Mechanism of microtubule-facilitated "fast track" nuclear import

Abstract

Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold. Moreover, MTAS functions depend on mutual exclusivity of binding of PTHrP to MTs and IMPβ1 such that, following MT-dependent trafficking toward the nucleus, perinuclear PTHrP can be displaced from MTs by IMPβ1 prior to import into the nucleus. This is the first molecular definition of an MTAS that facilitates protein nuclear import as well as the first delineation of the mechanism whereby cargo is transferred directly from the cytoskeleton to the cellular nuclear import apparatus. The results have broad significance with respect to fundamental processes regulating cell physiology/transformation.

FIGURE 1.

Efficient nuclear accumulation of PTHrP-(82–108) is dependent on intact MTs. A, schematic representation of the various GFP-PTHrP truncated derivatives used in this study with parathyroid hormone (PTH)-like, NLS, and nuclear export sequence (NES) domains indicated. B, CLSM images of live COS-7 cells transfected to express the indicated GFP fusion proteins and treated without or with NCZ as indicated. Left lower panels show fixed cells treated without or with NCZ and immunostained for tubulin. For GFP-PTHrP derivatives 1–141, 38–141, 1–108, 38–108, 66–108, and 82–108 that are predominantly nuclear/nucleolar in the absence of NCZ treatment, the cell cytoplasm is outlined with yellow dotted lines. Scale bars represent 20 μm. C, results for quantitative analysis of digitized images, such as those in B, to determine the Fnu, Fn, or Fc corrected for background or the extent of nucleolar and nuclear accumulation expressed as Fnu/c or Fn/c, respectively. Results represent the mean (n > 40), and error bars represent the standard error of the mean. Significant differences are denoted by the p values. †, unpaired t test; *, Mann-Whitney test.

FIGURE 1.

Efficient nuclear accumulation of PTHrP-(82–108) is dependent on intact MTs. A, schematic representation of the various GFP-PTHrP truncated derivatives used in this study with parathyroid hormone (PTH)-like, NLS, and nuclear export sequence (NES) domains indicated. B, CLSM images of live COS-7 cells transfected to express the indicated GFP fusion proteins and treated without or with NCZ as indicated. Left lower panels show fixed cells treated without or with NCZ and immunostained for tubulin. For GFP-PTHrP derivatives 1–141, 38–141, 1–108, 38–108, 66–108, and 82–108 that are predominantly nuclear/nucleolar in the absence of NCZ treatment, the cell cytoplasm is outlined with yellow dotted lines. Scale bars represent 20 μm. C, results for quantitative analysis of digitized images, such as those in B, to determine the Fnu, Fn, or Fc corrected for background or the extent of nucleolar and nuclear accumulation expressed as Fnu/c or Fn/c, respectively. Results represent the mean (n > 40), and error bars represent the standard error of the mean. Significant differences are denoted by the p values. †, unpaired t test; *, Mann-Whitney test.

FIGURE 2.

Efficient nuclear accumulation of PTHrP-(82–108) is dependent on MT dynamics but not on actin filaments. A, CLSM images of live COS-7 cells transfected to express the indicated GFP fusion proteins and treated without (No add.) or with Taxol or CD as indicated. Lower panels show fixed cells treated without or with Taxol or CD and immunostained for tubulin (left) or actin filaments (right). Scale bars represent 20 μm. B, results for quantitative analysis of the levels of nucleolar or nuclear accumulation (see legend to Fig. 1) in COS-7 cells expressing the indicated GFP fusion proteins without or with Taxol or CD pretreatment. Results represent the mean (n > 40), and error bars represent the standard error of the mean. Significant differences are denoted by the p values. *, Mann-Whitney test.

FIGURE 3.

Efficient MT-dependent nuclear accumulation of PTHrP requires KKKK⁹¹ and KKRR¹⁰⁶. A, schematic representation of the GFP-PTHrP-(66–108) region showing the previously identified NLS (amino acids 66–94). The two basic regions and the linker regions that were mutated in this study are also indicated with residues selected for mutation in bold. B, schematic representation of PTHrP showing the modular NLS (residues 66–108) and MTAS (82–108) sequences identified in this study. C, CLSM images of live COS-7 cells transfected to express the indicated GFP-PTHrP-(66–108) derivatives and treated without or with NCZ as indicated. Scale bar represent 20 μm. D, results for quantitative analysis for the levels of nucleolar or nuclear accumulation (according to Fig. 2) in COS-7 cells expressing the indicated GFP-PTHrP-(66–108) derivatives treated without or with NCZ. Results represent the mean (n > 60), and error bars represent the standard error of the mean. Significant differences are denoted by the p values. *, Mann-Whitney test.

FIGURE 4.

Dependent on KKKK⁹¹ and KKRR¹⁰⁶, PTHrP-(66–108) can direct efficient MT-dependent nuclear import of large cargo protein. A, CLSM images of live COS-7 cells transfected to express the indicated GFP-PTHrP-β-gal fusion proteins and treated without (No add.) or with NCZ or Taxol as indicated. The scale bar represents 20 μm. B, results for quantitative analysis for the levels of nucleolar or nuclear accumulation (according to Fig. 2) in COS-7 cells expressing the indicated GFP-PTHrP-β-gal fusion proteins without or with NCZ or Taxol pretreatment. Results represent the mean (n > 60), and error bars represent the standard error of the mean. Significant differences are denoted by the p values. *, Mann-Whitney test.

FIGURE 5.

MT-facilitated nuclear import of PTHrP in vivo is dependent on residues KKKK⁹¹. CLSM visualization of the return of nuclear fluorescence after photobleaching (see “Experimental Procedures”) in COS-7 cells expressing GFP-PTHrP-(66–108)-β-gal-WT (A) and GFP-PTHrP-(66–108)-β-gal-BASIC-1mut (B) treated without (No add.) or with NCZ (+NCZ). Scale bars represent 20 μm. C, changes in Fn and Fc after subtraction of background fluorescence plotted over time postnuclear bleach. D, quantification of the recovery over time of nuclear fluorescence after photobleaching expressed in terms of fractional recovery of Fn/c (Fn/c of respective time points divided by the prebleach value). The regression values for the exponential fits were 0.83 and 0.88 for WT with no addition and +NCZ, respectively, and 0.86 and 0.92 for BASIC-1mut with no addition and +NCZ, respectively. Pooled data represents mean, n ≥ 10, and error bars represent standard error of the mean for the initial rate of cytoplasmic decay (Fc s⁻¹) (E) or the half-time of return of fluorescence (t½) (F) for GFP-PTHrP-(66–108)-β-gal-WT and GFP-PTHrP-(66–108)-β-gal-BASIC-1mut for non-treated and NCZ-treated cells are shown. Significant differences between cells treated without and with NCZ are denoted by the p values. †, unpaired t test; *, Mann-Whitney test. NS, not significant.

FIGURE 5.

MT-facilitated nuclear import of PTHrP in vivo is dependent on residues KKKK⁹¹. CLSM visualization of the return of nuclear fluorescence after photobleaching (see “Experimental Procedures”) in COS-7 cells expressing GFP-PTHrP-(66–108)-β-gal-WT (A) and GFP-PTHrP-(66–108)-β-gal-BASIC-1mut (B) treated without (No add.) or with NCZ (+NCZ). Scale bars represent 20 μm. C, changes in Fn and Fc after subtraction of background fluorescence plotted over time postnuclear bleach. D, quantification of the recovery over time of nuclear fluorescence after photobleaching expressed in terms of fractional recovery of Fn/c (Fn/c of respective time points divided by the prebleach value). The regression values for the exponential fits were 0.83 and 0.88 for WT with no addition and +NCZ, respectively, and 0.86 and 0.92 for BASIC-1mut with no addition and +NCZ, respectively. Pooled data represents mean, n ≥ 10, and error bars represent standard error of the mean for the initial rate of cytoplasmic decay (Fc s⁻¹) (E) or the half-time of return of fluorescence (t½) (F) for GFP-PTHrP-(66–108)-β-gal-WT and GFP-PTHrP-(66–108)-β-gal-BASIC-1mut for non-treated and NCZ-treated cells are shown. Significant differences between cells treated without and with NCZ are denoted by the p values. †, unpaired t test; *, Mann-Whitney test. NS, not significant.

FIGURE 6.

IMP-β1 binding to PTHrP requires both KKKK⁹¹ and KKRR¹⁰⁶. GFP-PTHrP truncated proteins or GFP alone as indicated were incubated with increasing concentrations of biotinylated GST-human IMPβ1, and an ALPHAScreen assay was performed (see “Experimental Procedures”). Each data point represents the average of triplicate results of a single typical experiment. Sigmoidal curves were fitted using SigmaPlot software to determine the B_max and K_d values; the regression coefficients for the curve fits in all cases were >0.94 with the sole exception of the fits where low binding is in evidence (regression coefficients of 0.86 and 0.82 for GFP-PTHrP-(66–94) and GFP, respectively). ND, not able to be determined.

FIGURE 7.

PTHrP-(82–108) confers binding to intact MTs in vitro and in vivo dependent on KKKK⁹¹ and KKRR¹⁰⁶. A and B, visualization of the indicated GFP-fused proteins incubated with in vitro intact assembled MTs as indicated (see “Experimental Procedures”); corresponding transmission images of the MTs are shown below with the exception of the right panels in A where the transmission images are on the right. Where indicated, GST-IMPβ1 (+IMPβ) or GST-IMPα (+IMPα) were preincubated (30 min) with GFP-PTHrP fusion proteins prior to MT binding for 1 h followed by CLSM imaging. Where indicated in B, GST-IMPβ1 was added 10 min prior to CLSM imaging after (“post”) 1-h preincubation of GFP-PTHrP with MTs. C, results for quantitative analysis for the levels of MT fluorescence (MT-associated fluorescence (Fmt) after the subtraction of background fluorescence) due to binding of fluorescent proteins. Results represent the mean (n > 100), and error bars represent standard error of the mean from two separate experiments where significant differences between the GFP-PTHrP proteins are denoted by the p values. D–G, COS-7 cells expressing the indicated proteins were lysed in MT stabilizing buffer and subjected to ultracentrifugation. The MT pellet/associated proteins (P) were separated from supernatant (S) containing soluble proteins and tubulin as described under “Experimental Procedures” and washed (W) three times prior to resuspension. Fractions were then subjected to Western analysis using the indicated antibodies. Densitometric analysis for the protein levels in D and F is presented in E and G where the signal intensity above background for the GFP fusion proteins in S and P are expressed relative to the corresponding values for tubulin, and the values are then expressed as a percentage relative to that derived for the supernatant (100%) (in E) or the ratio of pellet/supernatant relative to the maximum value (for wild type, single expression) (in G). No add., no addition.

FIGURE 7.

PTHrP-(82–108) confers binding to intact MTs in vitro and in vivo dependent on KKKK⁹¹ and KKRR¹⁰⁶. A and B, visualization of the indicated GFP-fused proteins incubated with in vitro intact assembled MTs as indicated (see “Experimental Procedures”); corresponding transmission images of the MTs are shown below with the exception of the right panels in A where the transmission images are on the right. Where indicated, GST-IMPβ1 (+IMPβ) or GST-IMPα (+IMPα) were preincubated (30 min) with GFP-PTHrP fusion proteins prior to MT binding for 1 h followed by CLSM imaging. Where indicated in B, GST-IMPβ1 was added 10 min prior to CLSM imaging after (“post”) 1-h preincubation of GFP-PTHrP with MTs. C, results for quantitative analysis for the levels of MT fluorescence (MT-associated fluorescence (Fmt) after the subtraction of background fluorescence) due to binding of fluorescent proteins. Results represent the mean (n > 100), and error bars represent standard error of the mean from two separate experiments where significant differences between the GFP-PTHrP proteins are denoted by the p values. D–G, COS-7 cells expressing the indicated proteins were lysed in MT stabilizing buffer and subjected to ultracentrifugation. The MT pellet/associated proteins (P) were separated from supernatant (S) containing soluble proteins and tubulin as described under “Experimental Procedures” and washed (W) three times prior to resuspension. Fractions were then subjected to Western analysis using the indicated antibodies. Densitometric analysis for the protein levels in D and F is presented in E and G where the signal intensity above background for the GFP fusion proteins in S and P are expressed relative to the corresponding values for tubulin, and the values are then expressed as a percentage relative to that derived for the supernatant (100%) (in E) or the ratio of pellet/supernatant relative to the maximum value (for wild type, single expression) (in G). No add., no addition.

FIGURE 8.

Optimal nuclear accumulation of endogenous cellular PTHrP is dependent on functional MTs. A, CLSM images of COS-7 and U2 OS osteosarcoma cells treated without (No add.) or with NCZ or Taxol prior to fixation and immunostaining for PTHrP or T-ag as indicated. Scale bars represent 20 μm. B and C, results for quantitative analysis of images, such as those in A, for the levels of Fn and Fc (expressed relative to non-treated cells) (B) or the extent of nuclear accumulation of PTHrP or endogenous T-ag (Fn/c) (according to Fig. 1) (C). Results represent the mean (n ≥ 110), and error bars represent standard error of the mean, where significant differences are denoted by p values. *, Mann-Whitney test.