TRIM5 Cytoplasmic Bodies Are Highly Dynamic Structures

Supplemental Materials

This article contains the following supporting material:

• Supplementary Movie 1A - Long rage, microtubule dependent movement of TRIM5· cytoplasmic bodies.
  GFP-huTRIM5· expressing cells were imaged in the absence (A) of presence (B) of the microtubule disrupting drug nocodazole. Movies show a 60 frame sequence captured at 1.325 second intervals. The high degree of motility is clear in the absence of drug (A), while this mobility is markedly impaired by nocodazole (B).
• Supplementary Movie 1B - Long rage, microtubule dependent movement of TRIM5· cytoplasmic bodies.
  GFP-huTRIM5· expressing cells were imaged in the absence (A) of presence (B) of the microtubule disrupting drug nocodazole. Movies show a 60 frame sequence captured at 1.325 second intervals. The high degree of motility is clear in the absence of drug (A), while this mobility is markedly impaired by nocodazole (B).
• Supplementary Movie 2 - Movement of TRIM5· cytoplasmic bodies on the microtubule network.
  A live TE671 cell stably expressing CFP-labelled tubulin (red) and GFP-huTRIM5α (green) was imaged using fluorescence microscopy at 10 second intervals. The cytoplasmic body can be seen traveling on the microtubule network.

• Supplementary Movie 3A - A and B. Smaller TRIM5α cytoplasmic bodies combine to form larger bodies.
  HOS cells were microinjected with GFP-rhTrim5α and cells were imaged at 2 minute intervals. At the onset of expression, small cytoplasmic bodies quickly became apparent, and these smaller bodies could combine to form larger bodies. Movie 3A shows a GFP-rhTRIM5· expressing cell, while movie 3B shows an enlargement of the left side of the cell where numerous bodies can be observed coalescing into larger cytoplasmic bodies. The signal in 3B has been amplified relative to 3A to make smaller bodies more visible.
• Supplementary Movie 3B - A and B. Smaller TRIM5α cytoplasmic bodies combine to form larger bodies.
  HOS cells were microinjected with GFP-rhTrim5α and cells were imaged at 2 minute intervals. At the onset of expression, small cytoplasmic bodies quickly became apparent, and these smaller bodies could combine to form larger bodies. Movie 3A shows a GFP-rhTRIM5· expressing cell, while movie 3B shows an enlargement of the left side of the cell where numerous bodies can be observed coalescing into larger cytoplasmic bodies. The signal in 3B has been amplified relative to 3A to make smaller bodies more visible.
• Supplementary Movie 4 - Individual TRIM5· cytoplasmic bodies can give rise to smaller, separate cytoplasmic bodies.
  HOS cells were microinjected with GFP-rhTrim5α and cells were imaged at 2 minute intervals. At the onset of expression, small cytoplasmic bodies quickly became apparent. During acquisition, individual cytoplasmic bodies could be seen dividing into smaller, separate bodies (indicated by arrows).
• Supplementary Movie 5 - Fluorescence recovery of bleached TRIM5· cytoplasmic bodies.
  HeLa cells transfected with YFP-rhTRIM5· expression plasmid were imaged (first frame) and then the indicated cytoplasmic bodies were photobleached using Zeiss's time series and bleach software with the confocal laser at 100% intensity for 120 iterations. Post-bleach images were obtained periodically every 30 seconds for 15 minutes
• Supplementary Movie 6 - Photoactivation analysis of TRIM5α cytoplasmic bodies.
  A HeLa cell transiently transfected with PAGFP-TRIM5α is activated in a small discreet region, while the rest of the PAGFP-TRIM5α remains unactivated and therefore is not fluorescent. TRIM5α dynamics is examined by monitoring the behavior of this fluorescent subpopulation of PAGFP-TRIM5α. Following activation, live-cell images were acquired every 10 minutes for 60 minutes.