1. Volume 8, Issue 8, Pages 1175-1187 (August 2015)
Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana 
Marta Zwiewka, Tomasz Nodzyński, Stéphanie Robert, Steffen Vanneste, Jiří Friml 
Molecular Plant 
Volume 8, Issue 8, Pages (August 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
Acute Environmental Osmotic Stress Affects Endocytic Trafficking.
(A–I) Internal PIN2-GFP intensity increased under 100 mM NaCl and 75 mM mannitol (B, C, G, H), but decreased under deionized/MQ water treatment (D, I) compared with the control samples (A, F).
(E) Quantification of internalized PIN2-GFP signals. Error bars indicate SE (n = 65 cells).
(F–I) Maximal projection of PIN2-GFP expressing root cells (z-stack = 10 slices of 1 μm).
(J–R) Internalization rate of FM4-64-labeled PM increased under 100 mM NaCl and 75 mM mannitol stress conditions (K, L, P, Q) and decreased after treatment with deionized/MQ water (M, R) compared with control medium conditions (J, O).
(O–R) Maximal projection of cells labeled with FM4-64 (z-stack 10 slices of 1 μm).
(N) Quantification of internalized FM4-64 signals. Error bars indicate SE (n = 80 cells). Asterisks mark significant differences (Student’s t-test, * P < 0.01).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
PIN Protein Accumulation in BFA Bodies Is Influenced by External Osmotic Stress Conditions.
(A–D) Visualization of PIN1 and PIN2 accumulation in BFA bodies under different osmotic conditions via immunolocalization. 100 mM NaCl and 75 mM mannitol stress conditions (B, C) cause increased PIN protein accumulation in BFA bodies, whereas deionized/MQ water treatment (D) results in the formation of smaller BFA bodies compared with control conditions (A). Insets show higher magnifications of the indicated PIN1-expressing areas.
(E) Quantification of the PIN1-labeled BFA bodies area formed under different osmotic stress conditions. Error bars indicate SE (n = 45 cells). Asterisks mark significant differences (Student’s t-test, * P < 0.05).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3
Acute Exogenous Osmotic Stress Affects Recycling of PM Cargos.
(A) PIN1 and PIN2 accumulation in BFA bodies after treatment for 1 h under control conditions.
(B–E) PIN1 and PIN2 immunolocalization after incubation for 1 h with the fungal toxin followed by 30 min of drug wash-out with different osmotic solutions. More intensive recycling was observed in wash-outs with deionized/MQ water (C) and 0.5× MS (B) in comparison with 100 mM NaCl (D) and 75 mM sorbitol (E), respectively.
(F) Area quantification of PIN1-containing BFA bodies. Error bars indicate SE (n = 55 cells). Asterisks mark significant differences in BFA wash-out efficiency between control medium conditions and in different osmotic solutions (Student’s t-test, * P < 0.05).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4
The Increased Endocytosis in Response to Acute Hyperosmotic Stress Is Mediated by the Clathrin Machinery.
(A–D) FM4-64 accumulation in BFA bodies under control (A, C) and 100 mM NaCl stress conditions (B, D) in the INTAM-driver line (A and B) and INTAM>>RFP-HUB1 (C and D).
(E) Quantification of FM4-64 internal signal intensity. Error bars indicate SE (n = 80 cells). Asterisks mark a significant difference between the control driver line and the INTAM>>RFP-HUB1 line (Student’s t-test, *P < 0.01).
(F–I) Accumulation of PIN1 (red) and PIN2 (green) in BFA bodies in the presence (H, I) or absence (F, G) of the CME inhibitor Tyrphostin A23 (Tyr23; 50 μM) under control (F, H) and 100 mM NaCl stress conditions.
(J) Quantification of the number of BFA bodies per cell that contain PIN1 protein. Error bars indicate SE (n = 60 cells). Asterisks mark significant differences (Student’s t-test, * P < 0.05).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5
The Early Endocytic Machinery Participates in Plant Physiological Adaptation to Hyperosmotic Stress.
(A–F) Accumulation of PIN1 and PIN2 in BFA bodies under control (0.5× MS; A, C, E) and 100 mM NaCl stress treatments (B, D, F) for the wild-type (A, B) and the endocytosis-defective ben1-2 (C, D) and chc2-1 (E, F) mutants.
(G) Quantification of the number of PIN1-labeled BFA bodies per cell. Error bars indicate SE (n = 70 cells). Asterisks mark significant differences (Student’s t-test, * P < 0.05).
(H–J) ben1-2 and chc2-1 mutants show worse recovery and more impaired growth than the wild-type (H) when transferred to standard medium (0.5× MS) after 3 days of exposure to hyperosmotic stress (0.5× MS with 300 mM mannitol). Scale bar represents 1 cm.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6
Acute Hyperosmotic Stress Overcomes the Inhibitory Effect of Auxin on Endocytosis.
(A–C) FM4-64 accumulation in BFA bodies after pulse labeling of the membranes and subsequent cotreatment with NAA and BFA under osmotic stress conditions. BFA body formation in root meristematic cells (A), in the NAA-treated control (B), and in cells exposed to 100 mM NaCl treatment in the presence of auxin (C).
(D) Quantification of BFA body size, as visualized by FM4-64, formed under different osmotic stress conditions with and without auxin. Error bars indicate SE (n = 55 cells). Asterisks mark significant differences between control medium conditions and different osmotic solutions (Student’s t-test, * P < 0.01).
(E–G) Immunolocalization of PM proteins PIN1 and PIN2 in roots treated with BFA under 75 mM mannitol conditions in the presence of auxin (NAA 10 μM) (F, G) in comparison with root cells cotreated with auxin and BFA (E).
(H) Quantification of BFA body number per cell. Error bars indicate SE (n = 65 cells). Asterisks mark significant differences (Student’s t-test, * P < 0.05).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

8. Figure 7
The Endocytosis-Deficient abp1 Knock-Down Mutants Show Enhanced Cargo Internalization in Hyperosmotic Conditions.
(A–L) The abp1 conditional knock-down lines with decreased endocytosis (D, G, J) in comparison with wild-type (A) show reinstated internalization rates (E, F; H, I; K, L) as visualized by accumulation of PIN1 and PIN2 in BFA bodies.
(M) Quantification of BFA body size formed under deionized/MQ water treatment. Error bars indicate SE (n = 55). Asterisks mark significant differences (Student’s t-test, * P < 0.05).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions