Movies

Preparation of Viral DNA from Nucleocapsids

Submandibular ganglion cells, expressing G-CaMP2 delivered by PRV369

Movie 1. Time-lapse microscopy recording of mitochondria in axons of mock infected SCG neurons.

Mitochondria of mock infected SCG neurons were stained with 10 nM MitoTracker Red CMX Ros and imaged in axons at a rate of 1.2 frames/second for 180 seconds. Playback speed is 30 frames/second.

Movie 2. Time-lapse microscopy recording of mitochondria in axons of wild type PRV infected SCG neurons.

Mitochondria of PRV Becker infected SCG neurons (16 hpi) were stained with 10 nM MitoTracker Red CMX Ros and imaged in axons at a rate of 1.2 frames/second for 180 seconds.

Movie 3. Live cell imaging of mitochondria and PRV capsids in axons of infected SCG neurons.

SCG neurons were infected with PRV GS443, a wild type strain that expresses GFP-tagged capsids (GFP-VP26) and stained with 10 nM MitoTracker Red CMX Ros. PRV capsids (green) and mitochondria (red) were visualized in axons at 12 hpi on a Leica SP5 confocal microscope equipped for imaging multiple channels simultaneously. Images were captured at a rate of 1.2 frames/second for 180 seconds.

Movie 4. Time-lapse microscopy recording of mitochondria in axons of HSV-1 KOS infected SCG neurons.

Mitochondria of HSV-1 KOS infected SCG neurons (18 hpi) were stained with 10 nM MitoTracker Red CMX Ros and imaged in axons at a rate of 1.2 frames/second for 180 seconds. Playback speed is 30 frames/second.

Movie 5. Time-lapse microscopy recording of mitochondria in axons of PRV gB-null (HF22A) infected SCG neurons.

Mitochondria of PRV HF22A infected SCG neurons (16 hpi) were stained with 10 nM MitoTracker Red CMX Ros and imaged in axons at a rate of 1.2 frames/second for 180 seconds. Playback speed is 30 frames/second.

Movie 6. Time-lapse microscopy recording of intracellular Ca2+ flux in mock infected SCG neuron cell bodies.

Mock infected SCG neurons were labeled with the Ca2+-sensitive dye Oregon Green 488 BAPTA-1 AM (5 μM) and imaged at a rate of 1 frame every 10 minutes for 19 hours. Playback speed is 20 frames/second.

Movie 7. Time-lapse microscopy recording of intracellular Ca2+ flux in wild type PRV infected SCG neuron cell bodies.

PRV Becker infected SCG neurons were labeled with the Ca2+-sensitive dye Oregon Green 488 BAPTA-1 AM (5 μM) and imaged at a rate of 1 frame every 10 minutes between 3-22 hpi (19 hours total). Playback speed is 20 frames/second.

Movie 8. Time-lapse microscopy recording of mitochondria in axons of PRV Miro1 (GFP-Miro1 expressing) infected SCG neurons.

Mitochondria of PRV Miro1 infected SCG neurons (16 hpi) were stained with 10 nM MitoTracker Red CMX Ros and imaged in axons at a rate of 1.2 frames/second for 180 seconds. Playback speed is 30 frames/second.

Movie 9. Time-lapse microscopy recording of mitochondria in axons of PRV Miro1ΔEF (GFP-Miro1ΔEF expressing) infected SCG neurons.

Mitochondria of PRV Miro1ΔEF infected SCG neurons (16 hpi) were stained with 10 nM MitoTracker Red CMX Ros and imaged in axons at a rate of 1.2 frames/second for 180 seconds. Playback speed is 30 frames/second.

PRV 341 was used to infect dissociated SCG neurons. Axons at sites distal from the cell body were imaged by epifluorescence microscopy at 8.5 h postinfection. GFP-Us9, VP26-mRFP, and an overlay of the two fluorescence channels are displayed as indicated. Imaging was done using sequential fluorescence channel acquisition achieving 0.89 frame/s. The movie is encoded to play back at 10 frames/s.

PRV 341 was used to infect dissociated SCG neurons. Axon segments proximal to the cell body were imaged by epifluorescence microscopy at 7 h postinfection. GFP-Us9, VP26-mRFP, and an overlay of the two fluorescence channels are displayed. The cell body is out of frame to the upper left side of the image. Imaging was done using sequential fluorescence channel acquisition achieving 0.57 frame/s. The movie is encoded to play back at 10 frames/s.

PRV 442 was used to infect dissociated SCG neurons. Axon segments proximal to the cell body were imaged by epifluorescence microscopy at 7 h postinfection. GFP-Us9 (Y49-50A), VP26-mRFP, and an overlay of the two fluorescence channels are displayed. The cell body is out of frame to the lower left side of the image. Imaging was done using sequential fluorescence channel acquisition achieving 0.6 frame/s. The movie is encoded to play back at 10 frames/s.

PRV 348 was used to infect dissociated SCG neurons. An isolated distal axon segment was imaged by epifluorescence microscopy at approximately 7 h postinfection. GFP-Us9, gM-mCherry, and an overlay of the two fluorescence channels are displayed. Imaging was done using sequential fluorescence channel acquisition achieving 0.59 frame/s. The movie is encoded to play back at 10 frames/s.

PK15 cells infected with a mixture of PRV180, PRV443, and PRV543 at a MOI of 10. Imaging was initiated at 3 hpi, and frames were collected every 10 min.

Anterograde motion of two capsids taken from our recent PNAS publication

Anterograde transport of many capsids, with one suddenly reversing direction towards the later half of the recording

Anterograde moving capsids accumulating at a bifurcated axon terminal

Quicktime movie of a serial section block-face scanning electron micrograph (SBFSEM) stack of 65 serial sections from a PRV-infected mouse submandibular ganglion (one neuron shown), sectioned at 50 nm. The volume shown is at the inner edge of the nucleus, with the nuclear envelope at the right hand side of the image. This movie is a cropped substack from Movie 2. Movie was acquired in the laboratory of Winfried Denk (Max-Planck Institute, Heidelberg, Germany) by Becket Feierbach.

Quicktime movie of a serial section block-face scanning electron micrograph (SBFSEM) stack comprising 100 serial sections from a PRV-infected mouse submandibular ganglion (one neuron shown), sectioned at 50 nm. Lower half of the cell was not obtained during image acquisition. Movie was acquired in the laboratory of Winfried Denk (Max-Planck Institute, Heidelberg, Germany) by Becket Feierbach.

Quicktime movie of a serial section block-face scanning electron micrograph (SBFSEM) stack comprising 150 serial sections from a mouse submandibular neuron, sectioned at 50 nm. Movie was acquired in the laboratory of Winfried Denk (Max-Planck Institute, Heidelberg, Germany) by Becket Feierbach.

Movie of virion component movements in axons within the chamber ring, using the Leica SP5 confocal microscope. The explant outside the ring was infected with PRV expressing a mRFP-capsid fusion and a gD-GFP fusion. The axons inside the ring run along the longitudinal axis of the frame and were imaged approximately 15.5 hpi. Ring edge and explant are located just beyond top of frame. The images were acquired at 5 frames/sec and are played back at 8 frames/sec. Acquisition by Becket Feierbach.

Movie of virion component dynamics entering post-synaptic cells inside the isolator chamber using the Leica SP5 confocal microscope. The majority of the axons are running along the diagonal form the top right to the bottom left. The explant outside the ring was infected with PRV expressing a mRFP-capsid fusion and a gD-GFP fusion and were imaged approximately 17.5 hours post-infection. Four optical slices at 0.6mm Z axis intervals were collected at each time point, providing a 1.28 second time interval per frame. The 2-D projections are shown over time and played back at 8 frames/sec. Acquisition by Becket Feierbach.

A differentiated PC12 cell body with multiple neurites infected with PRV GS443 (GFP-capsid) for 12 hours. Green capsid puncta are readily observed moving in the anterograde direction, i.e. away from the cell body, in all visible neurites. The cell body was imaged for approximately 13.5 minutes. Each frame is a 2D projection representing a stack of approximately 15 optical sections, 0.5mm apart (6.36 seconds/frame). The playback rate is 7 frames/sec.

A differentiated PC12 cell body infected with PRV 368 (Us9-null, GFP-capsid) for 12 hours. No capsid puncta were observed moving in the anterograde direction in neurites over a 12.5 minute period. Each frame is a 2D projection representing a stack of approximately 15 optical sections, 0.5mm apart (6.88 seconds/frame). The playback rate is 7 frames/sec.

A differentiated PC12 cell body infected with PRV 368 (Us9-null, GFP-capsid for 12 hours. No capsid puncta were observed moving in the anterograde direction in neurites over a 15 minute period (capsids were not present beyond the proximal segment). However, several capsids are undergoing transneuronal, retrograde transport from the infected cell body to an uninfected cell above the field of view (see Movie 4). Despite an abundance of moving capsid puncta within the cell body, no other egress events are visible. Each frame is a 2D projection representing a stack of approximately 15 optical sections, 0.5mm apart (6.98 seconds/frame). The playback rate is 7 frames/sec.

Capsids from the same infected cell body in movie 3 are transported in a retrograde manner to an uninfected cell above. A capsid is shown to traffic back to the uninfected PC12 cell body, enter the cell, and move to a perinuclear region where capsids are accumulating, perhaps the microtubule organizing center (MTOC). Note that no capsids are moving in the anterograde direction inside the infected cell. Each frame is a 2D projection representing a stack of approximately 15 optical sections, 0.5mm apart (6.7 seconds/frame). The playback rate is 7 frames/sec.

Time-lapse recording of capsid and VP22 transport dynamics inside the microfluidic chamber. Cell bodies in the somal compartment were infected with PRV 181, a recombinant expressing an mRFP-VP26 (capsid) fusion and a GFP-VP22 (outer tegument) fusion. Time-lapse images were taken of axons within the microgrooves at 11-14 h postinfection using the Leica SP5 confocal microscope. The movie shows an mRFP punctum moving in the retrograde direction and an mRFP punctum co-transporting with GFP-VP22 punctum in anterograde motion. Images were acquired at 4 frames/s. The playback rate is 5 frames/sec. Acquisition by Wendy Liu.

Time-lapse recording of the transport of light and heavy particles inside the microfluidic chamber. Cell bodies in the somal compartment were infected with PRV 181, a recombinant expressing an mRFP-VP26 (capsid) fusion and a GFP-VP22 (outer tegument) fusion. Time-lapse images were taken of axons within the microgrooves at 11-14 h postinfection using the Leica SP5 confocal microscope. Green puncta of light particles containing GFP-VP22 without mRFP-VP26 and yellow punta of heavy particles containing both GFP-VP22 and mRFP-VP26 were seen moving in the anterograde direction. Both light and heavy particles displayed heterogeneity in GFP fluorescence. Images were acquired at 4 frames/s. The playback rate is 5 frames/sec. Acquisition by Wendy Liu.

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