Figure 15A - B Confocal images showing staining of Olfactory Marker Protein ( OMP ) and ß3 - tubulin along the dorsal aspect of the mouse olfactory epithelium . While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata , their dendrites , and axons , the axon bundles ( green arrow ) located above the olfactory epithelium exemplify the clear signal - to - noise ratio benefits of glyoxal fixation versus that of the PFA - fixative . Immunostaining with the ß3 - tubulin antibody stains the dendrites and axons in both PFA and glyoxal - fixed tissue , but strong staining of the cilia can only be observed in the glyoxal - fixed sections ( B ) . C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb . Identities of the axons are in part defined by the neuropilin - 1 ( Nrp1 ) and neuropilin - 2 ( Nrp2 ) expression levels , visualized here with antibodies raised against the two proteins . While complementary expression of the two molecules can be seen in the PFA - fixed sections ( C ) , the glyoxal - fixed sections ( D ) exhibit profoundly improved signal - to - noise ratios for Nrp - 1 ( red arrows ) , and in the case of Nrp - 2 , also the segmentation of the axon bundle into varying levels of Nrp - 2 ( green arrows ) . E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons . The axons of olfactory sensory neurons can be readily visualized with PFA staining ( green ) in the superficial olfactory nerve layer and terminating in glomeruli located below ( green arrows ) . While sections fixed with either PFA or glyoxal display adequate staining levels , the signal distribution of the Immunostaining - fixed tissue appears more irregular ( E ) , seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal - fixed sections ( F ) . The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral / tufted cells as well as PFA of periglomerular neurons . Immunostaining with Vesicular Glutamate Transporter 2 ( VGLUT2 ) allows visualization of these structures and is easily seen in the glyoxal - fixed section ( F ) , while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA - fixed panel ( red arrows in E ) . Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal , albeit weaker versus Glyoxal ( inset ) . Staining with ß3 - tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal - fixed tissue , but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli ( blue arrows ) and in the external plexiform layer located below the glomeruli .

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Figure 15A - B Confocal images showing staining of Olfactory Marker Protein (OMP) and ß3-tubulin along the dorsal aspect of the mouse olfactory epithelium. While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata, their dendrites, and axons, the axon bundles (green arrow) located above the olfactory epithelium exemplify the clear signal-to-noise ratio benefits of glyoxal fixation versus that of the PFA-fixative. Immunostaining with the ß3-tubulin antibody stains the dendrites and axons in both PFA and glyoxal-fixed tissue, but strong staining of the cilia can only be observed in the glyoxal-fixed sections (B).C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb. Identities of the axons are in part defined by the neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) expression levels, visualized here with antibodies raised against the two proteins. While complementary expression of the two molecules can be seen in the PFA-fixed sections (C), the glyoxal-fixed sections (D) exhibit profoundly improved signal-to-noise ratios for Nrp-1 (red arrows), and in the case of Nrp-2, also the segmentation of the axon bundle into varying levels of Nrp-2 (green arrows).E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with PFA staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the Immunostaining-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as PFA of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.


Figure 2Morphological changes of vacuolar membranes and peroxisomal GFP - SKL during microautophagic degradation of peroxisomes after glucose adaptation . Double fluorescence of methanol - grown cells of P . pastoris cells at various times after the switch to glucose medium . ( a ) 0 h , ( b ) 30 min , ( c ) 2 h .

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Figure 2Morphological changes of vacuolar membranes and peroxisomal GFP-SKL during microautophagic degradation of peroxisomes after glucose adaptation. Double fluorescence of methanol-grown cells of P. pastoris cells at various times after the switch to glucose medium. (a) 0 h, (b) 30 min, (c) 2 h.


Figure 3Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes . The three panels in each figure are as follows ( a - e , left ) FM4 - 64 ; ( center ) GFP - SKL ; ( right ) superimposed FM4 - 64 and GFP - SKL signals . ( a , f ) stage 0 ; ( b , g ) early stage 1 ; ( c , h ) late stage 1 ; ( d , i ) stage 2 ; ( e ) stage 3 . ( d , arrowhead ) During the capture of these fluorescence images , this peroxisome - containing vesicle had just pinched off from the outer vacuolar membrane . Therefore , the FM4 - 64 - stained vesicle membrane and GFP - SKL could not be superimposed in this picture .

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Figure 3Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes. The three panels in each figure are as follows (a-e, left) FM4-64; (center) GFP-SKL; (right) superimposed FM4-64 and GFP-SKL signals. (a, f) stage 0; (b, g) early stage 1; (c, h) late stage 1; (d, i) stage 2; (e) stage 3. (d, arrowhead) During the capture of these fluorescence images, this peroxisome-containing vesicle had just pinched off from the outer vacuolar membrane. Therefore, the FM4-64-stained vesicle membrane and GFP-SKL could not be superimposed in this picture.


Figure 4Kinetics of morphological changes in vacuoles and peroxisomalGFP - SKL during microautophagic degradation of P . pastoris in glucose adaptation . At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point . Cells were counted in three randomly selected fields , and standard deviations are shown as vertical bars .

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Figure 4Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.


Figure 5Macroautophagic degradation of peroxisomes after transfer of methanol - grown P . pastoris cells to ethanol medium . ( a - e ) Electron micrographs of intermediates seen during macroautophagy . ( a ) After 1 h of ethanol adaptation , peroxisomes ( P ) were often surrounded by autophagosomal membranes , higher magnifications of which are in d and e . ( b ) After 1 h of ethanol adaptation , the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane . ( c ) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole . Magnified pictures of samples obtained at the ( d ) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes , and at the ( e ) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes . ( f ) Kinetics of morphological changes in vacuoles and peroxisomal GFP - SKL . Other conditions are described in Materials and Methods , and are the same as in Fig . 4 . ( g ) Fluorescence images taken after 2 h of ethanol adaptation of wild - type P . pastoris cells .

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Figure 5Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.


Figure 6Effect of inhibitors on transport of peroxisomal GFP - SKL to , and degradation in , vacuoles during ( a ) glucose adaptation and ( b ) ethanol adaptation . Wortmannin and PMSF were dissolved in DMSO , and added to the glucose medium . ( Filled circle ) Control ( 2 % DMSO ) ; ( open circle ) 1 mM wortmannin ( with 2 % DMSO ) ; ( filled square ) 3 mM PMSF ( with 2 % DMSO ) ; ( filled triangle ) 10 mg / ml cycloheximide .

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Figure 6Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.


Figure 7Microautophagic degradation of peroxisomes is not observed in a vacuolar protease - negative strain ( STK7 ) of P . pastoris . Kinetics of morphological changes in vacuoles and peroxisomal GFP - SKL during ( a ) glucose adaptation and ( b ) ethanol adaptation . ( c ) Fluorescence images taken after 2 h of glucose adaptation . Arrows show GFP - SKL in vacuolar matrix . Some cells showed diffusion of GFP - SKL into the vacuolar matrix .

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Figure 7Microautophagic degradation of peroxisomes is not observed in a vacuolar protease-negative strain (STK7) of P. pastoris. Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during (a) glucose adaptation and (b) ethanol adaptation. (c) Fluorescence images taken after 2 h of glucose adaptation. Arrows show GFP-SKL in vacuolar matrix. Some cells showed diffusion of GFP-SKL into the vacuolar matrix.


Figure 8 ( a ) Visualization of alcohol oxidase activity in colonies after ( a ) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild - type ( strain PPY12 ) and pag mutant cells ( strains STK1 - STK6 , Materials and Methods ) of P . pastoris . ( b ) Western - blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time . Antibodies used were AOX , alcohol oxidase ; F1 , mitochondrial F1β subunit ; G6PDH , glucose - 6 - phosphate dehydrogenase . Growth curve of the wild - type strain PPY12 and the pag4 mutant ( strain STK4 ) after shift from methanol to glucose .

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Figure 8(a) Visualization of alcohol oxidase activity in colonies after (a) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild-type (strain PPY12) and pag mutant cells (strains STK1-STK6, Materials and Methods) of P. pastoris. (b) Western-blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time. Antibodies used were AOX, alcohol oxidase; F1, mitochondrial F1β subunit; G6PDH, glucose-6-phosphate dehydrogenase. Growth curve of the wild-type strain PPY12 and the pag4 mutant (strain STK4) after shift from methanol to glucose.


Figure 9Fluorescence images of ( a ) pag2 ( strain STK2 ) and ( b ) pag3 ( strain STK3 ) mutants of P . pastoris after a 3 h shift to glucose . ( b ) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant .

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Figure 9Fluorescence images of (a) pag2 (strain STK2) and (b) pag3 (strain STK3) mutants of P. pastoris after a 3 h shift to glucose. (b) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant.


Figure 10Fluorescence images of the pag4 ( strain STK4 ) mutant of P . pastoris after ( a ) 0 , ( b ) 3 , or ( c ) 6 h of glucose adaptation . The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation .

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Figure 10Fluorescence images of the pag4 (strain STK4) mutant of P. pastoris after (a) 0, (b) 3, or (c) 6 h of glucose adaptation. The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation.


Figure 11Microautophagy of peroxisomes in a pex1 - ts mutant ( strain SJH242 ) of P . pastoris expressing GFP - SKL from the constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) promoter at the restrictive temperature for peroxisome assembly ( 34 ° C ) . ( a ) Kinetics of peroxisome degradation at 34 ° C . Strain SJH242 was induced on methanol at 23 ° C , and glucose adaptation was performed at 34 ° C . ( b ) Changes in vacuole morphology were seen even in the absence of normal peroxisomes . At 0 h , SJH242 cells were grown at 34 ° C when GFP - SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies . After 2 h , ∼ 20 - 30 % vacuoles showed complicated membrane structures representative of stage 2 cells .

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Figure 11Microautophagy of peroxisomes in a pex1-ts mutant (strain SJH242) of P. pastoris expressing GFP-SKL from the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter at the restrictive temperature for peroxisome assembly (34°C). (a) Kinetics of peroxisome degradation at 34°C. Strain SJH242 was induced on methanol at 23°C, and glucose adaptation was performed at 34°C. (b) Changes in vacuole morphology were seen even in the absence of normal peroxisomes. At 0 h, SJH242 cells were grown at 34°C when GFP-SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies. After 2 h, ∼20-30% vacuoles showed complicated membrane structures representative of stage 2 cells.


Figure 1 A Deletion of siaA , siaB , siaC , or siaD regulates biofilm formation . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) B The intracellular levels of c - di - GMPin PAO1 , ΔsiaC , ΔsiaB and the corresponding complemented strains were detected by LC - MS / MS E Bacterial two - hybrid assay reveals an interaction between SiaC and SiaD . The recombinant strains harboring different vectors were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . NA represents the empty vector pBT or pTRG . The strain expressing LGF2 and Gal11P was used as positive controls F Pull down assays confirmed the interaction between SiaC and SiaD . Cell lysates of P . aeruginosa containing pMM67EH - siaD - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads G SPR measurements of SiaC ( left ) or SiaCP ( right ) binding at varying concentrations to SiaD . SiaC specifically interacted with SiaD with a KD of 24 . 7nM , whereas SiaCP failed to interact with SiaB . Shown are measured binding responses ( black ) and curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant H Production of c - di - GMP by either SiaD or SiaD with SiaC at the indicated time - point was determined by HPLC

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Figure 1 A Deletion of siaA, siaB, siaC, or siaD regulates biofilm formation. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down)  B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS  E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls  F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads  G SPR measurements of SiaC (left) or SiaCP (right) binding at varying concentrations to SiaD. SiaC specifically interacted with SiaD with a KD of 24.7nM, whereas SiaCP failed to interact with SiaB. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant  H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC


Figure 2 A Bacterial two - hybrid assay reveals an interaction between SiaB and SiaC . The recombinant strains harboring different proteins were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . NA represents the empty vector pBT or pTRG . The strain expressing LGF2 and Gal11P were used as positive controls . B Pull - down assays confirmed the interaction between SiaB and SiaC . Cell lysates of P . aeruginosa containing pMM67EH - siaC - Flag were incubated with GST or GST - SiaB individually , and protein complexes were captured by glutathione beads . C SPR measurements of SiaC binding at varying concentrations to SiaB . SiaC specifically interacted with SiaB with a KD of 40 . 1 nM . Shown are measured binding responses ( black ) and curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant . D SiaB - mediated regulation of biofilm formation is dependent on SiaC . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . E Bacterial two - hybrid assayreveals an interaction between SiaC and SiaA PP2C - like domain . NA represents the empty vector pBT or pTRG . The recombinant strains harboring different proteins were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . The strain expressing LGF2 and Gal11P were used as positive controls . F Pull - down assays confirmed the interaction between SiaC and SiaA . Cell lysates of P . aeruginosa containing pMM67EH - siaA - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads . G Pull - down assays confirmed the interaction between SiaC and SiaA PP2C - like domain . Cell lysates of P . aeruginosa containing pMMB67EH - siaA386 - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads . H The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls.  B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads.  C SPR measurements of SiaC binding at varying concentrations to SiaB. SiaC specifically interacted with SiaB with a KD of 40.1 nM. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant.  D SiaB-mediated regulation of biofilm formation is dependent on SiaC. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).  E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls.  F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.  G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.  H The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


Figure 3 B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate . Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides . C Determination of modification sites by collision induced dissociation ( CID ) analysis . The MS / MS spectrum of modified L63LYLNTSSIK72 is shown . The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation , suggesting phosphorylation of Thr68 . D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro . SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP . Phosphorylated products were separated by SDS - PAGE . E Over - expression of SiaCT68A restored the ability of ΔsiaA to form biofilm . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . G Production of c - di - GMP at the indicated time - point by SiaD with SiaC or SiaCT68A was determined by HPLC . To evaluate the effect of SiaB - mediated phosphorylation on SiaD DGC activity , SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate. Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides.  C Determination of modification sites by collision induced dissociation (CID) analysis. The MS/MS spectrum of modified L63LYLNTSSIK72 is shown. The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation, suggesting phosphorylation of Thr68.  D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro. SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP. Phosphorylated products were separated by SDS-PAGE.  E Over-expression of SiaCT68A restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).  G Production of c-di-GMP at the indicated time-point by SiaD with SiaC or SiaCT68A was determined by HPLC. To evaluate the effect of SiaB-mediated phosphorylation on SiaD DGC activity, SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation.


Figure 4 C SiaCE33A partially restored the biofilm formation of ΔsiaA . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . D Triple mutation of E61A - Q64A - R67A is important for the function of SiaB . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 C SiaCE33A partially restored the biofilm formation of ΔsiaA. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).  D Triple mutation of E61A-Q64A-R67A is important for the function of SiaB. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


Figure 5 E Leu110 and Phe174 residues are important for SiaB function . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . Data represent the means and SDs of three biological replicates . * * , P < 0 . 01 based on one - way ANOVA test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 E Leu110 and Phe174 residues are important for SiaB function. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **, P<0.01 based on one-way ANOVA test.


Figure 1D , E Images of FRET efficiency in mock - treated ( - ant . A , D ) and Antimycin A - treated ( + ant . A . , E ) ubi - AT1 . 03NL wing discs at time point 2 h . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . F Line graphs showing the FRET efficiency in individual mock - treated ( - ant . A , n = 26 ) and ant . A - treated ( + ant . A . , n = 19 ) ubi - AT1 . 03NL wing discs before ( 0 h ) and after 2 h of treatment . Paired t - test , ns = not significant , * * * p ≤ 0 . 001 . G - J Time - controlled knock - down of metabolic enzymes in the dorsal compartment of ubi - AT1 . 03NL wing discs using the apGalts driver . Images of FRET efficiency in control ( G ) , apGalts > PfkRNAi ( H ) , apGalts > GapdhRNAi ( I ) , and apGalts > Glo1RNAi ( J ) wing discs after RNAi induction for 48 h ( G ) , 120 h ( H , J ) , and 93 hours ( I ) . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control ( n = 27 ) , apGalts > PfkRNAi ( n = 7 ) , apGalts > GapdhRNAi ( n = 10 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Loss of Pfk , Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , ns = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . L Time - controlled knock - down of ecdysoneless ( ecd ) in the dorsal compartment of ubi - AT1 . 03NL wing discs using apGalts > ecdRNAi . Image of FRET efficiency in apGalts > ecdRNAi wing disc 48 h after RNAi induction . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . M Line graph showing the FRET in the dorsal and ventral compartment of apGalts > ecdRNAi ( n = 28 ) wing discs . Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , * * * p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm. F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001. G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001. L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001.


Figure 2A - D Adult wing phenotype of knock - down of Gapdh ( C765 > GapdhRNAi ) , over - expression of full - length Ecd ( C765 > ecd ) , over - expression of C - terminally truncated form of Ecd ( C765 > ecdDN ) , and knock - down of ecd ( C765 > ecdRNAi ) in the whole wing disc ( see Appendix Fig S3A for the expression pattern of C765 - Gal4 ) . Images of adult wings of control ( A ) , C765 > GapdhRNAi ( A ' ) , C765 > ecd ( B ) , C765 > ecdDN ( C ) , and C765 > ecdRNAi ( D ) male flies . ( A ' ' ) Overlay of the wings shown in A and A ' . ( C ' ) Overlay of control wing and wing shown in C and C ' . Loss of Ecd throughout the wing discs produces adults with vestigal wings ( D ) . In contrast , the effects of ecdDN over - expression or Gapdh knock - down are less severe on tissue survival , giving rise to smaller wings with altered proportions . Images consist of a series of tiles that were stitched together by the microscope software . Scale bar = 500 μm . E , F Tukey box - and - whiskers plot showing quantifications of the area ( E ) and shape ( aspect ratio : major / minor axis , F ) of control , C765 > GapdhRNAi , C765 > ecd , and C765 > ecdDN wings . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to ± 1 . 5 times the interquartile range . Sample size is indicated for each perturbation . Loss of Gapdh or over - expression of EcdDN leads to smaller wings that are increased along the anterior - posterior ( A / P ) axis compared to the proximal - distal ( P / D ) axis . Statistical analysis was performed using one - way ANOVA , followed by Dunnett ' s multiple comparisons test . * * * p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D Adult wing phenotype of knock-down of Gapdh (C765>GapdhRNAi), over-expression of full-length Ecd (C765>ecd), over-expression of C-terminally truncated form of Ecd (C765>ecdDN), and knock-down of ecd (C765>ecdRNAi) in the whole wing disc (see Appendix Fig S3A for the expression pattern of C765-Gal4). Images of adult wings of control (A), C765>GapdhRNAi (A'), C765>ecd (B), C765>ecdDN (C), and C765>ecdRNAi (D) male flies. (A'') Overlay of the wings shown in A and A'. (C') Overlay of control wing and wing shown in C and C'. Loss of Ecd throughout the wing discs produces adults with vestigal wings (D). In contrast, the effects of ecdDN over-expression or Gapdh knock-down are less severe on tissue survival, giving rise to smaller wings with altered proportions. Images consist of a series of tiles that were stitched together by the microscope software. Scale bar= 500 μm. E, F Tukey box-and-whiskers plot showing quantifications of the area (E) and shape (aspect ratio: major/minor axis, F) of control, C765>GapdhRNAi, C765>ecd, and C765>ecdDN wings. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to ± 1.5 times the interquartile range. Sample size is indicated for each perturbation. Loss of Gapdh or over-expression of EcdDN leads to smaller wings that are increased along the anterior-posterior (A/P) axis compared to the proximal-distal (P/D) axis. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. ***p≤0.001.


Figure 3 A - B ' Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A , A ' ) and apGalts > ecdRNAi ( B , B ' ) wing discs , stained for the Hedgehog ( Hh ) pathway components Smoothened ( Smo , A , B ) , and Ci155 ( A ' , B ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 5 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > ecdRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 001 ) wing discs . Thus , loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm . C - E ' Time - controlled knock - down of metabolic enzymes in the dorsal compartment of the wing disc . IF of apGalts > PfkRNAi , apGalts > GapdhRNAi , and apGalts > Glo1RNAi wing discs , stained for Smo ( C , D , E ) and Ci155 ( C ' , D ' , E ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts > PfkRNAi ( n = 5 ) , apGalts > GapdhRNAi ( n = 5 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > PfkRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 01 ) , apGalts > GapdhRNAi ( Smo , Ci155 : p ≤ 0 . 05 ) , or apGalts > Glo1RNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 05 ) wing discs . Thus , loss of Pfk , Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 120 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A-B' Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A, A') and apGalts>ecdRNAi (B, B') wing discs, stained for the Hedgehog (Hh) pathway components Smoothened (Smo, A, B), and Ci155 (A', B'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=5) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>ecdRNAi (Smo: p≤0.001, Ci155: p≤0.001) wing discs. Thus, loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm. C-E' Time-controlled knock-down of metabolic enzymes in the dorsal compartment of the wing disc. IF of apGalts>PfkRNAi, apGalts>GapdhRNAi, and apGalts>Glo1RNAi wing discs, stained for Smo (C, D, E) and Ci155 (C', D', E'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts>PfkRNAi (n=5), apGalts>GapdhRNAi (n=5), and apGalts>Glo1RNAi (n=6) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>PfkRNAi (Smo: p≤0.001, Ci155: p≤0.01), apGalts>GapdhRNAi (Smo, Ci155: p≤0.05), or apGalts>Glo1RNAi (Smo: p≤0.001, Ci155: p≤0.05) wing discs. Thus, loss of Pfk, Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 120 h after RNAi induction. Scale bars= 50 μm.


Figure 4A - D ' ' ' IF of NIH3T3 cells stably expressing Smo - mEos2 treated with mock ( control , A - A ' ' ' ) , the Smo agonist ( + SAG , B - B ' ' ' ) or the glycolytic inhibitors 3 - bromopyruvate ( + 3 - BP , C - C ' ' ' ) and 2 - deoxyglucose ( + 2 - DG , D - D ' ' ' ) . DAPI staining , shown in gray , was used to visualize cell nuclei . Acetylated tubulin ( AcTub ) , used as a ciliary marker , is shown in magenta , Smo - mEos2 is shown in green . Zoomed - in images of cilia marked by white arrows in A ' ' , B ' ' , C ' ' , and D ' ' are shown in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . Scale bars = 5 μm , but 2 μm in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . E , F Box plot showing quantification of Smo - mEos2 ciliary localization in NIH3T3 cells upon 3 - BP ( E ) and 2 - DG ( F ) treatment , respectively . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to the minimum and maximum values . Sample size is indicated for each perturbation . Treatment with glycolytic inhibitors induces Smo - mEos2 localization to the primary cilium . t - test , * * * p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''. E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium . t-test, ***p≤0.001.


Figure 5 A - B Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A ) and apGalts > ecdRNAi ( B ) wing discs , stained for Hh . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 4 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analysis ( t - test ) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts > ecdRNAi wing discs . Thus , loss of Ecd does not affect Hh production and spreading . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm . C - F Time - controlled knock - down of ecd in the dorsal compartment of dispatched mutant wing discs ( dispS037707 , apGalts > ecdRNAi , see Fig 1C for the expression pattern of apGalts ) . IF of control ( C ) , apGalts > ecdRNAi ( D ) , dispS037707 ( E ) and dispS037707 , apGalts > ecdRNAi ( F ) wing discs expressing the decapentaplegic ( dpp ) reporter gene dpp - lacZ ( dppZ ) , stained for dppZ . Next to the images are quantifications of the respective stainings ( n = 4 ( C , F ) , n = 5 ( D ) , n = 3 ( E ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts > ecdRNAi ( p ≤ 0 . 01 ) ( C vs D ) , and dispS037707 and dispS037707 , apGalts > ecdRNAi ( p ≤ 0 . 01 ) wing discs ( E vs F ) . Loss of Ecd elevates the expression of dppZ in a broader than normal stripe . However , lack of Ecd function alone is not sufficient to trigger dpp expression , which instead depends on the presence of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm . G - J IF of dispS037707 ( G , I ) and dispS037707 , apGalts > ecdRNAi ( H , J ) wing discs , stained for Smo ( G , H ) and Ci155 ( I , J ) . Next to the images are quantifications of the respective stainings ( n = 2 ( G , H ) , n = 3 ( I ) , n = 4 ( J ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Although statistical analyses ( t - test ) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707 , apGalts > ecdRNAi wing discs , loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A-B Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A) and apGalts>ecdRNAi (B) wing discs, stained for Hh. Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=4) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analysis (t-test) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts>ecdRNAi wing discs. Thus, loss of Ecd does not affect Hh production and spreading. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm. C-F Time-controlled knock-down of ecd in the dorsal compartment of dispatched mutant wing discs (dispS037707, apGalts>ecdRNAi, see Fig 1C for the expression pattern of apGalts). IF of control (C), apGalts>ecdRNAi (D), dispS037707 (E) and dispS037707, apGalts>ecdRNAi (F) wing discs expressing the decapentaplegic (dpp) reporter gene dpp-lacZ (dppZ), stained for dppZ. Next to the images are quantifications of the respective stainings (n=4 (C, F), n=5 (D), n=3 (E)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts>ecdRNAi (p≤0.01) (C vs D), and dispS037707 and dispS037707, apGalts>ecdRNAi (p≤0.01) wing discs (E vs F). Loss of Ecd elevates the expression of dppZ in a broader than normal stripe. However, lack of Ecd function alone is not sufficient to trigger dpp expression, which instead depends on the presence of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm. G-J IF of dispS037707 (G, I) and dispS037707, apGalts>ecdRNAi (H, J) wing discs, stained for Smo (G, H) and Ci155 (I, J). Next to the images are quantifications of the respective stainings (n=2 (G, H), n=3 (I), n=4 (J)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Although statistical analyses (t-test) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707, apGalts>ecdRNAi wing discs, loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


Figure 6 A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing discs 48 h or 120 h after RNAi induction , respectively . V / D indicates the boundary between the ventral and dorsal compartments . Next to the maximal projections are shown the sum projections of cross - sections along the A / P axis . Loss of Ecd or Gapdh alters the plasma membrane potential . Scale bars = 50 μm . D - E ' ' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A ( gA ) . ( D ) Quantification of DiBAC4 ( 3 ) signal intensity in mock - treated ( - gA , n = 7 ) and gA - treated ( + gA , n = 5 ) wing discs . ( D ' , D ' ' ) Representative images of DiBAC4 ( 3 ) assay in mock - ( D ' ) and gA - treated ( D ' ' ) wing discs . Next to the maximal projections are shown sum projections of cross - sections along the A / P axis . ( E ) Quantification of PAC - NAE signal intensity in mock - ( n = 7 ) and gA - treated ( n = 9 ) wing discs , shown as fold change from control ( mock - treated ) . ( E ' - E ' ' ) Representative images of PAC - NAE uptake in mock - ( E ' ) and gA - treated ( E ' ' ) wing discs . GA treatment strongly reduces the uptake of PAC - NAE . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm.D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.


Figure 2 ( B ) Example of bead survival distributions for the 1G4 TCR binding NYE 9V . The survival probability at 1 s is shown as a dotted vertical line and is used to calculate the off - rate under force : − ln ( survival at 1 s ) / 1s . ( C ) Off - rates under force obtained from ( B ) are fitted with Bell ' s model ( solid line ) . Data information : The number of independent experiments performed on different days that were combined to produce the estimated off - rates are : 7 ( 1G4 / 9V ) ( D ) Off - rates under force for additional pMHCs interacting with the 1G4 TCR fitted to Bell ' s model ( solid line ) . ( E ) Correlation of the dissociation constant ( KD ) measured previously by SPR and the extrapolated zero - force flow chamber off - rate ( k0off ) from fitting Bell ' s model for the 1G4 TCR . ( F ) Correlation of the SPR dissociation constant ( KD ) with the fitted force sensitivity parameter ( xβ ) for the 1G4 TCR . Data information : Error bars ( SE ) in E , F , are obtained from the fit . The number of independent experiments performed on different days that were combined to produce the estimated off - rates are : 7 ( 1G4 / 9V ) , 8 ( 1G4 / 6V ) , 8 ( 1G4 / 4E5E8E ) , 10 ( 1G4 / 3Y ) , 6 ( 1G4 / 4D ) ( G ) Off - rates under force for pMHCs interacting with the A6 TCR fitted to Bell ' s model ( solid line ) . ( H ) Correlation of the dissociation constant ( KD ) measured previously by SPR and the extrapolated zero - force flow chamber off - rate ( k0off ) from fitting Bell ' s model for the A6 TCR . ( I ) Correlation of the SPR dissociation constant ( KD ) with the fitted force sensitivity parameter ( xβ ) for the A6 TCR . Data information : Error bars ( SE ) in H , I are obtained from the fit . The number of independent experiments performed on different days that were combined to produce the estimated off - rates are : 9 ( A6 / Tax WT ) , 9 ( A6 / 7Q ) , 8 ( A6 / 7R ) , 8 ( A6 / 5H ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Example of bead survival distributions for the 1G4 TCR binding NYE 9V. The survival probability at 1 s is shown as a dotted vertical line and is used to calculate the off-rate under force: −ln(survival at 1 s)/1s. (C) Off-rates under force obtained from (B) are fitted with Bell's model (solid line). Data information: The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 7 (1G4/9V)(D) Off-rates under force for additional pMHCs interacting with the 1G4 TCR fitted to Bell's model (solid line). (E) Correlation of the dissociation constant (KD) measured previously by SPR and the extrapolated zero-force flow chamber off-rate (k0off) from fitting Bell's model for the 1G4 TCR. (F) Correlation of the SPR dissociation constant (KD) with the fitted force sensitivity parameter (xβ) for the 1G4 TCR. Data information: Error bars (SE) in E,F, are obtained from the fit. The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 7 (1G4/9V), 8 (1G4/6V), 8 (1G4/4E5E8E), 10 (1G4/3Y), 6 (1G4/4D)(G) Off-rates under force for pMHCs interacting with the A6 TCR fitted to Bell's model (solid line). (H) Correlation of the dissociation constant (KD) measured previously by SPR and the extrapolated zero-force flow chamber off-rate (k0off) from fitting Bell's model for the A6 TCR. (I) Correlation of the SPR dissociation constant (KD) with the fitted force sensitivity parameter (xβ) for the A6 TCR. Data information: Error bars (SE) in H,I are obtained from the fit. The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 9 (A6/Tax WT), 9 (A6/7Q), 8 (A6/7R), 8 (A6/5H).


Figure 3 ( A ) The response force ( F ) on the TCR / pMHC interaction when pulled at acting on the spring attached to the C - terminus of the MHC which is being pulled with constant velocity ( ν ) . A second spring is attached to the C - terminus of the TCRβ and fixed in space . Insets : the TCR is shown in grey , the MHC in blue , and the peptide in red . The sites where the springs are attached are marked in orange . The direction of pulling is marked by a dashed line .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) The response force (F) on the TCR/pMHC interaction when pulled at acting on the spring attached to the C-terminus of the MHC which is being pulled with constant velocity (ν). A second spring is attached to the C-terminus of the TCRβ and fixed in space. Insets: the TCR is shown in grey, the MHC in blue, and the peptide in red. The sites where the springs are attached are marked in orange. The direction of pulling is marked by a dashed line.


Figure 4 ( B ) The concentration of productively signalling TCRs ( CN ) over the antigen concentration for antigens with the indicated zero - force off - rates ( koff ) . Shown is the effect of forces at 100 pN ( left ) and 10 pN ( right ) . The horizontal dashed line is the threshold value of CN required for activation in the model ( λ ) . The vertical dashed line shows the antigen concentration ( P ) required to elicit activation by the antigen with the largest value of koff . ( C ) The antigen concentration required to elicit activation ( P ) over koff for the indicated molecular forces on the TCR / pMHC interaction . ( D ) Antigen sensitivity quantified as the value of P from ( C ) over the applied force for a higher - affinity antigen with koff = 0 . 5 s - 1 . ( E ) Antigen discrimination quantified by the discrimination power ( α ) over the applied force . Discrimination power is defined as the ratio of the logarithm of the fold - change in P over the fold - change in koff for a higher - affinity ligand ( koff = 0 . 5 s - 1 ) and a lower - affinity ligand ( koff = 101 . 5 s - 1 ) from panel ( C ) . ( F ) Experimental value of P ( symbols ) over antigen affinity fitted by the kinetic proofreading model with force ( dashed and solid lines ) . The value of P is defined as the concentration of pMHC required for 15 % upregulation of CD69 above baseline and is shown as the mean with SEM from 4 independent experiments . ( G ) The fitted value of the applied force with SEM . ( H ) The fitted value of λ with SEM . Data information : An F - test is used to produce a p - value for the null hypothesis that the applied force or λ is the same across the three conditions . All data fitting ( F - H ) used N = 2 . 46 and kp = 2 . 15 s − 1 ( average of CD58 and ICAM - 1 conditions )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) The concentration of productively signalling TCRs (CN) over the antigen concentration for antigens with the indicated zero-force off-rates (koff). Shown is the effect of forces at 100 pN (left) and 10 pN (right). The horizontal dashed line is the threshold value of CN required for activation in the model (λ). The vertical dashed line shows the antigen concentration (P) required to elicit activation by the antigen with the largest value of koff.(C) The antigen concentration required to elicit activation (P) over koff for the indicated molecular forces on the TCR/pMHC interaction. (D) Antigen sensitivity quantified as the value of P from (C) over the applied force for a higher-affinity antigen with koff = 0.5 s-1. (E) Antigen discrimination quantified by the discrimination power (α) over the applied force. Discrimination power is defined as the ratio of the logarithm of the fold-change in P over the fold-change in koff for a higher-affinity ligand (koff = 0.5 s-1) and a lower-affinity ligand (koff = 101.5 s-1) from panel (C).(F) Experimental value of P (symbols) over antigen affinity fitted by the kinetic proofreading model with force (dashed and solid lines). The value of P is defined as the concentration of pMHC required for 15% upregulation of CD69 above baseline and is shown as the mean with SEM from 4 independent experiments. (G) The fitted value of the applied force with SEM. (H) The fitted value of λ with SEM. Data information: An F-test is used to produce a p-value for the null hypothesis that the applied force or λ is the same across the three conditions. All data fitting (F-H) used N = 2.46 and kp = 2.15 s−1 (average of CD58 and ICAM-1 conditions) taken


Figure 1 B : Silver nitrate staining of proteins pulled - down using the control peptide ( left ) or the FFAT peptide ( right ) , after SDS - PAGE . The two major differential bands are highlighted by arrows C : Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction . Score : protein score based on the sum of the ion scores of all peptides identified ; Cov . ; percentage of the protein sequence covered by identified peptides ; Pep . : number of unique peptide sequences identified E : Western blot analysis of proteins pulled - down using the control ( Ctrl ) peptide ( left ) or the FFAT ( FFAT ) peptide ( right ) . The Input , FT ( flow - through ) , and Elution fractions correspond to HeLa cell total protein extract , unbound proteins and bound proteins , respectively . Representative illustration of at least two independent experiments

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified  E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments


Figure 5 A : List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif ( see Appendix Table S1 for the full list ) . Proteins co - precipitated with GFP - MOSPD2 and GFP - MOSPD2 RD / LD proteins were identified by mass spectrometry ; proteins were filtered for their ability to bind MOSPD2 and not the RD / LD mutant . The sub - cellular localization of the proteins is indicated [ 53 , 65 - 69 ] B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagged STARD3 ( B ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F ) antibodies B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD3NL ( C ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed usin anti - ORP1 ( C ) , and anti - GFP ( B - F ) antibodies B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge ORP1L ( D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) , anti - ORP1 ( D ) ) , anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F ) antibodies B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD11 ( E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F ) antibodies B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) an HA - tagged PTPIP51 ( F ) ( WT and FFAT - deficient ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti - HA ( F ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F ) antibodies G : Immunoprecipitation ( anti - Flag ) experiment between Flag - tagged STARD3NL ( WT and FFAT - deficient ) and endogenous MOSPD2 . Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti - MOSPD2 , anti - STARD3NL and anti - actin antibodies

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A: List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif (see Appendix Table S1 for the full list). Proteins co-precipitated with GFP-MOSPD2 and GFP-MOSPD2 RD/LD proteins were identified by mass spectrometry; proteins were filtered for their ability to bind MOSPD2 and not the RD/LD mutant. The sub-cellular localization of the proteins is indicated [53,65-69]  B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagged STARD3 (B) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD3NL (C) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed usin anti-ORP1 (C), and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge ORP1L (D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E), anti-ORP1 (D) ), anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD11 (E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) an HA-tagged PTPIP51 (F) (WT and FFAT-deficient). Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti-HA (F) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies G: Immunoprecipitation (anti-Flag) experiment between Flag-tagged STARD3NL (WT and FFAT-deficient) and endogenous MOSPD2. Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti-MOSPD2, anti-STARD3NL and anti-actin antibodies


Figure 8 A : GFP - tagged MOSPD2 ( green ) expressing cells were labelled with an anti - MOSPD2 antibody ( magenta ) . The superposition of the green and magenta signals indicates that the anti - MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10 µm B : Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells ( WT ) and in HeLa cells expressing a control shRNA ( shCtrl ) and two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Quantification of MOSPD2 protein level is shown below . Means and error bars ( SD ) are shown . n : three independent experiments C : Endogenous MOSPD2 ( green ) was labelled in control HeLa cells ( WT ) , in cells expressing a control shRNA ( shCtrl ) , and in cells expressing two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Scale bar : 10 µm D : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing the ER marker GFP - ER ( magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10 µm E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing Flag - STARD3NL ( E ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay . Black rectangles indicate the positions of late endosomes ( E ) . Scale bars : 10 µm E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressin Flag - STARD3NL ∆ FFAT ( F ) ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10 µm G : Pearson correlation coefficients between endogenous MOSPD2 and Flag - STARD3NL ( WT or ΔFFAT ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - Flag - STARD3NL : 27 ; MOSPD2 - Flag - STARD3NL ΔFFAT : 26 ; from three independent experiments ) . Means and error bars ( SD ) are shown . Mann - Whitney test ( two - tailed P value ; * * * * : P < 0 , 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 A: GFP-tagged MOSPD2 (green) expressing cells were labelled with an anti-MOSPD2 antibody (magenta). The superposition of the green and magenta signals indicates that the anti-MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm B: Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells (WT) and in HeLa cells expressing a control shRNA (shCtrl) and two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Quantification of MOSPD2 protein level is shown below. Means and error bars (SD) are shown. n: three independent experiments  C: Endogenous MOSPD2 (green) was labelled in control HeLa cells (WT), in cells expressing a control shRNA (shCtrl), and in cells expressing two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Scale bar: 10 µm  D: Endogenous MOSPD2 (green) staining in HeLa cells expressing the ER marker GFP-ER (magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressing Flag-STARD3NL (E (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Black rectangles indicate the positions of late endosomes (E). Scale bars: 10 µm  E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressin Flag-STARD3NL ∆FFAT (F) (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm G: Pearson correlation coefficients between endogenous MOSPD2 and Flag-STARD3NL (WT or ΔFFAT) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-Flag-STARD3NL: 27; MOSPD2-Flag-STARD3NL ΔFFAT: 26; from three independent experiments). Means and error bars (SD) are shown. Mann-Whitney test (two-tailed P value; ****: P< 0,0001)


Figure 9 A : Western blot analysis of MOSPD2 , VAP - A and VAP - B proteins in control HeLa cells ( WT ) and in HeLa cells transfected with a control siRNA ( siCtrl ) , and with siRNA targeting MOSPD2 ( siMOSPD2 ) , VAP - A and VAP - B ( siVAP - A / VAP - B ) , and MOSPD2 , VAP - A and VAP - B ( siMOSPD2 / VAP - A / VAP - B ) B - F : TEM images of control HeLa cell ( B : HeLa - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm B - F : TEM images of control HeLa cell C : HeLa / siCtrl - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm B - F : TEM images o MOSPD2 ( D : siMOSPD2 - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm B - F : TEM images o VAP - A and VAP - B ( E : siVAP - A / VAP - B - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm B - F : TEM images o MOSPD2 , VAP - A and VAP - B ( F : siVAP - A / VAP - B / MOSPD2 ) - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm G - I : Quantification by stereology of ER - mitochondria ( G contacts . The percentages of mitochondria perimeter in contact with the ER ( G are shown as means and error bars ( SEM ) . ( G ) 200 ( HeLa ) , 208 ( siCtrl ) , 199 ( siMOSPD2 ) , 202 ( siVAP - A / VAP - B ) , 199 ( siMOSPD2 / VAP - A / VAP - B ) mitochondria from 8 , 10 , 8 , 12 and 12 cells , respectively , were analyzed G - I : Quantification by stereology o ER - endosome ( H contacts The percentages o ) , endosome perimeter in contact with the ER ( H are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : P < 0 , 001 ; * * * * : P < 0 . 0001 ) G - I : Quantification by stereology o endosome - endosome ( I ) contacts . The percentages o endosome perimeter in contact with an endosome ( I ) are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : P < 0 , 001 ; * * * * : P < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9 A: Western blot analysis of MOSPD2, VAP-A and VAP-B proteins in control HeLa cells (WT) and in HeLa cells transfected with a control siRNA (siCtrl), and with siRNA targeting MOSPD2 (siMOSPD2), VAP-A and VAP-B (siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (siMOSPD2/VAP-A/VAP-B)  B-F: TEM images of control HeLa cell (B: HeLa -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images of control HeLa cell C: HeLa/siCtrl -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images o MOSPD2 (D: siMOSPD2 -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images o VAP-A and VAP-B (E: siVAP-A/VAP-B -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images o MOSPD2, VAP-A and VAP-B (F: siVAP-A/VAP-B/MOSPD2) -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  G-I: Quantification by stereology of ER-mitochondria (G contacts. The percentages of mitochondria perimeter in contact with the ER (G are shown as means and error bars (SEM). (G) 200 (HeLa), 208 (siCtrl), 199 (siMOSPD2), 202 (siVAP-A/VAP-B), 199 (siMOSPD2/VAP-A/VAP-B) mitochondria from 8, 10, 8, 12 and 12 cells, respectively, were analyzed G-I: Quantification by stereology o ER-endosome (H contacts The percentages o ), endosome perimeter in contact with the ER (H are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001) G-I: Quantification by stereology o endosome-endosome (I) contacts. The percentages o endosome perimeter in contact with an endosome (I) are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


Figure 1Negative stain TEM image of B . cereus NVH 0075 - 95 endospore , showing spore body ( SB ) , exosporium ( E ) , and endospore appendages ( Ena ) , which emerge from the endospore individually or as fiber bundles . At the distal end , Enas terminate in a single or multiple thin ruffles ( R ) . cryoEM images ( left ) and negative stain 2D class averages of single S - Ena ( E ) and L - Ena ( F ) fibers . S - Ena show ~ 100 Å diameter and a helical or staggered appearance with a ~ 37 Å rise , whereas L - Ena have a ~ 80 Å diameter and appear as stacked disks of ~ 40 Å height and ~ 60 Å translation along the fiber . L - and S - Ena show , respectively , a single or multiple ruffle ( s ) ( black arrow , labeled ' R ' ) at the distal end of fiber .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Negative stain TEM image of B. cereus NVH 0075-95 endospore, showing spore body (SB), exosporium (E), and endospore appendages (Ena), which emerge from the endospore individually or as fiber bundles. At the distal end, Enas terminate in a single or multiple thin ruffles (R).cryoEM images (left) and negative stain 2D class averages of single S-Ena (E) and L-Ena (F) fibers. S-Ena show ~100 Å diameter and a helical or staggered appearance with a ~37 Å rise, whereas L-Ena have a ~80 Å diameter and appear as stacked disks of ~40 Å height and ~60 Å translation along the fiber. L- and S-Ena show, respectively, a single or multiple ruffle(s) (black arrow, labeled 'R') at the distal end of fiber.