Volume 7 issue 8 August 2006
RNA interference effector proteins localize to mobile cytoplasmic puncta in Schizosaccharomyces pombe
Jon B. Carmichael, Cezar Stoica, Henry Parker, J. Michael McCaffery, Andrew J. Simmonds, and Tom C. Hobman

Supplementary materials - Legends for Movies

Movie 1.GFP-Ago1 is associated with highly mobile cytoplasmic punta. S. pombe ( Δago1 ) expressing GFP-Ago1 were grown to mid-log phase and immobilized on slides containing 0.7% agarose:water (w/v). Twenty serial sections of 0.2µm thickness, perpendicular to the long edge of the cell, were captured at 0.3 seconds/section using a spinning disc confocal microscope. The maximum projection images representing the reconstitution of the sections are played at 2 frames per second resulting in a time compression of 12 times. Dcr1-GFP and GFP-Rdp1 associate with the same mobile elements (data not shown).

Movie 1 (.avi)

Movie 2. Movement of GFP-Ago1 containing cytoplasmic puncta is inhibited by depletion of cellular ATP. S. pombe ( Δago1 ) expressing GFP-Ago1 grown to mid-log phase, treated with deoxyglucose (200µg/ml) for one hour and immobilized on slides containing 0.7% agarose:water (w/v). Twenty-two serial sections of 0.2µm thickness, perpendicular to the long edge of the cell, were captured at 0.3 seconds/section using a spinning disc confocal microscope. The maximum projection images representing the reconstitution of the sections are played at 2 frames per second resulting in a time compression of 12 times. Note the increase in size and numbers of cytoplasmic puncta due to loss of ATP. Similar results were obtained with Dcr1-GFP and GFP-Rdp1 .

Movie 2 (.avi)

Movie 3. Movement of GFP-Ago1 containing cytoplasmic puncta requires intact microtubules. S. pombe ( δago1 ) expressing GFP-Ago1 were grown to mid-log phase, treated with the microtubule destabilizing agent TBZ (15µg/ml) for one hour and immobilized on slides containing 0.7% agarose:water (w/v). Sixteen serial sections of 0.2µm thickness, perpendicular to the long edge of the cell, were captured at 0.3 seconds/section using a spinning disc confocal microscope. The maximum projection image representing the reconstitution of the sections are shown played at 2 frames per second resulting in a time compression of 10 times. Note the increase in size and numbers of cytoplasmic puncta due to microtubule destabilization. Similar results were obtained with Dcr1-GFP and GFP-Rdp1.

Movie 3 (.avi)