1. Volume 6, Issue 6, Pages 1849-1862 (November 2013)
Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment (PVC) Function in Arabidopsis 
Tomasz Nodzyński, Mugurel I. Feraru, Sibylle Hirsch, Riet De Rycke, Claudiu Niculaes, Wout Boerjan, Jelle Van Leene, Geert De Jaeger, Steffen Vanneste, Jiří Friml 
Molecular Plant 
Volume 6, Issue 6, Pages (November 2013)
DOI: /mp/sst044
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 pat3 Mutant Displays Aggregation of Plasma Membrane Cargos.
(A–H) Plasma membrane cargos PIN1–GFP (A, B), PIN2–GFP (C, D), PIP2a–GFP (E, F), and AUX1–YFP (G, H) show pronounced intracellular aggregations in pat3 cells (B, D, F, H) in comparison to the control (A, C, E, G). To better visualize the intracellular aggregations of PIN2–GFP (C, D), PIP2a–GFP (E, F), and AUX1–YFP (G, H), dark treatment was used for 3 h for the first two cargos and 8 h, respectively.
(I) Some pat3 seedlings arrest on medium without sucrose (arrowhead). Both the arrested and WT-looking seedlings are 6-day-old pat3 mutants.
(J) Histogram showing the percentage of normal (1), arrested (2), and non-germinated (3) seedlings in pat3 population compared to wild-type grown on sucrose-free media; values are the average of two biological replicates (number of seedlings, n > 120 per replicate). Error bars represent SEs for two biological repeats. Bars = 10 µm (A–H) and 1 mm (I).
Molecular Plant 2013 6, DOI: ( /mp/sst044)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 pat3 Mutant Shows Defects in Late Endocytic Pathway.
(A, B) Long-time uptake of the endocytic tracer FM4-64 (4 µM; 3 h) reveals aberrant membrane structures in pat3 (B) in comparison to the control (A).
(C, D) Morphology of the lytic compartments stained with LysoTracker Red (2 µM; 1 h) is altered in pat3 (D) displaying a more aggregated pattern compared to the control (C).
(E, F) PVC marker SYP22–YFP reveals a more aggregated pattern in pat3 (F) than in the control (E).
(G–K) Transmission Electron Microscopy (TEM) analysis of pat3 (H–K) cells reveals multilayered vacuole-like structures that are not present in the wild-type (G).
(L) The percentage of cells containing multilayered vacuole-like structures is depicted on the graph; values are the average of two biological replicates (number of cells analyzed, n > 170 per replicate). Error bars represent SEs for two biological repeats. Bars = 10 µm (A–F) and 1 µm (G–K).
Molecular Plant 2013 6, DOI: ( /mp/sst044)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 The PVC-Dependent Degradation Pathway Is Impaired in pat3.
(A, B) Intracellular aggregations in pat3 labeled by PIN1–GFP (green), FM4-64 (red; 4 µM) after 3 h of staining and visualized as co-localization of both signals (yellow) (B) in comparison to the PIN1–GFP control (A).
(C, D) Intracellular aggregations in pat3 labeled by PIN1–GFP (green), acidotropic dye LysoTracker Red (2 µM) after 1 h of staining (red), and visualized as co-localization of both signals (yellow) (D) in comparison to the PIN1–GFP control (C).
(E, F) Co-treatment for 1 h with 50 µM BFA and 4 µM FM4-64 reveals that the protein agglomerations in pat3 cells (F) labeled by PIN1–GFP (green) are distinct from BFA bodies (overlay PIN1–GFP with FM4-64 signal in yellow) and are not present in the control (E), where only BFA bodies are visible (yellow).
(G–J) Wortmannin induces swelling of the PIN1–GFP-labeled intracellular aggregations in pat3 after a 1-h treatment (33 µM) (J) as compared to the treated PIN1–GFP control (I) and untreated pat3 (H) and PIN1–GFP (G).
(K, L) Electron micrographs of wortmannin-treated (1 h, 33 µM), wild-type PIN1–GFP root vasculature cells (K) and pat3 cells showing strongly vacuolated PVC with luminal inclusion (L).
(M, N) 3-h treatment with 16.5 µM wortmannin mimics the subcellular phenotype of pat3 in the wild-type (M) and further enhances the swelling of the intracellular aggregations in pat3 (N). Bars = 10 µm.
Molecular Plant 2013 6, DOI: ( /mp/sst044)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Mapping of pat3 Identifies a Mutation in VPS35A, Coding a Subunit of the Retromer Complex.
(A) Locus information of the VPS35A gene and the gene model. Sequencing of At2g17790, VPS35A, revealed mutations in a splice site of the 6th intron for pat3-1 and the 15th intron in the case of pat3-2. Sites of the three allelic mutations are indicated by vertical arrowheads (black); the mutated nucleotides in the intron splice sites of the two EMS alleles are depicted by red letters.
(B) RT–PCR amplification of transcripts from wild-type (W) pat3-1 (1), pat3-2 (2), and pat3-3 (3) cDNA shows an intron retention in pat3-1 (1) visualized by PCR product size shift. Primers for the RT–PCR amplifications used to detect the intron splicing defects are indicated on the VPS35A gene model and on the corresponding electrophoresis gel images in the upper, right-hand corner. Primers used for the RT–PCR of the long gene fragment are shown as black dashed arrows; primers flanking the 6th and the 15th introns are shown in blue and red, respectively.
(C–H) The cellular phenotypes of the pat3 alleles (D–F) and their F1 heteroallelic crosses (G, H) in comparison to the PIN1–GFP wild-type control (C).
Bars = 10 µm.
Molecular Plant 2013 6, DOI: ( /mp/sst044)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Mutation in VPS29, Encoding a Subunit of the Retromer Complex, Causes Ectopic Accumulation of Proteins in the PVC.
(A–D) Plasma membrane cargos AUX1–YFP (A, B) and PIP2a–GFP (C, D) show intracellular aggregations in vps29-3 cells (B, D) in comparison to the control (A, C). To visualize the intracellular aggregations of AUX1–YFP, dark treatment of 8 h was used prior to imaging.
(E, F) Intracellular aggregations of PIN1–GFP (green) in vps29-3 (F) are co-labeled by 4 µM FM4-64 (red) after 3 h of staining (co-localization visible as yellow merged signals) in comparison to the PIN1–GFP in wild-type background (E).
(G, H) PVC marker SYP22–YFP shows a more aggregated pattern in vps29-3 (H) than in the control (G).
Bars = 10 µm.
Molecular Plant 2013 6, DOI: ( /mp/sst044)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Disruption of VPS35 Homologs B and C Does Not Lead to Trafficking Defects of PIN1–GFP.
(A–D) PIN1–GFP does not accumulate aberrantly in vps35b (C) and vps35c (D) showing normal trafficking as in wild-type (A) in contrast to intracellular aggregation observed in vps35a/pat3-3 (B). Bars = 10 µm.
Molecular Plant 2013 6, DOI: ( /mp/sst044)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions