1. Plasmodium falciparum STEVOR phosphorylation regulates host erythrocyte deformability enabling malaria parasite transmission
by Bernina Naissant, Florian Dupuy, Yoann Duffier, Audrey Lorthiois, Julien Duez, Judith Scholz, Pierre Buffet, Anais Merckx, Anna Bachmann, and Catherine Lavazec
Blood
Volume 127(24):e42-e53
June 16, 2016
©2016 by American Society of Hematology

2. The cytoplasmic domain of STEVOR contributes to infected erythrocyte stiffness.
The cytoplasmic domain of STEVOR contributes to infected erythrocyte stiffness. (A) Schematic representation of STEVOR recombinant proteins overexpressed in the Full-Ty1 and in the Trunc-Ty1 lines. Signal peptide (orange, SP), PEXEL/HT motif (black bar), N-terminal semiconserved region (light blue, C1), hypervariable region (dark blue, V), transmembrane domain (yellow, TM), C-terminal conserved cytoplasmic region (light blue, C2), and Ty1-tag (green star) are indicated. (B,E) Retention rates in microsphilters of stage III GIE (B) and asexual stages (E) from the control (light gray), the Full-Ty1, the Trunc-Ty1, and the Null lines cultivated with (+BSD, dark gray) or without (−BSD, medium gray) BSD. Error bars denote the standard error of the mean. Outliers are shown as open circles. Stars represent highly significant differences in retention rates (***P < .001; **P < .01). n, number of experiments; ns, nonsignificant differences in retention rates. (C,F) Western blot analysis of STEVOR expression in stage III GIE (C) and asexual stages (F) from the Full-Ty1 and the Trunc-Ty1 lines cultivated with (+BSD) or without (−BSD) BSD. Immunoblots were probed with a rabbit polyclonal antibody directed against STEVOR (PFC0025c) and with a rat polyclonal antibody directed against HSP70 to normalize expression. Quantitation of signal intensities was realized using Quantity One software (Bio-Rad). (D,G) Immunofluorescence analysis of stage III GIE (D) and asexual stages (G) from the Full-Ty1 and the Trunc-Ty1 lines. Infected erythrocytes were stained with anti-Ty1 antibodies followed by anti-mouse Alexa 488–conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 µm.
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology

3. STEVOR proteins interact with components of the ankyrin complex.
STEVOR proteins interact with components of the ankyrin complex. (A) Schematic representation of STEVOR recombinant protein overexpressed in the Full-myc line. Black bars represent recombinant his-tagged Stevor-Cter and N-terminal (Stevor-Nter) domains. (B) Immunoprecipitation of stage III V GIE lysates from the Full-myc and the Null lines with anti-myc and anti-GST antibodies. Immunoblot was probed with a rabbit polyclonal antibody directed against STEVOR (PFL2610w). (C) MS analysis of immunoprecipitates from the Full-myc and the Null line with anti-myc and anti-GST antibodies. *Average number of peptides for 4 independent experiments. (D) Pulldown assays of recombinant spectrin dimer (Sigma-Aldrich) (top and middle panel) or recombinant Band 3 (bottom panel) with recombinant his-tagged STEVOR C-terminal (Stevor-Cter) or N-terminal (Stevor-Nter) domains on nickel beads. Input represents 10% of recombinant proteins used for pulldowns. Immunoblot were probed with a rabbit anti-spectrin α antibody (top panel), a rabbit anti-spectrin β antibody (middle panel) or a rabbit anti-band 3 antibody (bottom panel).
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology

4. cAMP-mediated changes in GIE deformability are linked to STEVOR expression.
cAMP-mediated changes in GIE deformability are linked to STEVOR expression. (A-B) Retention in microsphilters of stages III (A) and stages V (B) GIE from the B10 (dark gray) and the A12 (medium gray) wild-type clones preincubated at 37°C during 30 minutes with (+cAMP) or without (–cAMP) 100 µM 8Br-cAMP. (C-D) Retention in microsphilters of stages III (C) and stages V (D) GIE from the control (dark gray) and the Null (medium gray) parasite line. GIEs were preincubated at 37°C 30 minutes with (+cAMP) or without (−cAMP) 100 µM 8Br-cAMP. Error bars denote the standard error of the mean. Outliers are shown as open circles. Stars represent significant differences in retention rates (***P < .001; **P < .01; *P < .05). ns, nonsignificant differences in retention rates.
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology

5. The cytoplasmic domain of STEVOR is phosphorylated by PKA
The cytoplasmic domain of STEVOR is phosphorylated by PKA. (A) Schematic representation of STEVOR proteins and alignment of flanking sequences of the 3 NetPhos-predicted serine phosphorylation sites for all members of the STEVOR family.
The cytoplasmic domain of STEVOR is phosphorylated by PKA. (A) Schematic representation of STEVOR proteins and alignment of flanking sequences of the 3 NetPhos-predicted serine phosphorylation sites for all members of the STEVOR family. Serine S123, S165, and S324 (in red) are numbered regarding the consensus sequence. (B) In vitro PKA phosphorylation assay for 3 peptides corresponding to a typical PKA phosphorylation substrate (Kemptide) and to the predicted-phosphorylation site on the C-terminal domain of STEVOR (Stevor S324). As a negative control, an alanine residue was substituted to the serine 324 (Stevor A324). The 3 peptides were incubated with a recombinant bovine PKA in the presence of 32[P]ATP, with or without PKA inhibitors H89 or KT5720. Phosphorylation signal (mean ± standard deviation [SD]) under different conditions is reported relative to kemptide phosphorylation from 3 independent experiments. a.u., arbitrary units. (C) In vitro PKA phosphorylation assay for the the Stevor S324 and the Stevor A324 peptides incubated with 40-hpi schizont extracts (control line) in the presence of 32[P]ATP, with or without H89. Phosphorylation signal (mean ± SD) under different conditions is reported relative to Stevor S324 peptide phosphorylation from 3 independent experiments. (D) Immunoprecipitation of 40-hpi schizont lysates from the Full-myc line with anti-myc, anti-GST, and anti-phospho-PKA antibodies. Immunoblot was probed with a rat antibody directed against STEVOR (PFC0025c). (E) Immunoprecipitation of stage III GIE lysates from the Full-Ty1 line with anti-GST and anti-phospho-PKA antibodies. Immunoblot was probed with a rabbit antibody directed against STEVOR (PFC0025c). (F) Immunoprecipitation of 40-hpi schizont lysates from the Full-myc line with anti-GST and anti-myc antibodies. Immunoblot was probed with a rabbit anti-phospho-PKA antibody.
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology

6. Immature GIE stiffness is dependent on STEVOR S324 phosphorylation.
Immature GIE stiffness is dependent on STEVOR S324 phosphorylation. (A) Schematic representation of STEVOR recombinant protein overexpressed in the Full-Ty1, the FullS324A-Ty1, and the FullS324E-Ty1 lines. Red bar, The serine 324 in the Full-Ty1 line is mutated in alanine 324 in the FullS324A-Ty1 line and in glutamate 324 in the FullS324E-Ty1 line, respectively. (B) Western blot analysis of STEVOR expression in stage III GIE from the Full-Ty1, the FullS324A-Ty1, the FullS324E-Ty1 and the Null lines cultivated with (+BSD) or without (−BSD) blasticidin. Immunoblots were probed with a rabbit antibody directed against STEVOR (PFC0025c) and with a rat antibody directed against HSP70 to normalize expression. Quantitation of signal intensities was realized using Quantity One software (Bio-Rad). (C) Immunofluorescence analysis of stage III GIE from the Full-Ty1, the FullS324A-Ty1, and the FullS324E-Ty1 lines. Infected erythrocytes were stained with anti-Ty1 antibodies followed by anti-mouse Alexa 488–conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 µm. (D) Retention rates in microsphilters of stage III GIE from the control (light gray), the Full-Ty1, the FullS324A-Ty1, the FullS324E-Ty1, and the Null lines cultivated with (+BSD, dark gray) or without (−BSD, medium gray) BSD. Error bars denote the standard error of the mean. Outliers are shown as open circles. Stars represent highly significant differences in retention rates (***P < .001). ns, nonsignificant differences in retention rates.
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology

7. The switch in deformability in mature stages occurs upon STEVOR S324 dephosphorylation.
The switch in deformability in mature stages occurs upon STEVOR S324 dephosphorylation. (A) Immunoprecipitation of stage III and stage V GIE lysates from the Full-Ty1 line with anti-Ty1, anti-GST, and anti-phospho-PKA antibodies. Immunoblots were probed with a rabbit antibody directed against STEVOR (PFC0025c). (B) Retention rates in microsphilters of stage V GIE from the control (light gray), the Full-Ty1 (medium gray), and the FullS324A-Ty1 (dark gray) lines. (C) Retention rates in microsphilters of stage V GIE from the control (light gray) and the FullS324A-Ty1 (dark gray) lines preincubated at 37°C 30 minutes with (+cAMP) or without (−cAMP) 100 µM 8Br-cAMP. (D) Retention rates in microsphilters of stage V GIE from the control (light gray), the Null (medium gray), and the FullS324A-Ty1 (dark gray) lines preincubated at 37°C 30 minutes with (+Sildenafil) or without (Untreated) 100 µM sildenafil citrate. Error bars denote the standard error of the mean. Outliers are shown as open circles. Stars represent significant differences in retention rates (***P < .001; **P < .01; *P < .05). ns, nonsignificant differences in retention rates.
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology

8. Model for STEVOR phosphorylation-mediated regulation of GIE deformability.
Model for STEVOR phosphorylation-mediated regulation of GIE deformability. (A) Regulation of deformability in immature and mature GIE from the control line. (B) Deformable phenotype in immature GIE from the FullS324-Ty1 line. (C) Stiff phenotype in sildenfil-treated mature GIE from the control line.
Bernina Naissant et al. Blood 2016;127:e42-e53
©2016 by American Society of Hematology