum dots
Excitation = 488 nm, peak emission = 580 nm
Peptide Synthesis, Conjugation and Labeling
The SV-40 NLS peptide (PKKKRKVKC) was synthesized by SynPep Inc. with purity >98%. The peptide was modified to introduce a cysteine group at its carboxyl terminus to allow for specific conjugation. The cysteine-modified NLS peptide was conjugated to the fluorescently labeled streptavidin protein using the heterobifunctional crosslinker SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (Pierce Biotechnology). The peptide concentration was 2.5-fold higher than that of the fluorescently labeled streptavidin protein (10 μM) so that on average more than one peptide is conjugated to a streptavidin molecule.
Conjugates were purified using YM-30 centrifugal filter (Millipore, MA) units to remove excess peptide. The conjugates were recovered as retentate on the filter membrane. The retentate was washed three times in 1× PBS buffer, followed by centrifugal separation at each stage to ensure removal of conjugating agents and excess peptide.
For conjugating Tat peptides to nanoparticles, we used the reaction between Tat peptide-biotin and streptavidin protein on particle surface. The amount of streptavidin molecules on bead surface was provided by the manufacturer (Molecular Probes, OR). For the 43 nm nanoparticles, there are about 5–7 streptavidin molecules per bead, and for the 90 nm nanoparticles, there are about 30 streptavidin molecules per bead (Invitrogen). For both 43 and 90 nm nanoparticles, Tat peptide was conjugated using 1:1 stoichiometric ratio of Tat-biotin and streptavidin (but not the beads), with a conjugation of ~1.31 μM. Due to the high affinity of the reaction and the limited availability of Tat-biotin molecules compared with biotin binding sites (4 biotin binding sites per streptavidin), it is expected that all the Tat peptides were conjugated to the nanoparticle surface. Therefore, on average there were at least 5 Tat peptides per bead. The 90 nm streptavidin-coated beads were first labeled with Alexa-647 (Molecular Probes) using a concentration 5 times that of the streptavidin (1.31 μM) on the surface of the beads. The excess dye was removed for the mixture using YM-100 centrifugal filters. The retentate was washed three times in 1× PBS buffer followed by centrifugal separation at each stage to remove any excess dye.
For conjugating biotin-modified Tat peptide to the streptavidin-coated QDs (each has about 4–6 streptavidin molecules according to the vendor), Tat peptide was reacted at twice the concentration of streptavidin (1 μM), resulting in approximately 8–12 Tat peptides per QD. The Tat-conjugated QDs and beads were not further purified and used directly for nuclear import assays.
Nuclear Import Assay and WGA Blockage
The nuclear import assay developed by Adam et al.1,2 was adapted in this study. Specifically, HeLa cells were permeabilized using 40 μg/mL digitonin in transport buffer for 5 min at 4 °C. The concentration of digitonin (40–80 μg/mL) and the incubation time (5–15 min) for permeabilization were chosen based on previous studies.2,10 For nuclear import in digitonin-permeabilized cells, the permeabilized cells were washed with transport buffer (formulated according to Adam et al.1,2) and then incubated with 1 μM of the Tat-conjugated, fluorescently labeled streptavidin for 30 min at 37 °C. In addition, experiments were conducted to validate the transport of NLS conjugated macromolecules in the presence of 50% rabbit reticulocyte lysate (RRL) in import buffer (Promoega Corp., WI) with 0.5 mM ATP, 0.2 mM GTP, 5 mM creatine phosphate, 1 unit creatine phosphokinase (Calbiochem, MA). The transport buffer with import factor was formulated according to the published protocol.1,2 The cells were imaged without further washing. The nuclear import assay for streptavidin-coated fluorescent beads was carried out with similar conditions except that a much lower bead concentration was used and that 1% bovine serum albumin (BSA) was added to the transport buffer to reduce non-specific interactions of the beads with either the cytoplasm or the membranes in digitonin-permeabilized cells. The 90 nm beads were sonicated before incubation with cells to eliminate aggregation. For measurement of the rate of QD import into the nuclei of digitonin-permeabilized cells, an estimated 0.5 nM QD concentration was used.
In using WGA as a blocker to inhibit the nuclear import process, permeabilized cells were first incubated in transport buffer with WGA (1 mg/mL) alone for 10 min, then with both WGA (1 mg/mL) and the Tat protein conjugate (1 μM) for 30 min. The import of Tat-conjugated, dye-labeled streptavidin was then imaged.
Imaging was carried out using a Zeiss LSM 510 confocal microscope. Confocal microscopy, with its ability to control slice thickness and intracellular focal volume, helps distinguish between cargos inside the cell nucleus and those on the surface. For the Alexa 647-labeled streptavidin protein and 90 nm beads (Polysciences Inc.), fluorescence imaging was performed with excitation at 638 nm and emission detection at 660 nm using a long pass filter. For the 43 nm fluorescent beads (Molecular Probes), excitation was at 488 nm and emission detection was performed using a band pass filter of 510–530 nm. QDs were excited at 488 nm and emission detection was at 565 nm using a long pass filter. Table 1 summarizes the excitation and emission wavelengths for all the cargos used in this study.
Determination of Nuclear Import Kinetics
To determine the kinetic rate constant of nuclear import, the fluorescence signal intensity of Tat-conjugated QDs was quantified as a function of time over a defined region of interest (ROI) at room temperature. This curve was normalized and fitted to a first-order exponential function of the form y = 1 − exp(−kt) to obtain the characteristic parameter k describing the rate of import of the QD-linked Tat peptide, where t is time in seconds, and y is the normalized average intensity of the fluorescence signal in the nuclei of digitonin-permeabilized cells.
Results and Discussion
Nuclear Import of Tat-Conjugated, Fluorescently Labeled Streptavidin
As a first step to characterize Tat-based nuclear import, we studied the Tat-based delivery of fluorescently labeled streptavidin into the nuclei of digitonin-permeabilized cells. Digitonin permeabilization has been used in nuclear import assays to characterize the delivery of various cargos into permeabilized cells. Digitonin interacts with cholesterol-rich membranes and leaves the nuclear envelope intact,7 allowing for the direct study of nuclear import using a variety of approaches. Streptavidin was chosen as a cargo of interest as it has 4 binding sites for biotin, allowing for conjugation of multiple peptides. Streptavidin also has a molecular weight greater than 50 kDa, preventing it from simply diffusing across the nuclear membrane.1 Streptavidin was labeled with Alexa-647 to minimize autofluorescence from the cells during imaging. The digitonin-permeabilized cells were incubated with the Tat-conjugated, fluorescently labeled streptavidin as described in the “Experimental procedures” section.
To test if Tat-based nuclear import uses the same mechanism as NLS, Tat-streptavidin delivery assays were carried out in a transport buffer without the addition of cytoplasmic extracts or an ATP regenerating system. It has been shown1,2 that during permeabilization of cells with digitonin, nuclear import factors and other cytosolic factors are removed from the cells. Thus to reconstitute the nuclear import system for classical NLS-based import, it is essential to add back the cytoplasmic extract along with an ATP regenerating system.1 To directly compare the import of Tat–streptavidin with that of NLS-mediated import under the same experimental conditions, a construct of the NLS peptide conjugated to fluorescently labeled streptavidin was prepared according to the conjugation protocol described in the Experimental Procedures section and incubated under the same conditions as for the Tat-streptavidin complex.
WGA Does Not Block Tat-Mediated Nuclear Import
Assays were performed to study the effect of WGA blockage on Tat-mediated nuclear import. WGA is a generic blocker of the nuclear import process in digitonin-permeabilized cells.14,22,24 It binds specifically to the glycosylated proteins of the NPC and thus competes off the nuclear import machinery or blocks its interaction with the pore complex.2,7 In our study, the WGA treatment of digitonin-permeabilized cells was carried out with a WGA concentration 20-fold higher than the concentrations reported in previous studies7 to ensure effective blockage. As shown in Fig. 2c, even with this high concentration, WGA was not able to block the Tat peptide-mediated nuclear import of dye-labeled streptavidin. This clearly indicates that, compared with NLS-mediated import, the Tat peptide uses a different mechanism in interacting with the nuclear envelope and its pore complexes. Since WGA only binds to glycosylated proteins of the nuclear pore complex, it is possible that Tat interacts with different proteins within the pore complex for nuclear import. It is also possible that the interaction between Tat peptide and the NPC or the nuclear envelope has either a higher affinity than WGA blockage or significantly different interaction than the classical NLS pathway.
Transport of Macromolecules Through Nuclear Pore Was Not Compromised
To confirm the functional integrity of nuclear membrane and its ability to import NLS conjugated cargo after digitonin permeabilization, we carried out an experiment using NLS conjugated streptavidin protein. This conjugate was introduced into digitonin-permeabilized cells in the presence of ATP and other nuclear transport factors present in rabbit reticulocyte lysate.1,2,16 As shown in Fig. 2d, NLS conjugated streptavidin molecules accumulated in cell nucleus, which clearly indicates that the transport of macromolecules to the nucleus in digitonin-permeabilized cells was not compromised. Similar results have been reported in the literature.1
Tat-Mediated Import of 43 nm Fluorescent Beads into Digitonin-Permeabilized and Live Cells
To demonstrate the import of large beads into the nucleus under normal physiological conditions and to compare the results with digitonin-permeabilized cells, delivery assays were performed in which we imaged the import of 43 nm streptavidin-coated, Tat-conjugated beads into the nuclei of live HeLa cells. For this assay, we used the same concentration of fluorescent beads as that used in the digitonin-permeabilized cells. The Tat-conjugated beads were incubated with live cells without any permeabilization. The images were collected 1.5 h after initial incubation. Due to the presence of more barriers to transport (such as the plasma membrane and cytoskeletal structures) in live cells, the live-cell images of bead delivery were taken after 1.5 h of incubation in contrast to the incubation time of 0.5 h for the studies with digitonin-permeabilized cells.
As demonstrated by Fig. 3c, accumulation of 43 nm beads is clearly visible in the nucleoplasm of live HeLa cells, in agreement with the results obtained previously12,25 with iron oxide nanoparticles in a similar size range. This confirms that large cargos (>40 nm) can be imported to the nuclei of living cells by Tat peptide. Note that the localization of the beads within the cell nucleus is different in live and permeabilized cells: in permeabilized cells, Tat-conjugated beads showed significant accumulation in the nucleoli (Figs. 3a and 3b), whereas in live cells, there was a widespread distribution in the nucleoplasm, with less accumulation in the nucleolus (Fig. 3c).
To drive this point home, in Figs. 3d and 3e, the fluorescent signal (Fig. 3d) and the white light image of cells (Fig. 3e) were shown separately, and the overlay of the two is shown in Fig. 3f. The confocal image in Fig. 3d shows clearly the nucleoli in the image plane, and the fluorescence signal shown in Fig. 3d in the same image plane indicates that there was only a small amount of 43 nm particles, if any, entered the nucleolus. Evidently, the fluorescence images show that the nanoparticles were indeed in the cell nucleus, for otherwise the images would not correlate so well with the shape of the cell nuclei and the locations of the nucleoli.
Tat-Mediated Import of 90 nm Fluorescent Beads into Digitonin-Permeabilized Cells
An attempt was made to deliver 90 nm beads into the nuclei of live HeLa cells. However, we found that in cell culture media large clumps of beads were formed on the surface of cells, resulted in a significant amount of non-specific binding of the beads on cell surface as well as non-specific uptake of beads into cells via the endocytic pathway. Consequently, it was difficult to visualize nuclear localization of 90 nm beads in living cells since the large aggregates of beads either trapped in endosomes and other intracellular compartments, or stayed on cell surface. In contrast, with digitonin-permeabilized cells, the absence of cell culture media reduced significantly the formation of large aggregate, and digitonin helped overcome the plasma membrane barrier, thus eliminating the trapping of beads into endosomes.
The observed import of 90 nm beads into the nucleus in this study raises several important questions concerning Tat-peptide-based cargo delivery and its interaction with the NPC. For example, what is the biochemical basis of the interaction between Tat-peptide conjugates and the nuclear envelope, and does the Tat peptide use an alternative pathway to import cargo from the cytoplasm to the nucleus? This observation revealed that the particle-size limit on nuclear import might have been underestimated; a detailed biochemical study of the underlying mechanism(s) of Tat-based nuclear import is left to the future work.
Rate of Import of Tat-Conjugated Cargo
Quantitative measurement was carried out to characterize the uptake kinetics of Tat conjugated macromolecules in the nuclei of digitonin-permeabilized cells and determine whether the Tat-peptide mediated import process has characteristics similar to, or different from, that of NLS-based import. Initial experiments were performed using Tat-conjugated, fluorescently labeled streptavidin proteins at a low concentration (~50 nM). This method is similar to that used in studying the import of NLS–peptide linked cargo.16 However, photobleaching of the dye had a significant effect on the fluorescence intensity in the cell nuclei and thus the overall kinetic measurement. To overcome this limitation, we used quantum dots (QDs) as a model cargo, which has high signal intensity and essentially no photobleaching.4 The use of QDs allowed us to determine the kinetic rates of import at a nanomolar concentration of the Tat-conjugated QDs (physiological levels). Further, QDs with ~20 nm diameter represent a macromolecular cargo with an intermediate size between individual streptavidin proteins and 43 nm streptavidin-coated nanoparticles. We conjugated Tat peptide to streptavidin-coated QDs (~20 nm in diameter) and delivered the resulting QD construct into digitonin-permeabilized cells to observe cargo import as a function of time. This approach has the potential to perform single molecule imaging of the import process without significantly perturbing the physiological condition of the cell.
The increase in fluorescence intensity of the cell free area (on the glass slide) was only observed right after addition of QDs; the background fluorescence reached a plateau in approximately 35 s after addition of QDs. This background signal was possibly due to non-specific interaction of Tat-conjugated QDs with the glass surface, since the Tat peptide is positively charged. In a control experiment using QDs without Tat peptide, we did not observe any increase in non-specific binding of QDs with the glass slide.
There are a few interesting features of the results shown in Fig. 6. The curve shown in Fig. 6 has the characteristics of a receptor-mediated process, although Tat peptide-based nuclear import does not require the presence of cytoplasmic components such as importin, and blockage with WGA does not inhibit nuclear import. Further, comparison of the curve in Fig. 6 for Tat-mediated import with that of NLS-mediated import16 reveals similar characteristics, although the concentration of the probe used in this study is 1000-fold lower than that used in studying the kinetics of NLS-mediated import. Our data suggest that the kinetic rate constant for Tat peptide-mediated import is much higher than that of NLS-mediated import, since we used a much lower probe concentration with comparable saturation times.
In summary, in this study we performed nuclear import assays with different cargos using the Tat peptide and compared the results with NLS-mediated cargo delivery into the cell nucleus. Specifically, the Tat-mediated import process was studied using a range of cargo molecules, including dye-labeled streptavidin protein, 43 and 90 nm beads and ~15–20 nm QDs. The results of this study revealed significant differences between Tat- and NLS-mediated nuclear import. For example, Tat peptide mediated import is not inhibited by WGA blockage and does not require supplementation with ATP, whereas WGA blockage and ATP depletion have been shown to inhibit the nuclear import of NLS-linked cargo. The results also demonstrate the ability of Tat peptide to import 43 and 90 nm beads in digitonin-permeabilized cells, suggesting the significant difference in the import process of Tat-linked cargo and its interaction with nuclear envelope compared with NLS-mediated import. To quantify the rate of nuclear import of Tat-linked cargo, we used Tat-conjugated QDs for imaging the import process as a function of time. The measured kinetic curve (amount of QDs in the nucleus as a function of time) indicates that the characteristics of Tat-mediated import can be modeled as a first-order kinetic process, which is similar to that obtained with the NLS peptide. However, our results suggest that Tat-mediated import has much faster kinetics than the NLS mediated nuclear import. Taken together, our results demonstrate significant differences between the Tat- and NLS-mediated nuclear import pathways. These results have significant implications in developing new approaches for delivery of macromolecules into the nuclei of cells.
The molecular mechanism of how Tat peptide imports cargo into the cell nucleus remains elusive. Clearly, the import process of Tat peptide conjugates is not diffusion-driven.25 It has been suggested9 that Tat peptide conjugated with beta-galactosidase is imported into the cell nucleus by a novel mechanism which does not require the presence of cytosolic factors but requires ATP. However, our experimental results indicate that Tat peptide-mediated nuclear import does not require ATP or cytosolic factors. Clearly, Tat peptide carries cargo into the cell nucleus using a mechanism different from that of NLS. Even more striking is that Tat peptide can import beads of 90 nm into the cell nucleus, challenging the current understanding of import through the NPC. Although establishing the exact mechanism for Tat peptide-mediated nuclear import is beyond the scope of this report, we believe that the discoveries made in this study will help develop a fundamental understanding of Tat peptide-based nuclear import as well as new approaches to deliver nanoparticles into the nuclei of living cells.
The results of this study have demonstrated import of Tat conjugated nanoparticles to the nuclei of digitonin permeabilized cells. Additional studies are needed to confirm that large nanoparticles can be delivered into the nuclei of living cells by Tat peptide. For example, 3D multi-color confocal imaging studies with spectrally distinct fluorophores shall be performed to individually track the fate of Tat peptide and nanoparticles in permeabilized and living cells to provide a more detailed characterization of Tat mediated nuclear import process. It is also desirable to gain a mechanistic insight into the interactions of Tat peptide with nuclear membrane by which import of nanoparticles across nuclear membrane can occur. This may be achieved by combining fluorescence imaging with electron microscopy to characterize the nuclear import of Tat conjugated nanoparticles with higher spatial resolution.
This work was supported by NIH Roadmap Initiative in Nanomedicine through a Nanomedicine Development Center award, 1PN2EY018244 (GB), and by the Office of Science, Department of Energy grant DE-FG02-04ER63785 (GB).
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