1. Volume 4, Issue 2, Pages 264-278 (March 2011)
AtFH8 Is Involved in Root Development under Effect of Low-Dose Latrunculin B in Dividing Cells 
Xue Xiu-Hua , Guo Chun-Qing , Du Fei , Lu Quan-Long , Zhang Chuan-Mao , Ren Hai-Yun  
Molecular Plant 
Volume 4, Issue 2, Pages (March 2011)
DOI: /mp/ssq085
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 AtFH8(FH1FH2) Can Form Dimers.
(A) FPLC analysis of AtFH8(FH1FH2). Standard curve is the elution profile of gel filtration standard. The numbers above the standard curve indicate the molecular weight of respective elution peak. AtFH8 curve is the elution profile of AtFH8(FH1FH2). An elution peak corresponding to about 158 kDa is observed and then applied to SDS–PAGE to verify that it is AtFH8(FH1FH2). Inset is the result of SDS–PAGE.
(B) Sedimentation–velocity analytical centrifugation of AtFH8(FH1FH2). AtFH8(FH1FH2) sedimented at 2.7 and 4.6 S. Conversion of the parameters give an apparent mass of 58 and 117 kDa, which represented monomers and dimers, respectively.
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Both AtFH8(FH1FH2) and AtFH8(FH2) Can Bundle Actin Filaments.
Actin bundles examined by fluorescence microscopy (A–C) and electron microscopy (D–F). (A) and (D) Control actin filaments. (B) and (E) Actin bundles generated by AtFH8(FH1FH2). (C) and (F) Actin bundles generated by AtFH8(FH2). For (A–C), bar = 20 μm. For (D–F), bar  = 100 nm.
(G) AtFH8(FH1FH2) and AtFH8(FH2) bundle actin filaments in a concentration-dependent manner. The left panel shows the control sample and samples containing AtFH8(FH1FH2) and the right panel shows the samples containing AtFH8(FH2).
(H) The bundling activity of AtFH8(FH2) is weaker than AtFH8(FH1FH2). The percentage of pellet obtained by densitometry analysis of the SDS–PAGE gel in G is plotted against AtFH8(FH1FH2) or AtFH8(FH2) concentration. White column, AtFH8(FH1FH2); gray column, AtFH8(FH2).
(I) AtFH8(FH2) binds to actin filament side weaker than AtFH8(FH1FH2). Lanes 1–5 are high-speed cosedimentation samples with 200 nM AtFH8(FH2) and 0, 1, 2, 3, and 4 μM pre-polymerized actin filaments.
(J) Plot of relative gray scale of AtFH8(FH2) in Coomassie blue-stained SDS–PAGE gel against actin concentrations as described in Figure 2I. The gray scale of AtFH8(FH2) in pellets is indicated by black rhombuses and the supernatants are indicated by black squares; the intersection of the two indicates that 50% of AtFH8(FH2) binds to actin filaments. A representative of three independent experiments is shown.
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Actin Can Polymerize into Stellar Structures in the Presence of AtFH8(FH1FH2).
Actin was polymerized in the presence of 400 nM AtFH8(FH1FH2) and AtFH8(FH2) for 1 h. The samples were phalloidin stained and observed under fluorescence microscope and electron microscope.
(A–H) Actin structures observed by fluorescence microscopy and (I, J) electron microscopy. (A) Polymerized actin filaments. (E) Polymerized actin–profilin complex. (B) and (F) Actin bundles generated in the absence of profilin (B) and presence of profilin (F). (C, G) The typical stellar structures generated in the absence of profilin (C) and presence of profilin (G). (D) and (H) are the actin bundles or actin filaments generated by AtFH8(FH2) in the absence of profilin (D) or presence of profilin (H). (I) Actin filaments observed under electron microscope. (J) Actin bundles and stellar structures generated by AtFH8(FH1FH2).
(K) The fine structure of actin bundle generated by AtFH8(FH1FH2). Inset is the higher magnification of the indicated area.
(L) The samples containing the indicated proteins were incubated at room temperature for 1 h and resolved by SDS–PAGE. Supernatants and pellets are as indicated. From left lane to right lane, 0, 200, 400, 600, and 800 nM AtFH8(FH1FH2) or AtFH8(FH2) were added. +p, in the presence of profilin; –p, in the absence of profilin.
(M) Profilin weakens but does not block the ability of AtFH8(FH1FH2) to induce actin bundle formation during actin assembly while profilin can abolish the corresponding ability of AtFH8(FH2). The percentage of pellet obtained by densitometry analysis of the SDS–PAGE gel in (L) is plotted against AtFH8(FH1FH2) or AtFH8(FH2) concentration. Gray column with solidus, actin polymerized in the presence of AtFH8(FH1FH2); gray column, actin polymerized in the presence of AtFH8(FH2); white column with solidus, actin polymerized in the presence of AtFH8(FH1FH2) and profilin; white column, actin polymerized in the presence of AtFH8(FH2) and profilin. In (A-H), bar = 100 μm. In (J), bar = 2 μm. In (I) and (K), bar = 500 nm. In inset of (K), bar = 10 nm.
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
AtFH8 Localizes to the Nuclear Envelope and the Newly Formed Cell Wall in Transgenic Arabidopsis Root Cells.
Wild-type Arabidopsis and AtFH8 T–DNA insertion mutants were respectively transformed by inducible vectors carrying AtFH8–GFP chimeras and the PAtFH8:AtFH8–GFP vector. Gene expression was inducted by 100 nM estrogen and observed by confocal microscopy.
(A) Schematic domain composition of AtFH8–GFP fusion constructs. PRR, proline rich region; TM, transmembrane domain; FH1, formin homology 1 domain; FH2, formin homology 2 domain; GFP, green fluorescent protein. Numbers under each construct indicate the amino acids that it contains.
(B, C) The localization of full-length AtFH8 in wild-type Arabidopsis.
(D, E) The localization of the truncated AtFH8 lacking the N-terminus in wild-type Arabidopsis.
(F, G) The localization of the N-terminus of AtFH8 in wild-type Arabidopsis.
(H, I) The immunolocalization of AtFH8 in PAtFH8:AtFH8–GFP transgenic lines. The nuclear envelope (H) and the newly formed cell wall (I) immunolocalization of AtFH8 in the root tip cells. The solid arrowheads indicate the nuclear envelope, the arrows indicate the newly formed cell wall, and the open arrowheads indicate the nuclear sub-cellular localization. Bar = 10 μm.
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 The Histochemical Localization of AtFH8.
The histochemical localization of AtFH8 in the transgenic line expressing the promoter of AtFH8 fused with the β-glucuronidase (GUS) (PAtFH8:GUS). Seedlings stained for PAtFH8:GUS expression in 5-day-old wild-type Arabidopsis (A) and PAtFH8:GUS transgenic plants (B–F). (C) is the magnification of the black frame of (B), (E) is the magnification of the black frame of (D), and (F) is the magnification of the red frame of (D). In (A), (B), and (D), bar = 25 μm. In (C) and (E, F), bar = 100 μm.
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

7. Figure 6 AtFH8 Participates in Arabidopsis Root Development.
(A) Digital camera images of the primary root growth and lateral root initiation in 8-day-old wild-type, atfh8-1, and the PAtFH8:AtFH8–GFP complemented transgenic line treated with or without LatB. The primary root growth was slower and lateral root initiation was inhibited in atfh8-1 and the PAtFH8:AtFH8–GFP transgenic line complemented the phenotype.
(B, C) The primary root length and the lateral root numbers of 8-day-old wild-type, atfh8-1, and PAtFH8:AtFH8–GFP treated with 0, 30, 40, and 50 nM of LatB, showing significantly (*P < 0.05) reduced primary root length and lateral root numbers of atfh8-1 seedlings in a LatB concentration-dependent manner. Mean values ± 95% confidence intervals are given for each genotype (n = 30). The loss of AtFH8 decreases the dividing cells of the root tip treatment with 40 nM LatB (D, E). The length of the epidermal cells of maturation zone in wild-type, atfh8-1, and PAtFH8:AtFH8-GFP (D). The numbers of dividing cells in root tips of wild-type, atfh8, and PAtFH8:AtFH8–GFP in the presence and absence of LatB at 3, 4, 5, 6, and 7 d after sowing (E). Solid diamond, wild-type without LatB; solid rectangle, atfh8-1 without LatB; the solid triangle, PAtFH8:AtFH8–GFP without LatB; the diamond, wild-type treatment with LatB; rectangle, atfh8-1 treatment with LatB; triangle, PAtFH8:AtFH8–GFP treatment with LatB. The experiment was performed independently four times, with similar results. Error bars indicate SE (n = 30).
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
F-Actin Organization in the Root of 8-Day-Old Wild-Type and atfh8-1 Seedlings Marked by 35S:GFP–fABD2 Treated With or Without 40 nM LatB.
(A, E, I, M) Actin filaments in wild-type root without LatB. (A) The root apical meritem. (E) The cortex of the root vasculature. (I) The vasculature of the root. (M) Lateral root primordia.
(B, F, J, N) Actin filaments in atfh8-1 root without LatB. (B) The root apical meristem. (F) The cortex of the root vasculature. (J) The vasculature of the root. (N) Lateral root primordia.
(C, G, K, O) Actin filaments in wild-type root treated with 40 nM LatB. (C) The root apical meristem. (G) The cortex of the root vasculature. (K) The vasculature of the root. (O) Lateral root primordia.
(D, H, L, P) Actin filaments in atfh8-1 root treated with 40 nM LatB. (D) The root apical meristem. (H) The cortex of the root vasculature. (L) The vasculature of the root. (P) Lateral root primordia. The open arrowheads, actin bundles in pericycle of the root; the solid arrowheads, F-actin in lateral root primordia cells. In (A–L), bar = 20 μm, and in (M–P), bar = 10 μm.
Molecular Plant 2011 4, DOI: ( /mp/ssq085)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions