Maturation of late Golgi cisternae into RabERAB11 exocytic post-Golgi carriers visualized in vivo

Maturation of Golgi cisternae into post-Golgi carriers is directly visualized using the Aspergillus nidulans Ypt31/RAB11 homologue RabE as a reporter of post-Golgi identity. A microtubule-based conveyor belt fuels carriers to actin microfilaments radiating from the apex, which carry out the membrane-proximal transport step of exocytosis.

beam splitter. Experiment carried out with strain MAD5098. (B) Kymographs of GFP-HypB Sec7 and mCherry-RabE RAB11 channels derived from time-series constructed with projections of Z-stacks acquired every 3.3 sec and deconvolved. Left, the insets in the GFP and mCherry channels are shown merged, at double magnification. Strain and conditions were as in (A). (C) Quantitation of GFP-HypB Sec7 and mCherry-RabE RAB11 channels for n=7 cisternae maturing into post-Golgi RabE RAB11 carriers. (D) Example of one of these events with quantitation of the two channels.
Supplemental Figure S4. The abnormal hypB5 aggregate of RabE RAB11 membranes also contains RabA RAB5 . Colocalization of GFP-RabE and mCherry-RabA in a hypB5 cell, following a 50 min shift to a restrictive temperature. Under such conditions RabE RAB11 is present in abnormal membrane aggregates, and this experiment shows that in these there is detectable colocalization with the early endosomal marker mCherry-RabA. It should be emphasized that in wild-type cells RabA early endosomes and RabE carriers are distinct populations (Supplemental Figure S6 and S7) and thus that this colocalization of the two Rabs is caused by the abnormal membrane traffic resulting from HypB Sec7 impairment. Inverted greyscale images represent maximal intensity projections of Z-stacks, whereas colored images represent optical sections (Z-pass indicated). This experiment was carried out with strain MAD5078, using fructose as carbon source. Control experiments with strain MAD5082 demonstrated that abnormal RabE RAB11 aggregates also form in hypB5 cells that do not express mCherry-RabA (not shown). All images are at the same magnification. Figure S5. MT organization controls and effect of benomyl on RabE RAB11 carriers (A) TubA-GFP labeled microtubules in the tip regions of cells with the indicated genetic backgrounds, or in the presence of the also indicated cytoskeletal inhibitors. Images are projections of Z-stacks treated with the 'unsharp' filter of Metamorph, to enhance contrast. All panels are shown at the same magnification (myoE∆ cells are conspicuously wider than the wild-type). (B) Kymographs of GFP-RabE carriers in wild-type hyphal tips treated or not with benomyl. Note that benomyl results in complete MT depolymerization [panel (A)]. Figure S6. RabE RAB11 carriers and RabA RAB5 early endosomes are distinct populations (A) Kymograph capturing trajectories of RabA RAB5 -labeled early endosomes and GFP-RabE carriers. They were derived from movies in which the red and green channels were photographed simultaneously using a Dual-View beam splitter. (B) As above in a cell treated with latrunculin B to displace SPK RabE RAB11 carriers to the MT conveyor belt. (C) GFP-RabE and mCherry-RabA aggregated in different structures in cells treated with both latrunculin B and benomyl.

Supplemental Figure S7. RabE RAB11 carriers and PdnIns3P endosomes are distinct populations
Non-overlapping trajectories of GFP-FYVE 2 -labeled early endosomes and mCherry-RabE carriers captured with kymographs (strain MAD5072). These were derived from movies in which the red and green channels were photographed simultaneously using a Dual-View beam splitter. Kymographs corresponding to a tip-proximal region (where RabE RAB11 carriers are most abundant) and a tip-distal region are shown. In either case RabE RAB11 carriers are more abundant than early endosomes labeled with GFP-FYVE 2 .

Supplemental Figure S8 RabE RAB11 and mRFP-PH OSBP localize to static aggregates in a hyphal tip cell treated with both latB and benomyl
The pictures display a hyphal tip cell treated with both latB and benomyl for 28 min. Images represent the starting frame of a time-stack acquisition (71 frames, 1 frame every 3 sec) in which each time frame is a maximal intensity projection of a Z-stack. The kymograph at the bottom shows that GFP-RabE and mRFP-PH OSBP aggregates do not overlap over time.

Supplemental Table S1
A. nidulans strains used in this work

Supplemental Table S2
Oligonucleotides used for genetic manipulation and diagnostic PCRs Supplemental Movie S1 GFP-RabE carriers arriving at the prominent SPK in a cell undergoing apical extension. The GFP channel is shown in inverted grey scale. Strain MAD4120. Time is in min:sec:msec.

Supplemental Movie S2
mCherry-RabE colocalizing with MyoE-GFP in the SPK of several hyphae growing over a ~ 30 min period. Strain MAD4403 cultured on fructose. Time is in h:min:sec

Supplemental Movie S3
Late Golgi cisternae labeled with mRFP-PH OSBP mature into GFP-RabE post-Golgi carriers over time. These carriers are delivered to the SPK. Individual frames represent maximal intensity projections of deconvolved z-stacks of strain MAD4440 (1 stack every 4 sec). Time is in min:sec.

Supplemental Movie S4
Late Golgi cisternae labeled with mRFP-PH OSBP co-filmed with GFP-RabE with a time resolution of 250 msec. The boxed region contains two LGC captured during their transition to RabE RAB11 carriers that subsequently undergo movement towards the SPK. Frames represent middle planes of strain MAD4440. Time is in sec:msec

Supplemental Movie S5
Late Golgi cisternae labeled with HypB Sec7 -GFP co-filmed with mCherry-RabE. Individual frames represent maximal intensity projections of deconvolved Z-stacks of strain MAD5098 (1 stack every 3.3 sec). An example of a LGC that forms after 9 sec and matures into a RabE carrier is arrowed in the corresponding channels. Time is in min:sec.

Supplemental Movie S6
GFP-RabE carriers following trajectories of TubA-GFP MTs. An example of one such carrier moving to the SPK is indicated with a grey arrowhead. Strain MAD4263. Time is in sec:msec.

Supplemental Movie S7
Movie highlighting one example of a GFP-RabE carrier (grey arrowhead) that moves basipetally within the tip region, following the trajectory of a TubA-GFP MT in strain MAD4263. Time is in sec:msec

Supplemental Movie S8
Rapidly moving GFP-RabE structures in a hyphal tip of strain MAD4120 cell treated with latB. Time is in sec:msec Supplemental Movie S9 GFP-RabE structures rapidly moving on the MT conveyor belt in latB-treated MAD4263 cell that co-expresses GFP-labeled TubA to reveal the position of the MTs. Time is in sec:msec.
Supplemental Movie S10 GFP-RabE carriers arriving at the characteristic apical crescents of several myoE∆ hyphal tip cells. Strain MAD4409. Time is in sec:msec.

Supplemental Movie S11
Two parallel sequences with the same time resolution of GFP-RabE carriers showing decoration of actin cables in a myoE∆ tip (top sequence) alongside with a benomyl-treated myoE∆ tip (bottom). Note the complete absence of moving structures and actin cable decoration in the bottom sequence. Strain MAD4409. Time in sec:msec.