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Index of Online Supplemental Material for
J. Cell Biol. Vol. 147 No. 4 p.743, White et al.

All movies are in QuickTime format.

For details about the techniques used to produce and quantitate the movies, including simultaneous imaging of CFP and YFP, image processing methods, and image correlation analysis, please see Additional Materials and Methods.

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The movies have been adjusted for best display on a Macintosh monitor, gamma 1.8, white point correction 6500 or 9300K. You may need to adjust the brightness, contrast, and gamma correction for the best viewing. Fainter structures may only be visible after correcting for your monitor. Viewing is best in a darkened room.

All movies are a series of single confocal frames. "Gray" movies show each channel separately side-by-side. Channels in "Color" movies are combined red and green, with overlap in yellow.

Elapsed time is indicated as hours:minutes:seconds (.fractions).

Figure 2
A	Fig. 2 A.mov GFP-Rab6_HeLa_Fast.mov	GFP-Rab6 trafficking in a stably transfected HeLa cell Tubular and globular trafficking elements exit the Golgi and translocate outwards to the cell periphery along microtubules at 0.6-1 µm/s. Corner regions show particularly active trafficking. Trafficking elements often disperse into a diffuse network fluorescence, particularly via the corner regions. Bar, 5 µm. GFP-Rab6_HeLa_Fast.mov shows GFP-Rab6 trafficking at 150 ms/frame and indicates the confocal movies have not missed fast-moving trafficking elements.
B	Fig._2 B.mov	Cumulative projection of GFP-Rab6 trafficking A cumulative brightest point projection of Fig._2 A.mov shows the tracks followed by GFP-Rab6 trafficking elements, and emphasizes the high flux through the corner regions. Most trafficking elements translocate outward from the Golgi and about the cell periphery. Bar, 5 µm.
Figure 3
Fig._3_Grey.mov Fig._3_Color.mov FP-Rab6_on_MT_A_Grey.mov FP-Rab6_on_MT_A_Color.mov FP-Rab6_on_MT_B_Grey.mov FP-Rab6_on_MT_B_Color.mov		FP-Rab6 elements traffic along microtubules PtK2 cell were transiently cotransfected with CFP-Rab6 and YFP-tubulin. Movies Fig._3 and the additional movie A show examples of FP-Rab6 transport carriers translocating along microtubules in the cell periphery. The additional movies B shows FP-Rab6 association with microtubules throughout an entire cell. In additional movies B, individual FP-Rab6 TCs may be difficult to discern against the cytosolic pool of FP-Rab6. The color movies show CFP-Rab6 in green and YFP-tubulin in red. Bars: (Fig._3 A) 5 µm; (Fig._3 B) 10 µm.
Figure 4
A	Fig._4 A_Grey.mov Fig._4 A_Color.mov	Shiga toxin B-fragment traffics in GFP-Rab6 elements during initial Golgi->ER transport Cy3-labeled STB was bound to GFP-Rab6 HeLa cells at 4°C and internalized at 37°C for 20 min to accumulate the fragment in the Golgi, then shifted to 28°C to prolong Golgi exit. Other temperature shift protocols were performed as described with similar outcome. STB and GFP-Rab6 exit the Golgi in tubular and globular structures, translocate together in the cell periphery, and accumulate together in cell corner regions. The left panel in Fig._4 A_Color.mov is a pixel-by-pixel analysis showing STB signal in structures with a significant GFP-Rab6 signal (upper 75%). The color panel shows GFP-Rab6 in green and STB in red. Bar, 5 µm.
B	Fig._4 B_Grey.mov Fig._4 B_Color.mov	Shiga-toxin B-fragment traffics with GFP-Rab6 in peripheral corner regions during initial Golgi->ER transport A closeup view of a corner region from Fig. 4 A shows trafficking elements containing both STB and GFP-Rab6 exiting the Golgi and translocating to a peripheral corner region. The images are the original size. The left panel in Fig._4 B_Color.mov is a pixel-by-pixel analysis showing STB signal in structures with a significant GFP-Rab6 signal (upper 75%). The color panel shows GFP-Rab6 in green and STB in red. Frame width 5.3 µm.
C	Fig._4 C_Grey.mov Fig._4 C_Color.mov	Shiga toxin B-fragment traffics in GFP-Rab6 elements at later times of STB internalization After 5 h of internalization at 37°C, STB partitions between the ER and Golgi, but continues to traffic in GFP-Rab6 positive structures. The color movie shows GFP-Rab6 in green and STB in red. Bar, 5 µm.
D	Fig._4 D.mov	Shiga toxin B-fragment trafficking in GFP-Rab6 elements in the cell periphery at later times A closeup of a peripheral region from the HeLa cell in Fig. 4 C.
Figure 5A
KDELR-GFP_HeLa.mov KDELR-CFP_YFP-Rab6_Grey.mov KDELR-CFP_YFP-Rab6_Color.mov		FP-Rab6 is distinct from Golgi->ER transport defined by KDELR-FP trafficking KDELR-GFP_HeLa.mov shows the dynamics of KDELR-GFP stably expressed in a HeLa cell. KDELR-GFP trafficking shows clear differences with GFP-Rab6 trafficking; KDELR-GFP elements do not accumulate in peripheral corner regions, tubulation is less frequent, and the movement of globular elements occurs over a shorter range. Bar, 5 µm. KDELR-CFP_YFP-Rab6_Grey.mov shows KDELR-CFP and YFP-Rab6 in a transiently Cotransfected PtK2 cell. KDELR-CFP is present in the periphery but does not accumulate in corner regions together with YFP-Rab6. YFP-Rab6 tubular and globular trafficking elements do not accumulate KDELR-CFP. The color movie shows KDELR-CFP in red and YFP-Rab6 in green. Bar, 10 µm.
Figure 6A
Fig._6 A_Grey.mov Fig._6 A_Color.mov		Shiga toxin B-fragment segregates from KDELR-GFP trafficking structures during Golgi->ER transport STB internalized for 20 min at 37°C in a stably transfected HeLa cell traffics separately from KDELR-GFP, and accumulates in distinct regions of the cell. The color movie shows KDELR-GFP in green and STB in red. Bar: 10 µm.
Figure 7
A	Fig._7 A.mov	Inhibition of COPI function inhibits KDELR-GFP trafficking Stably transfected HeLa cells expressing KDELR-GFP were microinjected with anti-EAGE antibody to inhibit COPI function. In this experiment the cell on the lower left was injected. Motility is inhibited to 60% of the uninjected cell. Bar, 5 µm.
B	Fig. 7 B.mov	Inhibition of COP-I function does not inhibit GFP-Rab6 trafficking GFP-Rab6 HeLa cells were injected with anti-EAGE to inhibit COPI function. In this movie the cell in the upper left was injected. Motility in the injected cell is slightly increased compared to the uninjected cell. Bar, 5 µm.
Figure 8
A	Fig._8 A_Grey.mov Fig._8 A_Color.mov Fig._8 A_Additional_Grey.mov Fig._8 A_Additional_Color.mov	FP-Rab6 trafficking elements are intimately associated with the ER network PtK2 cells were cotransfected with the fluorescent ER resident CFP-Sec61ß and YFP-Rab6. YFP-Rab6 trafficking elements correlate with ER dynamics, and YFP-Rab6 stably accumulates at the ends of ER tubules. The color movies show YFP-Rab6 in green and CFP-Sec61ß in red. Bar, 4 µm.
B	Fig._8 B_Grey.mov Fig._8 B_Color.mov	Shiga toxin B-fragment enters the ER from peripheral corner regions Fig._8 B_Grey.mov shows STB after 90 min of internalization in a HeLa cell expressing GFP-Sec61ß. The correlation plot of the two fluorescence channels shows that the overlap of STB and ER increases over time (left panel). By the end of the movie, STB and GFP-Sec61ß show significantly similar patterns (Correlation plot and Fig._8 B_Grey.mov). The color movie shows GFP-Sec61ß in green and STB in red. Bar, 4 µm.

Correspondence to: jwhite{at}embl-heidelberg.de

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