Microtubule plus end–associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells

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• Supplemental Materials (PDF)
• Video 10 -
  HSV-1 capsid movement in NHDFs transfected with DCTN1-siRNA or expressing EB1-DN. NHDFs transfected with DCTN1 siRNA #2 (left) or expressing dominant-negative EB1 (EB1-DN; right) were infected with HSV-1 K26GFP (green). 3 hpi, nuclei were stained with Hoechst (blue). Cells were analyzed using time-lapse microscopy on a Leica DMI6000B-AFC widefield microscope with a 100× objective using an InVivo environmental chamber at 37°C. Images were captured at 1 fps for ∼330 s. The playback speed of the video is 15 fps (i.e., ∼15× real speed). In DCTN1-depleted cells or cells expressing dominant-negative EB1, viral particles remain at the cell periphery and fail to initiate long-range transport to the nucleus.
• Video 2 -
  Movement of mitochondria in control or EB1 siRNA–treated NHDFs. NHDFs were treated with control siRNA (left) or EB1 siRNA #1 (right ) and then treated with MitoTracker (green) as described in Materials and methods section Antibodies, WB, IF, and imaging. Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 0.3 fps for 3 min. The playback speed of the video is 15 fps (i.e., ∼50× real speed).
• Video 1 -
  Time-lapse of EB1-DN in NHDFs, and HSV-1 motility in ciliobrevin D–treated NHDFs. (A) NHDFs were transfected with pN1-EB1-mCherry (false-colored green for contrast) encoding dominant-negative EB1 (EB1-DN) using Lipofectamine 3000. 24 h after transfection, cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective. Images were acquired at 4 fps for 37 s. The playback speed of the video is 15 fps (i.e., ∼4× real speed). A representative cell is shown. (B) NHDFs were treated with 200-µM ciliobrevin D for 1 h and then infected with HSV-1 containing a GFP-tagged capsid protein, VP26 (K26GFP; green). 3 hpi, nuclei were stained with Hoechst (blue) and cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 1 fps for 155 s. The playback speed of the video is 15 fps (i.e., ∼15× real speed). A representative video illustrates the brief but abortive long-range motility of virus particles in the presence of dynein inhibitor.
• Video 6 -
  Movement of lysosomes in DMSO- or ciliobrevin D–treated NHDFs. NHDFs were treated with DMSO solvent control (left) or 200-µM ciliobrevin D (CbD; right) for 1 h and then stained with LysoTracker (green) as described in Materials and methods section Antibodies, WB, IF, and imaging. Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 2 fps for 50 s. The playback speed of the video is 30 fps (i.e., ∼15× real speed).
• Video 8 -
  HSV-1 capsid movement in control siRNA–treated NHDFs. NHDFs were treated with control, nontargeting siRNAs and then infected with HSV-1 containing a GFP-tagged capsid protein, VP26 (HSV-1 K26GFP; green). 3 hpi, nuclei were stained with Hoechst (blue). Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 1 fps for ∼400 s. The playback speed of the video is 15 fps (i.e., ∼15× real speed). Virus particles at different stages of transport and infection could be observed. Two representative videos are shown that illustrate these different stages (left and right). Many particles had reached and brightly lit the nucleus, while those remaining in the cytoplasm exhibited rapid, long-range movements characteristic of MT-based motility. Particles were also observed moving in and out of, and even forming, perinuclear clusters that represent the centrosome, while others could be seen approaching the nucleus.
• Video 4 -
  Movement of lysosomes in control or EB1 siRNA–treated NHDFs. NHDFs were treated with control siRNA (left) or EB1 siRNA #1 (right) and then treated with LysoTracker (green) as described in Materials and methods section Antibodies, WB, IF, and imaging. Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 2 fps for 48 s. The playback speed of the video is 30 fps (i.e., ∼15× real speed).
• Video 7 -
  Movement of mitochondria in DMSO- or ciliobrevin D–treated NHDFs. NHDFs were treated with DMSO solvent control (left) or 200-µM ciliobrevin D (CbD; right) for 1 h and then stained with MitoTracker (green) as described in Materials and methods section Antibodies, WB, IF, and imaging. Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 0.3 fps for 3 min. The playback speed of the video is 15 fps (i.e., ∼50× real speed).
• Video 9 -
  HSV-1 capsid movement in EB1- or CLIP-170–depleted NHDFs. NHDFs were treated with EB1 siRNA #1 (left) or CLIP-170 siRNA #1 (right) and then infected with HSV-1 K26GFP (green). 3 hpi, nuclei were stained with Hoechst (blue). Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an InVivo environmental chamber at 37°C. Images were captured at 1 fps for ∼400 s. The playback speed of the video is 15 fps (i.e., ∼15× real speed). In EB1- or CLIP-170–depleted cells, viral particles remain at the cell periphery and fail to initiate long-range transport to the nucleus.
• Video 5 -
  Movement of lysosomes in NHDFs depleted of CLIP-170 or DCTN1. NHDFs were treated with CLIP-170 siRNA #1 (left) or DCTN1 siRNA #2 (right) and then treated with LysoTracker (green) as described in Materials and methods section Antibodies, WB, IF, and imaging. Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 2 fps for 30–50 s. The playback speed of the video is 30 fps (i.e., ∼15× real speed).
• Video 3 -
  Movement of mitochondria in NHDFs depleted of CLIP-170 or DCTN1. NHDFs were treated with CLIP-170 siRNA #1 (left) or DCTN1 siRNA #2 (right) and then treated with MitoTracker (green) as described in Materials and methods section Antibodies, WB, IF, and imaging. Cells were analyzed using time-lapse microscopy on a DMI6000B-AFC widefield microscope with a 100× objective using an environmental chamber at 37°C. Images were captured at 0.3 fps for 3 min. The playback speed of the video is 15 fps (i.e., ∼50× real speed).