Fig 1.
Localization of RAB-7 and carboxy-DFFDA at the hyphal tip.
Panel A was intentionally overexposed to show the overall outline of the hyphal tip. Panel B shows the location of RAB-7, tagged with RFP. Arrows point to examples of PVCs. Panel C shows the location of carboxy-DFFDA, largely in the tubular vacuoles in the distal region. Panel D is the merged image for Rab-7-RFP and carboxy-DFFDA.
Fig 2.
Three-dimensional structure of the PVC.
The images are optical sections of a hyphal tip expressing RAB-7-RFP. 40 images were obtained over a depth of 10 microns. In panel A 10 optical sections are shown, each separated from the other by approximately 0.39 microns. Panel B shows an enlargement of the 4th optical section from panel A. Panel C shows the images as reconstructed in three dimensions, viewed from three different angles of rotation.
Fig 3.
Rapid changes in the shape of the PVC.
Panel A shows images of a single PVC, visualized with RAB-7-RFP, taken at intervals of 0.2 sec. For comparison, the arrows point to a small tubule which shows little change in shape or movement. In Panel B a series of optical sections was taken at each time point, over an interval of 48 sec. For each of the five images a single optical section is shown on top, and the three-dimensional reconstruction from that set of optical sections is shown below. The three-dimensional images are slightly tilted for better observation of the structure.
Fig 4.
Dynein is observed within the cavity of most PVCs.
Six frames from S5 Movie, obtained with a spinning disk confocal microscope, are shown. The PVCs are observed with VMA-1-GFP, and dynein is observed with DIC-mCherry. The arrows point to a dynein comet moving rapidly towards the hyphal tip.
Fig 5.
High resolution imaging of dynein within PVCs.
PVCs are observed with RAB-7-GFP and dynein with DIC-mCherry. Panels A, B and C are three-dimensional reconstructions from a series of 40 optical sections obtained over a depth of 10 microns. Panels D, E and F are from a single optical section.
Fig 6.
Effect of microtubule depolymerization on PVC structure.
Panels A and B show the bml-GFP strain in the absence (Panel A) and presence (Panel B) of 5 μg/ml of benomyl. Panels C-N show strain DIC-mCh, Rab-7-GFP. In Panels C, D, E the hypha was not exposed to benomyl. In Panels F, G, H the hypha was exposed to 5 μg/ml benomyl. In panel I, J, K the hypha was exposed to 10 μg/ml. In Panels L, M, N, the hypha was exposed to 5 μg/ml.