![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||
This item has the following additional materials available:
Videos 1 A,B: Microtubule dynamics in TIR-FM. NRK fibroblasts were microinjected with cDNA encoding either tau-GFP at 2h (A) or GFP-ß-tubulin at 24h (B) prior to imaging in TIR-FM at 2 frames/sec. The video sequences show regions indicated by white boxes from Fig. 1b (tau-GFP) and Fig. 1e (GFP-ß-tubulin), respectively. Both videos show distinct microtubules undergoing phases of growth, shortening and pause, typically called dynamic instability. Also, there are microtubules, which move sideways and bend. The dynamics seen with tau-GFP are very similar to what we observe with GFP-b-tubulin.
Videos 2 B,C: Transport, docking and fusion of vesicles along microtubules imaged in simultaneous dual-color TIR-FM.
(B) The nucleus of a NRK fibroblast was microinjected with cDNA encoding p75-GFP and tau-GFP and imaged in TIR-FM with a temporal resolution of ~5 frames/sec. The sequence was processed according to the protocol for ‘temporal’ pseudo-color TIR-FM to separate the fast moving vesicles (red) from the slow motion of the microtubule cytoskeleton (green) (experimental procedures). A region of the whole cell (grey scale image) is shown in the video sequence (same as Fig. 2B). The transport of membrane cargo along the microtubules occurred in the typical saltatory fashion, alternating between fast and slow movements. The vesicles frequently switched between differenmicrotubule tracks in the dense microtubule meshwork. Some vesicles reached the edge of the cell (see left margin at the end of the sequence). After stopping for various times on the microtubule track the vesicles appear to fuse with the plasma membrane while we see an increase and spread of the fluorescence intensity. White arrows appearing during the last 5 frames before each fusion start indicate the fusions. Note, that due to the ‘temporal’ pseudo-color processing, a bright green signal remains at the center of each fusion after dispersal of the cargo.
(C) A MDCK cell was microinjected with cDNA encoding p75-YFP and tau-CFP and imaged in simultaneous dual-color TIR-FM with a temporal resolution of ~ 5 frames/sec. The sequence was processed according to the protocol described below, including bleed-through correction and contrast enhancement (experimental procedures). A tubular vesicle loaded with p75-YFP (red) is seen to track along the continuoumeshwork of microtubule tracks (green) (see Fig. 2c). It bends while switching tracks, moves back a small distance, until it collapses and fuses along the microtubule track.
Together these data indicate that all vesicles are transported via microtubules to their site of fusion with the plasma membrane. All scale bars represent 2mm.
Videos 3 A,B: Collapse and Fusion of tubular vesicles. Sparsely plated MDCK cells were microinjected with cDNA encoding p75-GFP and imaged in TIR-FM after release of the Golgi block (experimental procedures) with a time resolution of ~5 frames/sec. The sequences show collapse and fusion of: a tubular vesicle that completely fuses in one single step (Fig. 3a), a tubular vesicle that fuses in two steps to the plasma membrane (Fig 3b).
The fusions are seen as a simultaneous rise in total and width2 of the fluorescence intensity. Note, that the center of both fusions is not at one end of the tubular vesicle, but rather close to the center of the tubule. All scale bars represent 1mm.
The long tubular vesicle (B) is entering the evanescent field with an upward movement while still being held on its bottom end. The swaying movements of the center suggest that it is partially attached to the cytoskeleton. At the onset of the first fusion the rest of the tubule undergoes an abrupt shortening which delivers membrane protein to the plasma membrane. However, this vesicle does not collapse fully into the fusion center. The first (partial) fusion and shortening is followed by a 3 - 4 sec long stationary phase. Just before the start of the second fusion, the center of the vesicle fluorescence moves about 0.5mm. During the second fusion event all remaining cargo is delivered to the plasma membrane.
Prepared by: the MBC Journal Production Manager
| ||||||||||||||||||
