EMBO/soda-nel huggingface dataset:
The E3 ligase Highwire promotes synaptic transmission by targeting the NAD synthesizing enzyme Nmnat (2019)
Figure 2

Figure 2. Excess presynaptic dNmnat drives defects in evoked neurotransmission at the NMJ in Hiw mutants, but has no role in synaptic overgrowth. (A) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing RNAis to knockdown either dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [#29402] ) or Wnd (hiwND8;Elav-Gal4 > UAS-Wnd-RNAi [#25396]). (B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=39, F=14.02, p<0.0001. WT vs hiw p = 0.0012 (**), WT vs hiw > Wnd-RNAi p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p = 0.9880 (NS)]. (C) Quantification of quantal content (±SEM). [WT n = 9 cells, hiw n = 9 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F= 2.508, p<0.0001. WT vs hiw p = 0.0361 (*), WT vs hiw > Wnd-RNAi p = 0.0001 (***), WT vs hiw > dNmnat-RNAi p = 0.9889 (NS)]. (D) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels A-C. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). (E) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 23, hiw n = 21 , hiw > Wnd-RNAi n= 16, hiw > dNmnat-RNAi n=19. 1-way ANOVA w/Tukey's multiple comparisons, DF=78, F=169.1, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Wnd-RNAi p = 0.9988 (NS), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.9695 (NS)].
The E3 ligase Highwire promotes synaptic transmission by targeting the NAD synthesizing enzyme Nmnat (2019)
Figure 3

Figure 3. hiw mutants exhibit dNmnat-dependent defects in release probability (Pr) at the larval NMJ. (A) Representative images of immunostained larval NMJs and active zones from WT, hiw mutant, and hiw mutant driving an RNAi against dNmnat [#29402]. In the top 3 panels, greyscale images of entire terminal were acquired by staining for nerve marker HRP. Presynaptic Brp puncta (green) and postsynaptic dGluRIII clusters (magenta) were imaged to quantify N in each genotype. (B) Quantification of NMJ synaptic terminal surface area in each genotype. [WT n = 14, hiw n = 12 , hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=22.427, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.2907 (NS)]. (C) Quantification of BRP puncta per NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.854, p = 0.0711. WT vs hiw p = 0.0573 (NS), WT vs hiw > dNmnat-RNAi p = 0.4529 (NS), hiw vs hiw > dNmnat-RNAi p = 0.5030 (NS)]. (D) Quantification of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.093, p = 0.4716. WT vs hiw p = 0.2168 (NS), WT vs hiw > dNmnat-RNAi p = 0.1882 (NS), hiw vs hiw > dNmnat-RNAi p = 0.9966 (NS)]. (E) Representative electrophysiological traces from a synaptic facilitation experiment in which trains of 5 pulses were used to evoke consecutive action potentials at 100 ms ISI. Genotypes for these experiments were: WT (elav>UAS-RFP), hiw mutant (hiw; elav>UAS-RFP), or hiw depleted of dNmnat (hiw; elav>dNmnat-RNAi). (F) Quantification of the facilitation index, in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse. [100 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 7.546, p =0.0023. WT vs hiw p =0.0020 (**), hiw vs hiw> dNmnat-RNAi p= 0.0229 (*), WT vs hiw> dNmnat-RNAi p= 0.5141 (NS). 50 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 5.587, p =0.0089. WT vs hiw p =0.0129 (*), hiw vs hiw> dNmnat-RNAi p= 0.0217 (*), WT vs hiw> dNmnat-RNAi p= 0.9546 (NS).]
The E3 ligase Highwire promotes synaptic transmission by targeting the NAD synthesizing enzyme Nmnat (2019)
Figure 4

Figure 4. dNmnat levels are increased near active zones in hiw mutant NMJ synaptic terminals. (A) Representative images of NMJ synaptic terminals in control (DVGlut-Gal4 > UAS-HA-dNmnat) and hiw mutant (hiwND9,DVGlut-Gal4 > UAS-HA-dNmnat) larvae. NMJs at larval muscle 4 were identified using HRP (blue in top panel) immunostaining. Insets highlight active zones within single boutons, which were identified by staining for the presynaptic active zone protein Brp (red). An anti-HA antibody was used to stain for an HA-tagged dNmnat (green) transgene to measure protein levels in control and hiw mutant active zones. (B) Quantification of HA-dNmnat levels across the entire NMJ synaptic terminal, normalized to total terminal area and presented as fold change over WT. [WT > HA-dNmnat n = 10 terminals, hiw > HA-dNmnat n = 10 terminals. Unpaired T-test, DF = 18, p = 0.0028 (**)]. (C) Quantification of HA-dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones (herein referred to as "terminals") versus active zones (AZs) only. [WT n = 10 terminals, hiw n = 9 terminals, each parsed into terminal minus AZs and AZs only. 2-way ANOVA with Tukey's multiple comparisons, DF = 34, p = 0.0005 for row factor (terminal versus AZs) and p < 0.0001 for column factor (WT vs hiw). WT terminal vs hiw terminal p = 0.9830 (NS). Hiw terminal vs hiw AZs p < 0.0001 (****). WT AZs vs hiw AZs p < 0.0001 (****).]
The E3 ligase Highwire promotes synaptic transmission by targeting the NAD synthesizing enzyme Nmnat (2019)
Figure 5

Figure 5. Excess dNmnat in a hiw mutant disrupts the ultrastructure of active zone T-bars.

(A) Representative electron micrographs of entire boutons in wild type, hiw;wnd, and hiw;wnd, Elav-GAL4 > dNmnat RNAi NMJs. (B) High magnification electron micrographs showing representative "T-bar" structures at active zones in NMJs of the indicated genotypes. Red arrows denote the beginning and end of the T-bar top, demonstrating changes in T-bar width in the genotypes tested. As illustrated, a significant reduction in T-bar size is observed in hiw;wnd, and this defect is suppressed in hiw;wnd, Elav-GAL4 > dNmnat RNAi. (C) Quantification of mean T-bar width [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=13.829, p < 0.0001. WT vs hiw;wnd p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.9318 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)]. (D) Analysis of cumulative frequency distribution of T-bar width shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.5636, p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1815, p = 0.1240 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.4121, p < 0.0001 (****)]. (E) Quantification of mean T-bar height [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=10.472, p < 0.0001. WT vs hiw;wnd p = 0.0008 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.6694 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)] (F) Analysis of cumulative frequency distribution of T-bar height shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.3343, p = 0.001 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1012, p = 0.7748 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.3484, p = 0.0008 (***)].
The E3 ligase Highwire promotes synaptic transmission by targeting the NAD synthesizing enzyme Nmnat (2019)
Figure 6

Figure 6. The NAD+ synthetic function of dNmnat is necessary to decrease evoked release. (A) Schematic of the NAD+ synthesis pathway in Drosophila. Red text indicates enzymes tested within the context of synaptic release at the larval NMJ. (B) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), or one of the catalytic dNmnat mutants (DVGlut-Gal4 > UAS-WR-dNmnat, DVGlut-Gal4 > UAS-H30A-dNmnat). (C) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=0.8596, p = 0.4383. UAS-RFP vs UAS-WR-dNmnat p = 0.8810 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9997 (NS)]. (D) Quantification of quantal content (±SEM) [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=1.154, p = 0.3365. UAS-RFP vs UAS-WR-dNmnat p = 0.8696 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9697 (NS)]. (E) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing an RNAi to knockdown NAD+syn (hiwND8;Elav-Gal4 > UAS-NAD+syn-RNAi). (F) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F=14.02, p<0.0001. WT vs hiw p <0.0001 (****), WT vs hiw > NADsyn-RNAi p = 0.5147 (NS)]. (G) Quantification of quantal content. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F= 6.635, p = 0.0047. WT vs hiw p = 0.0063 (**), WT vs hiw > NADsyn-RNAi p = 0.8942 (NS)]. (H) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels E-G. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). (I) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 25, hiw n = 21, hiw > NADsyn-RNAi n=24. 1-way ANOVA w/Tukey's multiple comparisons, DF=67, F=141.3, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Nadysn-RNAi p > 0.0001 (****)].
Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence (2020)
Figure 2

Figure 2. Bacterial growth and ndhC and ndhF expression in Newman WT strain and isogenic ndh mutants.

Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated.

(A) Bacterial growth was measured by optical density at 600 nm. Data are representative of five independent experiments.

(B) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth. Data are expressed as average ± SEM of three independent experiments. *** Denotes p < 0.001 compared to ndhC expression in WT strain grown in MHB-ca medium via two-way ANOVA with Tukey's posttest.
Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence (2020)
Figure 5

Figure 5. ndhC and ndhF expression, biofilm formation and redox balance of ndh mutants under biofilm growth conditions.

(A) PndhC and PndhF YFP reporter constructs were used to follow gene expression under the biofilm growth conditions. Data collected after 24 h of static incubation was normalized by optical density at 600 nm. Data are expressed as average ± SEM of three independent experiments. No significant differences were found when ndhC expression in WT strain was compared via one-way ANOVA with Dunnett's posttest.

(B) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.

(C) MKH2/MK ratios were measured in cells grown under the same conditions as for biofilm formation. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.

(D) NADH/NAD+ ratios were measured in cells grown under the same conditions as for biofilm formation. 100% corresponds to a NADH/NAD+ ratio of 0.09. Data are expressed as average ± SD of four independent experiments performed by duplicates. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.
Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence (2020)
Figure 7

Figure 7. Global regulators connecting virulence and fatty acid homeostasis in the ndh mutants.

(A) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of four independent experiments. *** Denotes p < 0.001, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.

(B) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. ** Denotes p < 0.005, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.

(C) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB-ca medium, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.

(D) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.

(E) Hemolytic activity of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of four independent experiments. ** Denotes p < 0.005 compared to ∆ndhC strain with the empty plasmid via Student's t-test.

(F) Biofilm formation of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown under biofilm producing conditions. Data are expressed as average ± SD of three independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to ∆ndhC strain with the empty plasmid via Student's t-test.
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 1

Figure 1: The calcium-binding protein caldendrin interacts with a 49 amino-acid region containing the first IQ motif of myosin Va in a Ca2+-dependent manner.

A Schematic showing overall domain organization of the caldendrin protein, including an unstructured N-terminal part, and four EF-hand binding motifs in the C-terminal part. Green boxes indicate functional EF-hands, blue indicates cryptic EF-hand. The dashed line indicates where the Nt and Ct constructs were separated. Co-immunoprecipitation experiments from extracts of HEK293T cells, co-expressing full-length GFP-myosin Va (myoVa fl) and indicated caldendrin-tagRFP (cald) constructs, show that caldendrin interacts with myoVa in a Ca2+-dependent manner via its N-terminus (Nt), and not via its C-terminus (Ct). Tubulin was used as a loading control for the input. MW = molecular weight marker.

B Pull down assay indicates that recombinant purified GFP-myoVa and untagged caldendrin (cald wt) directly bind to each other in a Ca2+-dependent manner. Note that there is no binding observed for a Ca2+-binding mutant of caldendrin (cald mut / Left panel). Right panel: neither wt nor mut caldendrin bind to GFP-coupled beads.

C-E Mapping of the binding region between myoVa and caldendrin using co-immunoprecipitation of cald-tagRFP and GFP-myoVa fragments expressed in HEK293T cells. Arrows in D indicate the size of the respective myosin fragment.

F Summary of the results from C - E. Caldendrin binds specifically to a fragment containing IQ-motif 1 (amino acids 742-791), but not to any other region of myoVa, in a Ca2+-dependent manner.
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 2

Figure 2: Binding of caldendrin and calmodulin to a 49 amino-acid fragment of the myosin Va protein including the first IQ motif is mutually exclusive.

A Schematic of caldendrin and calmodulin competition assay. A construct containing the GFP-tagged caldendrin binding peptide (aa 742 - 791) of myoVa (GFP-myoVa IQ1) was purified via GFP-trap beads from HEK293T cell lysate enriched with bacterially produced calmodulin (CaM). The CaM-associated construct (Input) was then incubated with lysate from HEK293T cells expressing either cald-tagRFP or tagRFP only.

B Western blot analysis of competition co-immunoprecipitation experiment in HEK293T cells. In the presence of Ca2+, caldendrin binds strongly to the IQ1 fragment (upper arrow), while tagRFP by itself does not bind (lower arrow). Detection using a calmodulin antibody indicated that Ca2+-dependent binding of caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control (tagRFP only). MW = molecular weight marker.

C Quantification of the normalized relative intensity of cald-tagRFP (left) and CaM bands (right) in western blot analysis of three independent experiments as shown in B. Shown is mean ± SEM. Upper: Ca2+ greatly increases cald-tagRFP binding to GFP-myoVa-IQ1. Lower: When Ca2+ is available, the simultaneous presence of cald-tagRFP leads to a reduced binding of CaM compared to the control (tagRFP).

D CD spectroscopy reveals distinct secondary structure elements of the myoVa IQ1-containing peptide in water and ammonium acetate solution. Far-UV CD spectra of the myosin peptide acquired either in ultrapure water (black) or in ammonium acetate buffer (red; 0.5nm step size, 3sec/point, 25μs sample period, 3 repeats each, T = 20.0 ± 0.3 °C).

E Pie chart indicating structure elements of the myoVa IQ1-containing peptide in ammonium acetate solution calculated from spectra shown in (D).

F Native MS analysis of ion saturation for CaM. Ca2+ carried along from purification is detected on CaM in native MS. Without further supplementation with ions, intact CaM shows primarily its high affinity calcium binding sites occupied (left), while in the same analysis myoVa peptide selectively and preferentially binds only to the fully Ca2+-saturated CaM species (right). Additional unassigned peaks represent unspecific adducts of Na+ and Mg2+ ions.

G Native MS analysis of ion saturation for caldendrin. Supplemented by an excess of 200 µM Ca2+ and 100 µM Mg2+, caldendrin displays myoVa peptide binding to both fully Ca2+-saturated and 2 Ca2+-bound species (right), which are also both detectable for the intact caldendrin protein in the same analysis (left).

H No ternary complex is observed when CaM and caldendrin are mixed together with excess myoVa peptide in native MS. The individual proteins bind individually to myoVa peptide indicating competition for the same or closely located epitope. Fraction of caldendrin degrades into a 28kDa fragment, which has lost it affinity towards myoVa peptide almost completely.
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 3

Figure 3: Caldendrin inhibits myosin Va processivity in in vitro F-actin gliding assay.

A Upper: Coomassie blue stained SDS-PAGE showing purified full-length myoVa (arrow) from HEK293T cells using a StrepTag-GFP-tag. Lower: Western blot of StrepTag-GFP-myoVa shows that myosin is associated with HEK293T-cell endogenous calmodulin (CaM) upon purification.

B Schematic representation of in vitro F-actin gliding assay. CaM = Calmodulin, cald = caldendrin, PLL-PEG = Poly-L-lysine-polyethylene-glycol

C Visualization of F-actin gliding caused by myoVa activity. The first (red) and last (green) frame of a 30 second time-lapse image stack were color-coded and overlaid to visualize movement. Yellow = actin filament did not move during 30 seconds of imaging. In the presence of only CaM (left), the majority of actin filaments are gliding. In the presence of both CaM and cald (right), a large number of filaments are immobile. Scale bar = 10 µm. Also see Movie EV1.

D Quantification of immobile filaments as shown in C. In the presence of cald, the fraction of immobile actin filaments is significantly increased compared to CaM only (* p = 0.031, n=6 experiments, Wilcoxon matched-pairs signed rank test. In each experiment, between 189 to 491 individual filaments were observed. Individual actin filament numbers per experiment: 275, 309, 189, 362, 390, 491).

E Relative frequency distribution of recorded gliding velocities. In the presence of cald, the velocity distribution is shifted towards slower velocities compared to CaM only (mean ± SEM, n=5 experiments, 8 filaments for each experiment and condition).

F Quantification of F-actin gliding velocity. In the presence of cald, the velocity of F-actin gliding is significantly reduced compared to CaM only (mean ± SEM, ** p = 0.0052, paired t-test, n=5 experiments).