Mäntylä E , Chacko JV , Aho V , Parrish CR , Shahin V , Kann M , Digman MA , Gratton E , Vihinen-Ranta M .

P41 GM103540 NIGMS NIH HHS , P50 GM076516 NIGMS NIH HHS

	Figure 1. Overview of image acquisition and analyses workflow. The laser scanning confocal microscopy (LSCM) line scans of viral capsids were studied by 3D particle segmentation analysis, and fluorescence correlation spectroscopy -based methods such as auto- and pair correlation (pCF) function, and number and brightness (N&B) analyses. The motion and interaction of capsids were investigated by two-color pCF overlay and pCF cross-correlation analyses.
	Figure 2. Intracellular capsid distribution. (A) 3D reconstruction of a confocal image showing distribution of A594 labeled capsids (red) and lamin C-EGFP (green) at ~1 h p.i. (B) 3D segmentation particle analysis of number of virus-containing cytoplasmic particles as a function of their volume, (C) an average volume and (D) total intensity as a function of the distance from the NE. The black dots represent the means and the error bars represent the ± standard error of mean (±SEM).
	Figure 3. Image correlation analysis of capsid movement. (A) Average fluorescence intensity image showing the position and fluctuations of lamin C-EGFP. (B) Cytoplasmic capsid intensity trajectories along the scanned line in the cytoplasm, at the nuclear envelope (NE) and in the nucleoplasm as a function of time and distance are shown. (C) Autocorrelation function (ACF) analysis of intracellular viral movement. (D) Pair correlation function (pCF) analysis showing a positive correlation across the lamin C-EGFP and viral capsid transit over NE (arrowhead). Pseudocolor coloring with intensity increasing from blue to red demonstrates the distribution of fluorescence intensity. X axis represents distance in pixels along the line scan and y axis is logarithmic time scale. (E) File averaged diffusion coefficients from the ACF analysis of capsid movement in the cytoplasm, at the NE and in the nucleoplasm (38 measurements from 14 cells) are shown as boxplots. The error bars represent the ± standard error of mean (±SEM).
	Figure 4. Number and brightness analysis of capsid dynamics. (A) ACF based file averaged analysis showing the number of all moving particles in the cytoplasm (dark gray), at the NE (light gray) and in the nucleus (white) (n = 15 cells). Number (B,C) and brightness (D,E) analyses of fast and slow moving particles in the cytoplasm, NE and nucleus. The black horizontal lines represent the median, the crosses represent the mean, and boxes represent the quartiles of data range including the median. (F) A histogram of the average brightness of the slow fluorescent viral particles as a function of distance from the nuclear lamina in all cells studied. (G) Single-cell fluorescence intensity map showing the number (red) and brightness (yellow) of cytoplasmic and nuclear capsids and distribution of lamin C (green, time-average of 100 line scans). (H) Histogram showing absolute brightness distribution of fast nuclear viral particles.
	Figure 5. Image pair-correlation analyses of importin β and capsid movement. (A) Confocal microscopy image of a CPV-A594 (red) infected cell stably expressing importin β-GFP (green) at ~1 h p.i (lower left). The image pCF analysis of intracellular movement of importin β and capsids (B). The intensity corresponds to the correlation integral of G(r, r + 4). (C) Overlay of importin β pCF (green) and capsid pCF (red) with the importin intensity image shown in (A, the lower left corner). (D) Normalized intensity line profile analysis of importin β pCF and capsid pCF overlay as a function of distance (pixels) from the NE (gray zone) showing simultaneous movement of importin β and capsids across the NE. White dashed line shows the positive correlation of capsid and importin β movements from cytoplasm to nucleoplasm. (E) Cross-correlation of importin β and capsid pCFs. (F) The overlay of cross-correlated pCF with importin β intensity image (gray). Scale bar, 10 µm.
	Figure 6. Schematic presentation of nuclear entry pathway of CPV. Upon release from the endosomal vesicles, CPV capsids are actively transported across the NE via importin β followed by disassembly in the nucleoplasm. However, the role of importin α in this process cannot be ruled out. During this transport, the motility of capsids fluctuates from cytoplasmic slow and fast, to nucleoplasmic slow and fast. The number of viral particles (purple gradient) is high distant from the nucleus and low near the NE. In the nucleus, higher amount of small particles is present suggesting for nucleoplasmic disassembly of the imported capsids.

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