Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase

Abstract

In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107-160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms.

Figure 1.

NPC density and nuclear growth during interphase. (A) An NRK cell stably expressing Pom121-3EGFP was tracked throughout the entire cell cycle. The signal from NPCs on the bottom surface of the nucleus is shown in inverted grayscale. Times relative to the first observed mitosis are indicated. (B) Individual landmark NPCs were tracked, and the area between them was measured as indicated. Overlay of the measured areas (right) illustrates the overall increase in area during the observation time. (C) NPC density through the cell cycle of individual cells (black). Each curve was fitted with a linear function using the least squares method, and the mean fit is shown in red (gray shading, SD of fit parameters). (D) Growth of nuclear surface area between tracked NPCs (n = 8). The area was normalized to the first time point, and each curve was fitted with a single exponential to derive the mean growth (red; gray shading, 95% confidence interval).

Figure 2.

Assembly of individual new NPCs in interphase. (A–C) Time series showing the assembly of individual NPCs labeled by members of different NPC subcomplexes: (A) Nup107–160 complex labeled by 3EGFP-Nup107 and 3EGFP-Nup133; (B) Nup93 complex labeled by Nup93-3EGFP; and (C) Pom121-3EGFP. (D) Time series showing the disappearance of an NPC labeled with Pom121-3EGFP. Red boxes on overview image indicate region of magnification. Circles highlight measured NPCs (red, new/disappearing NPC; black, old/reference NPC). Plots show background-subtracted and bleaching-corrected fluorescence intensities. For presentation purposes, images were smoothed with a Gaussian blur filter, kernel 1.5.

Figure 3.

Kinetics of NPC assembly in interphase. (A) Plots of NPC assembly kinetics. Curves were aligned after fitting with the sigmoid function along the half-maximal intensity. All measured data points are displayed in gray. Colored lines show a sigmoid curve with the mean characteristic time. Inset boxes represent individual fits for the examples shown in Fig. 2. (B) Boxplot of characteristic times for sigmoid fit. Boxes indicate 25% and 75% quartiles, red lines show median, and outliers are indicated by crosses. Blue lines show the median of corresponding mitotic fits (compare with C). The data in A and B were derived from seven, four, and seven cells for Pom121, Nup107, and Nup93, respectively. (C) Assembly curves for postmitotic NPC assembly (Dultz et al., 2008) were fitted with sigmoid curves for comparison (Fig. S2). P-values are for unpaired t tests.

Figure 4.

Order of NPC assembly. (A) A cell expressing Pom121-3EGFP and 3mCherry-Nup107 was imaged at a time resolution of 15 min. A newly appearing NPC is circled. Black data points represent the mean of 10 preexisting pores. Red boxes on overview image indicate region of magnification. (B) Kinetic traces of individual newly forming NPCs were fitted with sigmoid functions and aligned along the half-maximal intensity of Nup107. Data points for all series are shown as diamonds, and solid lines indicate the mean fit for each nucleoporin. (C) Order of appearance of the signals for Pom121 and Nup107 according to quantitative and unbiased criteria (compare with Materials and methods section Measurements of pore formation kinetics). Data are pooled from experiments with swapped fluorescent protein tags.