Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: 11-day-old plants expressing HIR2-mCherry fusion protein under the control of HIR2 promoter were imaged by scanning confocal microscopy. HIR2-mCherry signal was observed in the PM in lateral root cap cells (A-C) , in epidermal cells from the root differentiated zone (D-F) , in root hairs ( G ), in hypocotyl cells (H) and in cotyledon epidermal pavement cells (I) . ( A, D, G, H, I): Secant view of the cells. (B) and (E) : Tangential view in the middle of the root. (C) and (F) : XZ orthogonal projection from the Z-stack corresponding to A, B, and D, E, respectively. Scale bars represent 50 µm (A, B, D, E, H), 20 µm (C, F, I) or 10 µm (G).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Expressing, Control, Confocal Microscopy

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: Spinning disk microscopy analyses of root epidermal cells from pHIR2::HIR2-mCherry Arabidopsis transgenic lines co-expressing pREM1.2::YFP-REM1.2 (A) or pREM1.3::YFP-REM1.3 (B) . In majority, in (A) and (B), HIR2-mCherry PM nanodomains (left panels) and YFP-REM PM nanodomains (middle panel) do not co-localize (merge, right panel). Arabidopsis seedlings were grown for 11 days on MS/2 medium. Scale bars: 5 µm. (C) Quantification of the co-localization in PM nanodomains between HIR2-mCherry and YFP-REM1.2/YFP-REM1.3 proteins using Pearson’s correlation coefficient (PCC). Square regions of interest were analysed. As a control for random co-localization, the same regions where the YFP channel was rotated by 90° with respect to the mCherry channel were analysed (rot.). Scatter plots with indicated mean ± standard deviation. Letters indicate significant differences (p < 0.05, one-way ANOVA with post-hoc Tukey-Kramer multiple comparison test, n = 23, 28).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Microscopy, Transgenic Assay, Expressing, Control, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: (A) HIR2-mCherry expressed under the control of HIR2 promoter in Arabidopsis plantlets was immunopurified (IP) from root solubilized proteins using an anti-mCherry antibody coupled to magnetic beads, then, immunoblotting (IB) with an anti-mCherry antibody was performed on inputs and IP fractions. (B) Tandem mass spectrometry (MS/MS) spectrum corresponding to the fragmentation of a N-terminal peptide of HIR2-mCherry immunopurified fusion protein, with ion score Mascot 44 and sequence GNLFCCVLVK. ( C) Table of identified masses. Fixed modification (carbamidomethylation, 57.02146 Da ) and variable modification (myristoylation, 210.19837 Da) were considered. N-terminal and C-terminal fragments are indicated in red and blue characters, respectively (B, C). (D) Schematic representation of peptide fragmentation. Peptide characteristics are: Charge: +2, monoisotopic m/z: 710.41046 Da, MH + : 1419.81365 Da, retention time: 132.74 min.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Control, Magnetic Beads, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Modification

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: The subcellular localization of HIR2 point mutants fused to mCherry and expressed under the control of HIR2 promoter in N. benthamiana leaf epidermal cells (A-J) and in the root epidermal cells of Arabidopsis transgenic lines (K-T) was analyzed by scanning confocal microscopy. (A) and (K) : HIR2-mCherry. (B) and (L) : HIR2 G2A -mCherry. (C) and (M) : HIR2 C6A -mCherry. (D) and (N) : HIR2 C7A -mCherry. (E) and (O) : HIR2 C58A -mCherry. (F) and (P) : HIR2 C6A, C7A -mCherry. (G) and (Q) : HIR2 G2A, C6A, C7A -mCherry. (H) and (R) : HIR2 C6A, C58A -mCherry. (I) and (S) : HIR2 C7A, C58A -mCherry. (J) and (T) : HIR2 C6A, C7A, C58A -mCherry. Secant views are shown. Arabidopsis plantlets were grown 11 days on MS/2 medium. Scale bars represent 50 µm and 10 µm in panels (A-J) and (K-T), respectively. (U ) Schematic representation of HIR2 protein and the variants used in this work. HIR2 protein contains a N-terminal sequence composed of 7 amino acids, a SPFH domain (position 8 to 182) that was defined according to the Pfam website and a C-terminal domain (position 183 to 285). A sequence present in the very C-terminus of HIR2 displays homology to an oligomerization motif found in human Stomatin (underlined in red). Glycine 2 (G2) was shown to be myristoylated, cysteine 58 (C58) was proposed to be S-acylated and cysteines 6 and 7 (C6, C7) were predicted to be S-acylation sites according to GPS-Palm. Truncated variants of HIR2 and chimeric proteins are represented.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Control, Transgenic Assay, Confocal Microscopy, Sequencing

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: (A) Inhibition of S-acylation using 2-bromopalmitate (2-BP) disturbs HIR2 subcellular localization. The subcellular localization of HIR2-mCherry and GFP-LTi6b (control) expressed under the control of HIR2 and 35S promoters, respectively, was analyzed by scanning confocal microscopy in the roots of 11-day-old Arabidopsis seedlings treated with 2-BP or mock treated (mock). Scale bars represent 20 µm. (B) HIR2 is mono S-acylated and simultaneous mutations of C6 and C7 abolish HIR2 S-acylation. The S-acylation of WT HIR2, HIR2 C6A , HIR2 C7A , HIR2 G2A and HIR2 C6A, C7A proteins fused to the mCherry and expressed under the control of HIR2 promoter in Arabidopsis transgenic lines was assessed with a PEG-shift assay combined with anti-mCherry immunoblots. Following the cleavage of S-acyl groups with the thioester cleavage reagent hydroxylamine, a maleimide-functionalized polyethylene glycol (mPEG-Mal) was selectively coupled to free cysteines, which induced a mobility shift of 10 kDa for each S-acylation site. In the absence of hydroxylamine (-), no mass shift should be observed with a signal at ∼ 55 kDa, corresponding to the expected molecular weight of HIR2 and HIR2 mutated proteins fused to the mCherry. The mono S-acylation of WT HIR2, but also some HIR2 mutated proteins fused to the mCherry is indicated by the detection of a signal at ∼ 65 kDa in the presence of hydroxylamine (+). Non-S-acylated and mono S-acylated HIR2 and HIR2 mutated proteins fused to the mCherry are indicated by a star and an arrowhead, respectively. Arabidopsis seedlings were grown for 11 days on MS/2 medium.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Inhibition, Control, Confocal Microscopy, Transgenic Assay, Shift Assay, Western Blot, Mobility Shift, Molecular Weight

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: The S-acylation of WT HIR2, HIR2 C6A , HIR2 C7A , HIR2 G2A and HIR2 C6A, C7A proteins fused to the mCherry and expressed under the control of HIR2 promoter in Arabidopsis transgenic lines was assessed with a PEG-shift assay combined with anti-mCherry immunoblots. Following the cleavage of S-acyl groups with the thioester cleavage reagent hydroxylamine, a maleimide-functionalized polyethylene glycol (mPEG-Mal) was selectively coupled to free cysteines, which induced a mobility shift of 10 kDa for each S-acylation site. In the absence of hydroxylamine (-), no mass shift should be observed with a signal at ∼ 55 kDa, corresponding to the expected molecular weight of HIR2 and HIR2 mutated proteins fused to the mCherry. The mono S-acylation of WT HIR2, but also some HIR2 mutated proteins fused to the mCherry is indicated by the detection of a signal at ∼ 65 kDa in the presence of hydroxylamine (+). Arabidopsis seedlings were grown for 11 days on MS/2 medium. Non-S-acylated and mono S-acylated HIR2 and HIR2 mutated proteins fused to the mCherry are indicated by a star and an arrowhead, respectively. Three independent experiments corresponding to three biological replicates are shown. Experiment n°1 corresponds to the immunoblot presented in . The protein ladder transferred on the membranes is shown on the left of the immunoblots.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Control, Transgenic Assay, Shift Assay, Western Blot, Mobility Shift, Molecular Weight

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: Cell fractionation was performed on Nicotiana benthamiana leaves transiently expressing HIR2-mCherry (WT), HIR2 G2A -mCherry (G2A), HIR2 C6A -mCherry (C6A), HIR2 C7A -mCherry (C7A), HIR2 C6A, C7A -mCherry (C6A, C7A) and HIR2 G2A, C6A, C7A -mCherry (G2A, C6A, C7A) proteins under the control of HIR2 promoter. Cytosolic and microsomal protein fractions, noted CF and MF, respectively, were probed with an anti-mCherry antibody. After stripping, the transfer membranes were cut horizontally (dash lines) and re-probed with anti-H + -ATPase (upper part), and anti-cytosolic FBPase antibodies (bottom part). AHA and FBPase proteins were used as markers of membrane and cytosolic fractions, respectively. Two independent experiments corresponding to two biological replicates are shown. The protein ladder transferred on the membranes is shown on the left of the immunoblots.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Cell Fractionation, Expressing, Control, Stripping Membranes, Membrane, Western Blot

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: (A) The intracellularly-retained HIR2 C6A, C7A protein does not co-localize with the ER marker pHluorine-HDEL. HIR2 C6A, C7A -mCherry (left panel) and pHluorine-HDEL (middle panel) proteins were co-expressed in Nicotiana benthamiana leaf epidermal cells using HIR 2 and 35S promoters, respectively. Observation were performed by scanning confocal microscopy, maximum intensity projection of a Z-stack covering the cell surface is presented. The superimposed image for the two channels (merge) is shown in the right panel. (B) Close-up view of the region marked with a white rectangle in (A). (C) HIR2 C6A, C7A -mCherry protein is occasionally observed in round shape structures (left panel) that do not co-localize with the ER marker pHluorine-HDEL (middle panel) as seen in the merged images (right panel). (D – K) Contrary to the H + -ATPase AHA2, the trafficking of HIR2 to the PM does not require the COPII machinery. AHA2-mCherry (D-E) and HIR2-mCherry fusion proteins (F-G) are both localized in the PM when expressed in N. benthamiana leaf epidermal cells. (D) and (F) Secant views. (E) and (G) Maximum intensity projection of a Z-stack spanning from the cell surface to the position shown in (D) and (F), respectively. Note that AHA2 is also detected in some intracellular vesicles (E). (H) AHA2-mCherry is retained in the ER (left panel) when co-expressed with a dominant-negative (DN) form of Sar1 fused to the YFP (right panel) in N. benthamiana . (I) Close-up view of the region marked with a white rectangle in (H). (J) HIR2-mCherry is not retained in the ER and is mostly delivered to the PM (left panel) when co-expressed with Sar1DN-YFP (right panel) in N. benthamiana . In (H) and (J), a maximum intensity projection of a Z-stack covering the cell surface is presented. (K) Close-up view of the region marked with a white rectangle in (J). Scale bars represent 20 µm (A, D – H, J) and 5 µm (B, C, I, K).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Marker, Confocal Microscopy, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: Total Internal Reflection Fluorescence (TIRF) microscopy analysis was performed on the PM of Nicotiana benthamiana leaf epidermal cells (A-G) or Arabidopsis thaliana root epidermal cells (H-O) expressing HIR2 point mutants fused to mCherry and expressed under the control of HIR2 promoter. Note that the arrangement in nanodomains was only investigated for HIR2 mutated proteins that are still targeted/localized to the PM. (A) and (H) : HIR2-mCherry. (B) and (I) : HIR2 G2A -mCherry. (C) and (J) : HIR2 C6A -mCherry. (D) and (K) : HIR2 C7A -mCherry. (E) and (L) : HIR2 C58A -mCherry. (F) and (M) : HIR2 C6A, C58A -mCherry. (G) and (N) : HIR2 C7A, C58A -mCherry. (O) Coefficients of variance (CV) of fluorescence intensity were calculated from TIRF experiments performed as in (H) to (N) on Arabidopsis root epidermal cells. Error bars correspond to a confidence interval at 95%. Letters indicate significant differences (p-value < 0.05, one-way ANOVA with post-hoc Tukey multiple comparison test, n>25). Arabidopsis plantlets were grown 7 days on MS/2 medium. Scale bars represent 5 µm.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Fluorescence, Microscopy, Expressing, Control, Comparison

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: HIR2 C6A, C7A -mCherry and HIR2-EGFP proteins were co-expressed in N. benthamiana leaf epidermal cells using HIR2 and the 35S promoters, respectively. Observation were performed by scanning confocal microscopy. (A) HIR2-EGFP (left panel) and HIR2 C6A, C7A -mCherry (middle panel) are localized in the PM, where they form clusters that co-localize (merge, right panel). Upper panels: surface view at the top of the cell. Middle panels: secant view. Lower panels: maximum intensity projections of a Z-stack covering the cell surface. (B) Close-up view of the region marked with a white rectangle in (A). ( C) Signal intensity profiles of 8-bit images for EGFP channel (green line) and mCherry channel (magenta line) obtained from the linear ROIs (white line) in (B). Note the overlap of the signals illustrating the co-localization of both proteins within PM nanodomains. Scale bars: 50 µm (A) and 5 µm (B).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: The subcellular localization of HIR2-mEGFP (A, D) , HIR2 1-182 -mEGFP (B, E) and HIR2 1-254 -mEGFP (C, F) transiently expressed under the control of HIR2 promoter in N. benthamiana leaf epidermal cells was analyzed by scanning confocal microscopy. HIR2 1-182 and HIR2 1-254 are truncated proteins corresponding to the first 182 and 254 amino acids of HIR2, respectively. (A-C): Secant views. (D-F): Maximum intensity projections constructed from Z-stack of 25 focal planes spanning from the epidermis surface to the positions shown in (A-C). (G-I) : Secant views of root epidermal cells from Arabidopsis transgenic lines expressing HIR2-mCherry (G) , HIR2 1-182 -mCherry (H) and HIR2 1-254 -mEGFP (I) under the control of HIR2 promoter. Scanning confocal microscopy, scale bars represent 50 µm. Arabidopsis plantlets were grown 11 days on MS/2 medium. (J) Deletions in the C-terminal part of HIR2 disturb HIR2 oligomerization as revealed by co-immunopurifications. Immunopurifications (IP) using an anti-GFP antibody were performed on solubilized protein extracts from N. benthamiana leaves transiently co-expressing HIR2-mCherry with HIR2-mEGFP, HIR2 1-182 -mEGFP or HIR2 1-254 -mEGFP. Inputs and IP fractions were subjected to immunoblots (IB) with anti-GFP (top) and anti-mCherry antibodies (bottom).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Control, Confocal Microscopy, Construct, Transgenic Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: Immunopurifications (IP) using an anti-GFP antibody were performed on solubilized protein extracts from N. benthamiana leaves transiently co-expressing HIR2-mCherry with HIR2-mEGFP, HIR2 1-182 -mEGFP or HIR2 1-254 -mEGFP. Inputs and IP fractions were subjected to immunoblots (IB) with anti-GFP (top) and anti-mCherry antibodies (bottom). The expected molecular weights of HIR2-mCherry, HIR2-mEGFP, HIR2 1-182 -mEGFP and HIR2 1-254 -mEGFP proteins are 58.9 kDa, 59.3 kDa, 48.6 kDa and 56.3 kDa, respectively. Some signals can be observed for higher and lower molecular weights and probably correspond to not fully denatured and cleaved fusion proteins, respectively. Three independent experiments corresponding to three biological replicates are shown. Experiment n°1 corresponds to the immunoblots presented in . The protein ladder transferred on the membranes is shown on the left of the immunoblots.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: HIR2 C6A, C7A -mCherry protein was co-expressed with HIR2-mEGFP (A) , HIR2 1-182 -mEGFP (D) and HIR2 1-254 -mEGFP (G) in N. benthamiana leaf epidermal cells using HIR2 promoter. HIR2 1-182 and HIR2 1-254 are truncated proteins corresponding to the first 182 and 254 amino acids of HIR2, respectively. Observation were performed by scanning confocal microscopy. (A, D, G) : Upper panels: secant view; lower panels: maximum intensity projections constructed from Z-stack of 25 focal planes spanning from the epidermis surface to the positions shown in upper panels. ( B, E, H) Signal intensity profiles of 8-bit images for EGFP (full green line) and mCherry (dashed magenta line) obtained from the linear ROIs (white lines) shown in upper merge images of (A), (D) and (G). Note the almost complete overlap of the peaks in (B), shift of the peaks in (E) and broader peak corresponding to mCherry signal that partially co-localize with mEGFP peak in (H). ( C, F, I) XZ and YZ orthogonal projections from ROIs (white squares in lower merge images) of the Z-stacks corresponding to (A), (D), (G). The positions of the projections in XY is marked with white crosses in the insets. Scale bars represent 50 µm (A, D, G), and 10 µm (C, F, I).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Confocal Microscopy, Construct

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: The organization in the MP of HIR2-mEGFP (A, D) , HIR2 1-182 -mEGFP (B, E) and HIR2 1-254 -mEGFP (C, F) proteins expressed under the control of HIR2 promoter was analysed by TIRF microscopy. (A-C) : N. benthamiana leaf epidermal cells. (D-F) : Arabidopsis root epidermal cells. Arabidopsis seedlings were grown for 7 days on MS/2 medium. (G) Coefficients of variance (CV) of fluorescence intensity were calculated from TIRF experiments performed as in (D) to (F) on Arabidopsis root epidermal cells. Error bars correspond to a confidence interval of 95%. Letters indicate significant differences (p < 0.05, one-way ANOVA with post-hoc Tukey multiple comparison test, n>20). (H-M) Single-particle-tracking photoactivated localization microscopy (sptPALM) was performed on the PM of N. benthamiana leaf epidermal cells transiently expressing HIR2-mEOS2 and HIR2 1-182 -mEOS2 to determine the trajectories of each single protein. (H) Image reconstruction of several tens of single HIR2-mEOS2 and HIR2 1-182 -mEOS2 molecule trajectories indicated by different colors. (I) Vonoroï tessellation of HIR2-mEOS2 and HIR2 1-182 -mEOS2 molecule localization map from the same cells as in (H). The color code represents the local density of each molecule indicated by a black point. (J) Distribution of molecule instantaneous diffusion coefficient (D) (expressed in log (µm 2 /sec)) for HIR2-mEOS2 and HIR2 1-182 -mEOS2. (K) Quantification of the diffusion of HIR2-mEOS2 and HIR2 1-182 -mEOS2 molecules. (L) Mean square displacement (MSD) curves of HIR2-mEOS2 and HIR2 1-182 -mEOS2 molecules over the time. (M) Distribution of molecule local density for HIR2-mEOS2 and HIR2 1-182 -mEOS2. The subcellular localization of HIR2-mCherry (N) , HIR2 1-182 -mCherry (O) , HIR2 Nter -mEGFP (P) , HIR2 Nter-Cter -mCherry (Q) and HIR2 Cter -mCherry (R) transiently expressed under the control of HIR2 promoter in N. benthamiana leaf epidermal cells was analyzed by scanning confocal microscopy (secant views). HIR2 Nter and HIR2 Cter corresponds to the first 17 amino acids and the last 103 amino acids of HIR2, respectively, HIR2 Nter-Cter is a fusion of the first 17 amino acids of HIR2 to the last 103 amino acids. (S-V) TIRF microscopy analysis on the PM of N. benthamiana leaf epidermal cells expressing the same proteins than in (N) to (Q). (W) Coefficients of variance (CV) of fluorescence intensity were calculated from TIRF experiments performed as in (S-V). In this figure, error bars correspond to a confidence interval at 95% (G, W) and to standard deviation (K). Letters indicate significant differences (p-value < 0.05, one-way ANOVA with post-hoc Tukey multiple comparison test, n>20). In (K), **** represents p< 0.0001 in a t-test. Scale bars represent 5 µm (A-F ; S-V), 10 µm (H, N-R) and 0.102 µm (I).

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Control, Microscopy, Fluorescence, Comparison, Single-particle Tracking, Expressing, Diffusion-based Assay, Confocal Microscopy, Standard Deviation

Journal: bioRxiv

Article Title: Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

doi: 10.1101/2025.04.10.648198

Figure Lengend Snippet: (A-B) The production of apoplastic ROS was measured with a luminol-based assay in Nicotiana benthamiana leaf discs expressing the following proteins: HIR2-mCherry, HIR2 1-182 -mCherry and mCherry (negative control). (A) Leaf discs were treated or not with 100 nM flg22 and ROS accumulation was measured by following the emitted luminescence, every 1 min for 80 min. The emitted luminescence was normalized to the quantity of HIR2-mCherry, HIR2 1-182 -mCherry and mCherry, estimated via mCherry fluorescence, and expressed in relative light units (RLUs). Curves show mean values with discontinued lines representing SE. A representative experiment among three independent biological experiments is shown. (B) The average ROS accumulation at 17 min (maximum of the peak) was measured for three independent biological experiments performed as in (A). Error bars represent SE. Letters indicate statistically significant differences between the different proteins that were expressed (ANOVA, multiple comparisons post-hoc Tukey’s method at P-value<0.05). For each protein, 12 and 22 leaf discs for non-treated and flg22-treated conditions were analyzed, respectively.

Article Snippet: Following an ultracentrifugation at 100,000 × g for 1 h at 4 °C, solubilized proteins were recovered and mCherry fusion protein was immunopurified using RFP-Trap®_MA magnetic beads (Chromotek), following the instructions of the manufacturers, as previously described ( ).

Techniques: Expressing, Negative Control, Fluorescence

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Unique Aat amino acid sequences were detected using PSI-BLAST. These sequences were using to identify the strains that encoded them in the NCBI identical protein groups. As the aat genes were present on contigs of whole genome sequencing projects it was not possible to assess if a strain encoded a gene on the chromosome or plasmid. Instead contigs were used to identify complete the complete Aat system. This analysis does not include strains that might encode the aat genes or aap/cexE on a separate genomic element. However, a total of 827 complete Aat systems were identified in the same nucleotide accession. The positions of these genes were used to assess the organisation of the aat operon. From this assessment five different classes of aat operon organisation were defined.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Sequencing, Plasmid Preparation

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) Culture supernatant of ETEC H10407 and aat mutants harbouring pCfaD grown in LB supplemented with L-arabinose. Cells were removed from the culture supernatant and remaining protein precipitated with trichloroacetic acid. Protein samples were separated by tris-tricine SDS-PAGE and stained with Coomassie or transferred to nitrocellulose for western blotting with polyclonal antibodies against CexE. (B) Whole cell lysates of the aat mutants and parental strain separated by tris-tricine SDS-PAGE. (C) Whole cell lysates of ETEC H10407 grown in LB with or without azide. (D) Whole cell lysates of EAEC 042, aap, aatD and aatD complemented strains grown in DMEM high glucose. The positions of uCexE, mCexE, uAap, mAap, and preCexE are indicated as appropriate. CexE and Aap were detected by western blotting (α-CexE and α-Aap, respectively) and RNA polymerase (α-RNAP) was included as a loading control where appropriate.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: SDS Page, Staining, Western Blot, Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: His tagged CexE was isolated from cexE or cexE aatD double mutants harbouring pACYC- cexE -6His grown in the presence or absence of 17-ODYA and separated by SDS-PAGE. An azide linked Alexa Fluor 488 was conjugated to the alkyne moiety present in 17-ODYA by CuAAC. The incorporation of 17-ODYA into the target protein was detected by fluorescence (green bands) and the image was overlaid on the image of the SDS-PAGE gel. A known lipoprotein YraP was used as a positive control.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Isolation, SDS Page, Fluorescence, Positive Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: The membrane fraction of ETEC H10407 and aat mutant harbouring pCfaD were separated by SDS-PAGE. Transferred to nitrocellulose and probed for the cytoplasmic protein RNAP, lipoprotein NlpE and CexE using protein specific antibodies.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Membrane, Mutagenesis, SDS Page

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: E. coli BL21(DE3) was transformed with pET26b, or pET26b- cexE and pACYCDuet-1 or pACYC- aatD . Cultures were grown in LB and protein production was stimulated with IPTG. Whole cell lysate samples were taken and separated by tris-tricine SDS-PAGE. CexE was detected using a polyclonal antibody. RNAP was used as a loading control. The position of mCexE and uCexE are indicated.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Transformation Assay, SDS Page, Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) AatD from EAEC 042 and ETEC H10407 were aligned with Lnt from MG1655. AatD sequences identified by PSI-BLAST were aligned and used to create a Weblogo. The catalytic residues of the C-N hydrolase family are highlighted. (B) The structure of Lnt compared to the predicted structure of ETEC H10407 AatD. The catalytic residues of Lnt and AatD are highlighted in orange. (C) Magnified view of the catalytic site of Lnt with the predicted structure of AatD superimposed. (D) Residues are numbered as they appear in Lnt. E. coli BL21(DE3) was producing CexE in the presence of each of the four single site substitution mutants of AatD. CexE was detected using a polyclonal antibody.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques:

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) SignalP result of CexE sequence from ETEC H10407 (E3PPH2_ECOH1) (B) Weblogo of the 5 N-terminal residues of Aap and CexE sequences post Sec signal sequence cleavage. (C) ETEC H10407 cexE mutants transformed with pACYC184 (Δ cexE ) or pACYC- cexE -6His with either the wild-type sequence (WT cexE ) or one of the first five amino acids mutated to glutamic acid. CexE was detected by polyclonal antibodies and RNAP was used as a loading control. The percentage of mCexE was determined from 3 biological replicates.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Sequencing, Transformation Assay, Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Aap and CexE sequences were aligned using ClustalO. The alignment was then placed into WebLogo.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques:

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) CexE-6His was isolated from a cexE mutant and a cexE aatD double mutant and subjected to LC-MS/MS. CexE and pro-CexE were trypsinated and separated by HPLC. (B) The indicated peak was subjected to MS/MS to identify the amino acid sequence.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Isolation, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Heterologous acylation by AatD plasmid encoding mCherry with the CexE signal sequence followed by none, one (G), two (GG) or three (GGG) glycine residues post signal sequence was produced in an ETEC H10407 cexE mutant grown in the presence of 17-ODYA. The mCherry proteins were isolated using a C-terminal 8 His tag and azide linked Cy5 was incorporated into mCherry proteins using CuAAC. The acylation of mCherry was detected using fluorescence.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Plasmid Preparation, Sequencing, Produced, Mutagenesis, Isolation, Fluorescence

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: MCF-7 cell lines expressing biosensors that detect indicated PI species were treated with 250 nM doxorubicin for 24 hours, washed, and plated on untreated mCherry-MCF-7 “prey” cells. DOXO-NT cultures were imaged over days 3–8. Time course live-cell imaging of senescent MCF-7 cells that express ( A ) PLCD1-GFP marking PI(4,5)P2; ( B ) P4M-SidMx2-GFP marking PI(4)P; ( C ) 2xFYVE-GFP marking PI(3)P; ( D ) TAPP1-GFP marking PI(3,4)P2; ( E ) BTK-GFP marking PI(3,4,5)P3; ( F ) PLCD1(R40L)-GFP mutant that does not bind PI species (Negative Ctrl), throughout the entire process of engulfing mCherry-MCF-7 cells. Scale bar = 100 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: Expressing, Live Cell Imaging, Mutagenesis

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: (A ) Axial slices along the Z-axis and volume view reconstructions of a senescent MCF-7 cell expressing 2xFYVE-GFP and an mCherry expressing MCF-7 prey cell in a DOXO-NT culture. Top panel scale bar = 10 μm; bottom panel scale bar = 1 μm. ( B , C ) Representative image of a 2xFYVE-GFP cell engulfing a NIR-MCF-7 cell. Pixel intensity was determined across the distance of the yellow line and plotted in ( C ) for 5 individual cells. Scale bar = 60 μm. Underlying data can be found at . ( D ) Five 2xFYVE-GFP-MCF-7 predator cells were followed during engulfment of pHrodo red stained prey cells, and 2xFYVE-GFP intensity was measured at cytoplasm, membrane, and ring (as depicted in ) and pHrodo intensity was measured (right). The right graph shows the ratio of 2xFYVE-GFP intensity of the ring:cytoplasm (light green) over multiple hours surrounding pHrodo intensity increase for 5 cells. Error bars represent SEM. Underlying data can be found at . ( D ) Representative images of one 2xFYVE-GFP-MCF-7 predator cell engulfing pHrodo-labeled MCF-7 prey used for Fig 4D (right). ROIs used for quantification are shown in top panel. Scale bar = 100 μm. ( E ) Time course of a 2xFYVE-GFP-MCF-7 cell engulfing an MCF-7-mCherry prey cell. Dashed arrows indicate predator/prey contact; closed arrows indicate the overtopped prey cell with 2xFYVE concentration; open arrows indicate the prey cell no longer overtopped mCherry prey cell. Scale bar = 50 μm. ( F ) Axial slices along the Z-axis and volume view reconstructions of senescent 2xFYVE-GFP-MCF-7 predator cells engulfing mCherry-MCF-7 prey cells. Leftmost image shows the bottom axial slice. Closed arrows indicate 2xFYVE-GFP concentration at the prey cell. Scale bar = 10 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: Expressing, Staining, Labeling, Concentration Assay

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: (A ) Time course live-cell imaging of a senescent MCF-7 cell that expresses PLCD1-mCherry and P4M-SidMx2-GFP, throughout the entire process of engulfing NIR-MCF-7 prey cells in a DOXO-NT culture. Closed arrows indicate examples PLCD1-mCherry and P4M-SidMx2-GFP colocalization during engulfment. ( B ) Time course live-cell imaging of a senescent MCF-7 cell that expresses PLCD1-mCherry and 2xFYVE-GFP, throughout the entire process of engulfing a NIR-MCF-7 cell. Closed arrows indicate localization of PLCD1-GFP, but not 2xFYVE-mCherry, at mid stage engulfment. Open arrows indicate 2xFYVE-mCherry localization, but not PLCD1-GFP, at late-stage engulfment. Scale bar = 100 μm for A+B. ( C ) Volume view reconstruction of a senescent MCF-7 cell expressing PLCD1-GFP and P4M-SidMx2-mCherry that is engulfing 3 NIR-MCF-7 cells. Middle panels show separate color channels; bottom panel shows axial planes from bottom to top. Closed arrows indicate examples PLCD1-mCherry and P4M-SidMx2-GFP colocalization during engulfment, scale bar = 10 μm. ( D ) Volume view reconstruction of a senescent MCF-7 cell expressing PLCD1-mCherry and 2xFYVE-GFP, which is engulfing 2 NIR-MCF-7 cells. Middle panels show separate color channels; bottom panels show axial planes from bottom to top. Closed arrows indicate localization of PLCD1-mCherry, but not 2xFYVE-GFP, at mid stage engulfment. Open arrows indicate 2xFYVE-mCherry localization, but not PLCD1-GFP, at an internalized cell, scale bar = 10 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: Live Cell Imaging, Expressing

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: ( A ) CRISPR-Cas9 single-cell clone PIK3C2B knockouts of MCF-7 were screened by immunoblot. Clones indicated by asterisk were chosen for further testing. ( B ) Predator cell engulfment rates (left) and confluency as a measure of viability (right) for senescent MCF-7 parental cells and 3 PIK3C2B knockout clones were determined by time course imaging in a DOXO-NT culture. Underlying data can be found at . ( C ) Time course live-cell imaging of a senescent MCF-7 cell expressing 2xFYVE-mCherry and PIK3C2B-GFP throughout the entire process of engulfing an NIR-MCF-7 cell, scale bar = 100 μm. ( D ) Left: Merged (top) and separate (middle, lower) color channels of axial planes from bottom to top of a senescent MCF-7 cell expressing 2xFYVE-mCherry and PIK3C2B-GFP, engulfing NIR-MCF-7 cells. Right, volume view reconstruction of the same image, scale bar = 10 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: CRISPR, Western Blot, Clone Assay, Knock-Out, Imaging, Live Cell Imaging, Expressing