FCS analysis of nuclear transport proteins in vitro
In order to understand how intracellular environment affects protein mobility, it was first necessary to analyze dynamics of fluorescently labeled nuclear transport proteins (importin β, importin α, Ran and NTF2) in vitro
for comparison (). In each case autocorrelation data could be fitted with a single autocorrelation decay time (τD
) corresponding to the diffusion coefficient (D
) expected from the size of the monomeric protein, indicating that fluorescently labeled recombinant nuclear transport proteins behave as single molecules and do not form large aggregates in vitro
. NTF2 is known to dimerize in vitro
~ 1 μM) 52
. However, since FCS measurements were performed at nanomolar concentrations, the proportion of NTF2 dimer at these concentrations is too small to be detected by FCS. Furthermore, a two-fold difference in molecular mass between monomeric and dimeric NTF2 would result in ~ 1.25 fold difference in diffusion coefficient, which is difficult to resolve by FCS.
FCS analysis of nuclear transport proteins in vitro
Since FCS analyzes dynamic properties of fluorescently labeled proteins it was important to compare the properties of labeled and unlabeled proteins to determine if fluorescent labeling affects protein function. To analyze effects of fluorescent labeling on binding specificity importin β, importin α, Ran and NTF2 were differentially labeled with either Alexa Fluor 488 or Alexa Fluor 647 and tested for binding interactions in pairwise combinations, using dual channel cross correlation FCS in vitro 53
. The following specific interactions were demonstrated: importin β::importin α, importin β::RanQ69L, NTF2::RanE70A, RanQ69L::RCC1, RanE70A::RCC1, and RanT24N::RCC1 (data not shown). These results indicate that binding specificities of fluorescently tagged proteins are comparable to unlabeled proteins. To determine the effect of fluorescent labeling on nuclear transport function, fluorescent and non-fluorescent importin β, importin α, Ran and NTF2 were tested for their ability to mediate nuclear uptake of fluorescent cargo in a permeabilized cell assay (Figure S1
). In the absence of added protein cargo was not taken up into the nucleus. In the presence of either fluorescent or non-fluorescent importin β, importin α, Ran and NTF2, cargo was taken up into the nucleus. This indicates that fluorescently labeling does not interfere with the functions of importin β, importin α, Ran and NTF2 in nuclear transport.
Although fluorescent labeling does not completely inactivate importin β, importin α, Ran or NTF2 it is possible that a fraction of the protein(s) is inactivated. Fluorescent labeling was performed chemically, which means that fluorophores may be conjugated to different residues in different molecules. If fluorophore conjugation to some residues does not affect protein function while conjugation to other residues interferes with protein function a fraction of the labeled protein may be inactive. To test this possibility binding properties of labeled and unlabeled proteins were compared quantitatively using surface plasmon resonance analysis. In this technique one binding partner (ligand) is immobilized on a gold chip and the other partner (analyte) in solution is flowed across the chip in a microfluidic flow cell. If analyte binds to ligand the mass on the chip increases which is detected as a change in refractive index. Association of analyte with ligand over time and dissociation of analyte from ligand when buffer is passed through the flow cell are recorded as a sensorgram, which provides a quantitative measure of on rates and off rates. Comparing sensorgrams for labeled and unlabeled proteins provides a sensitive and quantitative way to determine if a fraction of the protein is inactivated by labeling.
Sensorgrams for different combinations of labeled and unlabeled proteins are shown in
. Panel A shows sensorgrams for unlabeled (blue) and labeled (red) importin β (analyte) binding to unlabeled importin α (ligand). The amplitudes of the sensorgrams are almost identical in both association and dissociation phases indicating that fluorescent labeling does not inactivate a significant fraction of importin β. The reciprocal experiment (with importin β as ligand) could not be performed because importin β is inactivated when immobilized on the chip. However we were able to repeat the experiment with immobilized labeled importin α as ligand (panel B). The amplitudes of the sensorgrams in panels A (unlabeled importin α as ligand) and B (labeled importin α as ligand) are comparable indicating that fluorescent labeling does not inactivate a significant faction of importin α. Panel C shows sensorgrams for unlabeled (blue) and labeled (red) Ran (analyte) binding to unlabeled NTF2 (ligand). The amplitude of the sensorgram for labeled Ran is slightly reduced compared to unlabeled Ran indicating that a small fraction (<25%) of Ran protein is inactivated by fluorescent labeling. As before, the reciprocal experiment (with Ran as ligand) could not be performed because Ran is inactivated when immobilized on the chip. However, the experiment was repeated with labeled NTF2 as ligand (PanelD). The amplitudes of the sensorgrams in panels C (unlabeled NTF2 as ligand) and D (labeled NTF2 as ligand) are comparable indicating that fluorescent labeling does not incativate a significant fraction of NTF2. These results indicate that conjugation with fluorophore does not significantly affect binding properties of nuclear transport proteins except for Ran, where conjugation with fluorophore interferes with binding to NTF2 in < 25% of labeled molecules. This means that fluorescent importin β, importin α, NTF2 and Ran should exhibit the same dynamic properties in live cells as their unlabeled counterparts.
FCS analysis of nuclear transport proteins in vivo
To measure protein dynamics in vivo, fluorescently tagged nuclear transport proteins were microinjected into the cytoplasm of B104 cells. After injected proteins reached steady state distribution within the cell, FCS measurements were performed with the observation volume positioned in nucleus, cytoplasm, or on the nuclear envelope (). Concentrations and autocorrelation decay times were determined by global fitting of FCS data from many injected cells. Immobile fractions were determined from photobleaching kinetics.
To distinguish effects of specific and nonspecific interactions on protein mobility, importin β, a protein with multiple specific interactions was compared with allophycocyanin (APC), a protein of similar molecular weight (100 kDa), but with no known specific interactions in mammalian cells. Both proteins exhibited normal single component diffusion in vitro
with diffusion coefficients of 67 for APC and 70 μm2
/s for importin b (). To study their dynamics in vivo
, the proteins were microinjected into either cell cytoplasm or the nucleus. APC remained in the injection compartment because the protein lacks nuclear localization signals and is too large to pass through the NPC by passive diffusion, whereas importin β equilibrates between nucleus and cytoplasm. FCS autocorrelation data for APC in vivo
, in either nucleus or cytoplasm, could be fitted with a single autocorrelation decay time (τD
= 1000 μs) corresponding to a slower diffusion coefficient (D
= 24 μm2
/s) than in vitro
. This is consistent with previous observations that diffusion of macromolecules in vivo
is non-specifically slowed by viscosity of cytoplasm relative to diffusion in vitro 46,54
. We conclude that non-specific factors (viscosity, macromolecular crowding, sieving effects of cytoskeleton, etc
.) reduce the diffusion coefficient in vivo
but do not necessarily produce apparent multicomponent or anomalous diffusion. The autocorrelation function for importin β had a more complex shape and could be fit with either a multicomponent diffusion model or an anomalous diffusion model. We attribute the complex shape of the measured autocorrelations to specific interactions of nuclear transport proteins with cellular component of different mobilities.
FCS analysis of allophycocyanin and importin β in vitro and in vivo
The in vivo autocorrelation data for nuclear transport proteins carries information about specific interactions of these proteins. Unfortunately, no method for rigorous analysis of such data is presently available. Nuclear transport proteins are known to form complexes that are immobile, have reduced mobilities, or are actively transported. Furthermore, the frequencies of binding/unbinding events, depending on the kinetic rates and concentrations, are broadly distributed. Despite this complexity, it is still possible to extract useful information from the data.
Complex autocorrelation functions can be analyzed by curve fitting with either anomalous or multicomponent diffusion models. One criticism of the latter is that many particles undergo binding/release reactions while passing through the FCS observation volume. Therefore apparent diffusion times cannot be interpreted as diffusion coefficients. On the other hand, multicomponent diffusion models can estimate the percentage of freely diffusing molecules while giving overall information about the dynamic properties of the remaining species.
Compared to the multi-component diffusion model, analysis with anomalous diffusion model is somewhat less informative. Since anomalous diffusion is an apparent phenomenon, a result of heterogeneous interactions of the protein of interest with the intracellular environment, the diffusion coefficient and the anomalous exponent produced by the analysis are only useful as overall descriptors of protein mobility and do not provide insight into specific interactions. Therefore FCS data was analyzed using the multicomponent normal diffusion model with up to three components.
Interpretation of in vivo FCS data is based on the assumption that dynamic properties of injected fluorescent proteins reflect the behavior of corresponding endogenous proteins. Concentrations of endogenous nuclear transport proteins in B104 cells measured by quantitative western blotting were: [importin β] = 9.7, [importin α] = 4.4, [Ran] = 8.2 and [NTF2] = 1.1 μM. By comparison, concentrations of exogenous fluorescent proteins in injected cells, determined from the amplitudes of the FCS autocorrelation functions, varied from cell to cell but were always in the low nanomolar range. Since concentrations of exogenous proteins are much lower than concentrations of corresponding endogenous proteins, exogenous protein is unlikely to affect the overall distribution of the corresponding endogenous protein.
Subcellular distributions of fluorescent proteins microinjected into B104 cells, determined by confocal microscopy (), were comparable to distributions of corresponding endogenous proteins, determined by immunofluorescence (data not shown, and reported previously in other cell types 16,30,42,55,56
), indicating that exogenous fluorescent proteins and corresponding endogenous proteins have comparable steady-state subcellular distributions. In addition, time-lapse analysis of uptake kinetics in injected cells (data not shown) revealed rapid nuclear uptake for each of the microinjected proteins, indicating that the labeled proteins are recognized by the nuclear transport system. Fuorescent importin β, NTF2, Ran, and Ran Q69L were concentrated in the vicinity of the nuclear envelope, as were the corresponding endogenous proteins, indicating that these proteins have affinity for nucleoporins or other components of the nuclear envelope. Fluorescent importin α, Ran E70A, and Ran T24N were not concentrated in the vicinity of the nuclear envelope indicating that this is not a general phenomenon due to fluorophore conjugation. Ran E70A accumulates in the nucleus because it can bind NTF2 for nuclear import but cannot bind karyopherins for nuclear export. RanQ69L does not accumulate in the nucleus because it cannot bind NTF2 for nuclear import but can bind karyopherins for nuclear export. The reason for nuclear accumulation of RanT24N is not clear. The protein is small enough to equilibrate between nucleus and cytoplasm by passive diffusion through the NPC but since it does not bind to either karyopherins or NTF2 it must bind to some other binding partner(s) concentrated in the nucleus. We conclude that subcellular distributions of microinjected fluorescent proteins and corresponding endogenous proteins are comparable, and that FCS measurements of microinjected fluorescent proteins accurately reflect the dynamic behavior of the corresponding endogenous proteins. Accordingly, relative proportions of components with different autocorrelation decay times (determined for fluorescent proteins by FCS) were used to calculate absolute concentrations of the endogenous counterparts, based on the total concentrations of each protein in the cell (determined by quantitative western blotting). In the case of mutant Ran proteins, relative proportions of different components are expressed as fractions since mutant proteins do not have endogenous counterparts.
Confocal imaging of cells injected with fluorescently labeled nuclear transport proteins
Dynamics of nuclear transport proteins in nucleus and cytoplasm
FCS analysis of nuclear transport proteins in the nucleus and cytoplasm is summarized in and , respectively. Autocorrelation data for each of the proteins were globally fitted with three autocorrelation decay times: fast (τD ~ hundreds of microseconds), medium (τD ~ milliseconds) and slow (τD ~ tens of milliseconds). Photobleaching data were fitted to single exponential kinetics to obtain the immobile fraction for each protein. The fact that FCS autocorrelation data for a particular protein can be fitted with discrete τD values does not imply that every molecule in the cell has dynamic properties characterized by one of the specific τD values. Each τD value should be considered to represent a range of dynamic behaviors.
FCS analysis of nuclear transport proteins in nucleus
FCS analysis of nuclear transport proteins in cytoplasm
Fast autocorrelation decay times (τD ~ hundreds of microseconds) generally represent freely diffusing protein, including various nuclear transport intermediates. Accordingly, fast component for importin β likely represents monomeric importin β as well as importin β::importin α::cargo and importin β::RanGTP. Fast component for importin α represents monomeric importin α as well as importin β::importin α::cargo and importin α::CAS::RanGTP. Fast component for NTF2 represents monomeric and dimeric NTF2 as well as NTF2::RanGDP. Fast component for Ran represents monomeric Ran as well as RanGDP::NTF2 and RanGTP::karyopherins. Mutant forms of Ran (E70A, T24N and Q69L) exhibited increased proportions of fast component compared to wild type Ran, which is consistent with identification of fast component as freely diffusing protein since mutant Ran proteins have reduced potential for specific interactions with other proteins. These results indicate that effective concentrations of freely diffusing nuclear transport proteins and nuclear transport intermediates represent a relatively small proportion of total concentrations in nucleus and cytoplasm, which implies that the majority of each protein is involved in non-nuclear transport molecular interactions.
Medium and slow components represent non-freely diffusing nuclear transport proteins in nucleus and cytoplasm, which can either correspond to medium and slow rates of diffusion of large complexes in cytoplasm or binding of fluorescent molecules to immobile binding partners in nucleus or cytoplasm, followed by dissociation with medium and slow off rates. Medium and slow autocorrelation decay times (1–30 milliseconds) would correspond to diffusion of relatively large objects (10–100 nm) in diameter) in cytoplasm. If one assumes that each fluorescent molecule binds only once to an immobile partner as it traverses the FCS observation volume, apparent off rates can be calculated from autocorrelation decay times (as shown in and ).
In both nucleus and cytoplasm, each nuclear transport protein exhibited significant photobleaching during FCS measurement, indicating that these proteins are bound to immobile (koff < 0.7 sec−1) structures. By this criteria, more than 60% of importin β (19.5 μM) in the nucleus is immobile. Confocal imaging of cells after FCS measurements were taken, revealed a photobleached area corresponding to the FCS observation volume that did not recover up to 45 minutes post-bleach. Furthermore, immobile importin β did not appear until at least 30 minutes after injection (data not shown). These results indicate that the pool of immobile importin β in nucleus equilibrates relatively slowly with the pool of freely diffusing importin β. More than 40% of importin α (5.3 μM) in nucleus is also immobile. In this case the photobleached area in importin α injected cells recovered rapidly and immobile importin α appeared rapidly after microinjection, indicating that immobile importin α in nucleus and cytoplasm equilibrates relatively rapidly with mobile importin α. Immobile fractions of importins α and β in nucleus may reflect binding to immobile nuclear structures. Immobile importins α and β in cytoplasm may reflect binding to immobile cytoskeletal elements such as microtubules (see below). Significant proportions of NTF2 (0.90 μM), and Ran (4.0 μM) were also immobile in the nucleus. In both cases the photobleached area recovered rapidly and the immobile component appeared rapidly after microinjection, indicating that immobile Ran and NTF2 equilibrate relatively rapidly with mobile protein in the nucleus and cytoplasm. Since RanQ69L in nucleus has a higher proportion of immobile component than the other two mutants, it is likely that the immobile fraction of Ran in the nucleus is predominantly Ran GTP.
To test the possibility that medium, slow and immobile components detected by FCS reflect binding of nuclear transport proteins to cytoskeletal elements, cells were treated with either nocodazole (to disrupt microtubules) or latrunculin (to disrupt microfilaments) before injecting fluorescent protein and performing FCS. Disruption of microtubules or microfilaments was confirmed by immunofluorescence with antibody β tubulin or phalloidin, respectively (data not shown). For proteins associated with cytoskeletal elements autocorrelation decay times could represent either rates of motor-driven transport on microtubules or microfilaments or rates of dissociation from the cytoskeleton. Motor driven transport rates would result in very slow autocorrelation times, which cannot be accurately measured because they are obscured by photobleaching effects. Accordingly, autocorrelation decay times affected by nocodazole (or latrunculin) are interpreted as off rates for dissociation from immobile cytoskeletal elements, with the understanding that rates of motor-driven transport may also contribute to slow autocorrelation decay times.
In the nucleus autocorrelation decay times and photobleaching kinetics were not affected by either nocodazole or latrunculin (data not shown) indicating that proteins in the nucleus are not associated with cytoskeletal elements. In cytoplasm, latrunculin did not affect autocorrelation decay times or photobleaching kinetics for importin α, NTF2 or Ran (data not shown), indicating that mobility of these proteins is not affected by microfilaments. Latrunculin did cause a slight increase in the proportion of importin β slow component at expense of medium component (data not shown). Since disruption of microfilaments by latrunculin is expected to increase rather than decrease mobility, this may be an indirect effect of latrunculin. In cytoplasm of nocodazole-treated cells concentrations of slow and immobile components for importin α and NTF2 were reduced relative to untreated cells with concomitant increases in medium components (). This indicates that in untreated cells importin α and NTF2 are associated with microtubules in the cytoplasm with slow off rates, and in the absence of microtubules associate with alternative immobile partners with medium off rates. In the case of importin β, nocodazole decreased medium and immobile components with concomitant increase in fast component, indicating that in untreated cells importin β associates with microtubules with medium off rate, and in the absence of microtubules is freely diffusing. In the case of Ran protein, nocodazole decreased fast and medium components with concomitant increase in slow and immobile components. Since nocodazole treatment is expected to increase mobility (as observed for importin α, importin β and NTF2), the effect on Ran protein probably does not indicate direct association with microtubules. Instead it suggests that Ran binds to slow or immobile binding partners that accumulate in nocodazole-treated cells.
FCS analysis of nuclear transport proteins in nocodazole treated cells
Dynamics of nuclear transport proteins at the nuclear envelope
When the FCS observation volume was positioned over the nuclear envelope significant numbers of immobile molecules were detected for each nuclear transport protein (~104 importin β molecules/pore, ~48 importin α molecules/pore, ~43 Ran molecules/pore and ~6 NTF2 molecules/pore) (). Immobile molecules could be associated with the inside of the pore, outside of the pore, nuclear envelope adjacent to the pore, or structures near the envelope. Nuclear uptake experiments (not shown) indicate that importin β gradually becomes concentrated in the vicinity of the nuclear envelope over a period of 30–45 minutes after injection, which is consistent with a pool of immobile molecules in the vicinity of the nuclear envelope, in slow equilibrium with nuclear and/or cytoplasmic pools. Other nuclear transport proteins (importin α, NTF2 and Ran) accumulate rapidly around the nuclear envelope, indicating more rapid equilibration with nuclear and/or cytoplasmic pools. Of the different Ran mutants, only Ran Q69L had a significant immobile fraction at the nuclear pore, suggesting that immobile Ran at the NPC is predominantly RanGTP. Immobile molecules of Ran E70A or Ran T24N mutant proteins were not detected at the NPC, consistent with their inability to be converted to RanGTP for export from the nucleus in association with importins or exportins.
FCS analysis of nuclear transport proteins in NPC
In the case of importin α, a significant number of NPC-associated molecules had fast (~30 molecules/pore; τD = 138 μs) and medium (~34 molecules/pore; τD = 1.7 ms) autocorrelation decay times. These probably do not represent molecules in transit through the pore because importin β, Ran and NTF2, which also traverse the pore, did not exhibit fast and medium components at the nuclear envelope. Fast and medium components of importin α in the vicinity of the nuclear envelope may reflect molecules that dissociate from cargo and/or CAS protein at the nuclear and cytoplasmic faces of the NPC, respectively, and diffuse freely or bind to immobile partners with medium off rates. Each of the mutant forms of Ran shows accumulation of fast component at the nuclear envelope, which may reflect the inability of these proteins to interact with immobile partners in the vicinity of the NPC. RanQ69L also exhibits a small amount of slow component at the nuclear envelope, which may reflect binding of RanGTP to immobile partners with a slow off rate.