Altered N-glycan composition impacts flagella-mediated adhesion in Chlamydomonas reinhardtii
- Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- Institute for Plant Biology and Biotechnology, University of Münster, Germany
- Institute of Integrative Biology, University of Liverpool, United Kingdom
- State Key Laboratory of Microbial Technology, and Marine Biotechnology Research Center, Shandong University, China
- Max Planck Institute for Dynamics and Self-Organization (MPIDS), Germany
- Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Germany
- Experimental Physics V, University of Bayreuth, Germany
- College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, China
- Institute of Plant Science and Resources, Okayama University, Japan
Figures
Figure 1 with 7 supplements
Altered N-glycosylation of FMG-1B does not change its flagellar localization.
(A) Diagram of N-linked glycan compositions of mutant strains characterized in Schulze et al., 2018 and used in the current study. While IMMan1A and the double mutant are mainly characterized by a …
Figure 1—figure supplement 1
Flagellar length is not altered in N-glycosylation mutants.
(a) Measurement of the flagellar length (in µm) among WT-Ins and three mutants. 50 flagella were measured in each experiment and this experiment has three biological repeats. Error bar: mean ± SD. p>…
Figure 1—figure supplement 2
N-Glycan structures are altered in mutants as compared to WT-Ins.
Whole-cell proteins (a) and isolated flagella (b) of WT-Ins and mutants were separated on a 7% SDS-PAGE, transferred to nitrocellulose and probed with anti-HRP, which is specifically binding to …
Figure 1—figure supplement 3
N-glycosylation mutants show increased Concanavalin-A affinity.
Whole-cell proteins of WT-SAG, WT-Ins and mutants were separated on a 7% SDS-PAGE, transferred to nitrocellulose and probed with the lectin Concanavalin A, which binds specific N-glycan epitopes. To …
Figure 1—figure supplement 4
Change of N-glycan pattern of FMG-1B in IM strains as compared to WT-Ins.
(a) Diagram of the topology of FMG-1B, the major component of the glycocalyx in C. reinhardtii. The identified N-linked glycosylation sites are marked. (b) Comparison of FMG-1B proteotypic N-glycopep…
Figure 1—figure supplement 5
Genetic crossing of original IM strains (mt+) with CC124 (mt-) to obtain mutants lacking IFT46::YFP.
(a) Screening the progenies of IMMan1A mutants with WT-CC-124 to obtain the mutant without IFT46::YFP background, which grew in TAP plate with Paromomycin added and died in TAP plate with Hygromycin …
Figure 1—figure supplement 6
The altered N-glycan did not change the localization FMG-1B in flagella.
(A) The mutants of IMMan1A-P15, IMXylT1A -P37, IMMan1A X IMXylT1A -P37 were mixed with equal amount of WT-Ins expressing IFT46::YFP, then immune stained with an antibody against N-glycan epitope, …
Figure 1—figure supplement 7
Quantitative mass spectrometry of isolated flagellar from WT-Ins and N-glycosylation mutants.
(a) Experimental procedures of flagella isolation and following analyses. (b) Immunoblot of isolated flagella probed with antibodies against chloroplast marker protein (Cytf, cytochrome f) and …
Figure 2 with 1 supplement
Altered N-glycosylation diminishes flagellar polystyrene bead attachment and -transport.
(A) Percentage of flagella with at least one polystyrene bead bound. Cells were incubated with polystyrene beads (0.7 µm in diameter) and subsequently analyzed by light microscopy. (B) Percentage of …
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Figure 2—source data 1
Raw data of attachment and movement of microbeads to and along flagella.
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig2-data1-v2.xlsx
Figure 2—video 1
Attachment and movement of a microsphere to and along flagella.
To obtain a kinetic measure of surface motility, cells were mixed with beads as above for 5 min and randomly observed under the light microscope. Each bead adhered to a flagellum was monitored for …
Figure 3 with 2 supplements
Quantification of the flagella-mediated adhesion using atomic force microscopy.
(A) Diagram of experimental procedures for force measurement: the cell adhered to the surface (1); the cell attached to the AFM cantilever (2); the cell was pulled up from the surface by AFM …
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Figure 3—source data 1
Raw data of AFM mesurement (force, average energy).
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Analysis of the force and energy required to overcome the adhesion of C. reinhardtii flagella to the surface from AFM force curves.
The curve shows the relation of pulling force and pulling distance during AFM probe retracting. The lowest point of the curve represents the maximal adhesion force of flagella which was used in Figur…
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Figure 3—figure supplement 1—source data 1
Exemplary data of AFM measurement.
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Detachment distance and total energy of the flagella adhesion quantified by atomic force microscopy.
(A) Flagella detachment distances of WT-Ins, IMMan1A, IMXylT1A, and IMMan1AxIMXylT1A were generated from force curves. (B) Total energy of flagellar adhesion of WT-Ins, IMMan1A, IMXylT1A, and IMMan1A…
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Figure 3—figure supplement 2—source data 1
Raw data of AFM measurement (detachment distance, total energy).
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig3-figsupp2-data1-v2.xlsx
Figure 4 with 3 supplements
Assessing flagella adhesion forces using micropipette force microscopy.
Flagella-mediated adhesion forces acquired for WT-SAG and a xylosyltransferase 1A mutant generated in the genetic background of WT-SAG (CRISPRXylT1A_1). Micropipette force measurements of the same …
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Figure 4—source data 1
Raw data of micropipette force measurement.
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig4-data1-v2.docx
Figure 4—figure supplement 1
Xylosyltransferase 1A mutant generated via CRISPR/Cas9 supports findings in IMXylT1A.
(a) Schematic representation of the XylT1A gene including the site targeted by CIRSPR/Cas9. (b) Parallel reaction monitoring was employed to prove the knock out of XylT1A on proteomic level in two …
Figure 4—figure supplement 2
Immunoblot proving the presence of FMG-1B in flagella of CRISPRXylT1A.1 and CRISPRXylT1A.2.
30 µg of protein per sample were separated by SDS-PAGE and transferred to a nitrocellulose membrane in biological quadruplicates. (a) Ponceau staining of the membrane reveals equal loading between …
Figure 4—figure supplement 3
Study of the effect of DMSO on the flagella adhesion forces using micropipette force microscopy.
Micropipette force measurements of the same cells were performed for WT-SAG under blue and red light in the (+) presence or (-) absence of DMSO. The concentration of DMSO corresponds to the one used …
Figure 5 with 1 supplement
IFT and gliding are unaffected in IMMan1AxIMXylT1A.
(A) Adhesion forces acquired for WT-Ins and the double mutant IMMan1AxIMXylT1A in the absence or presence of ciliobrevin D via AFM, respectively. Three biological replicates were performed with …
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Figure 5—source data 1
Raw data of IFT tracking and gliding velocity by TIRF microscopy.
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Adhesion force increases in temperature sensitive dhc1-b mutant at restrictive temperature.
(A) The unique inserted cassette of AphVIII in MAN1A gene was identified in the double mutant and the progenies of the cross between the IMMan1A IMXylT1A with dynein-1bts using PCR. (B) …
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Figure 5—figure supplement 1—source data 1
Raw data of AFM measurement proving the influence of dynein -1b.
- https://cdn.elifesciences.org/articles/58805/elife-58805-fig5-figsupp1-data1-v2.xlsx
Tables
Table 1
MS parameters.
Relevant parameters used to acquire IS-CID and not fragmented TopN MS spectra as well as PRM data.
| TopN without IS-CID | In-Source CID HCD | Parallel reaction monitoring | ||
|---|---|---|---|---|
| Eluent compositions | Peptide trapping: 0.05% trifluoroacetic acid (TFA) in ultrapure water (A1), 0.05% TFA in 80% acetonitrile (B1) Peptide separation: 0.1% formic acid (FA) in ultrapure water (A2), 0.1% FA in 80% acetonitrile (B2) | LC parameters | ||
| Trap Column | C18 PepMap 100, 300 µM x 5 mm, 5 µm particle size, 100 Å pore size; Thermo Scientific | |||
| Peptide trapping (eluents A1+B1) | 2.5% B1 at 5 μL/min for 5 min | 2.5% B1 at 10 μL/min for 3 min | ||
| Flow rate | 300 nL/min | 250 nL/min | ||
| Separation Column | Acclaim PepMap C18, 75 µm x 50 cm, 2 µm particle size, 100 Å pore size; Thermo Scientific | |||
| Gradient for peptide separation (eluents A2+B2) | 2.5% B2 over 5 min, 2.5–45% B2 over 40 min, 45–99 % B2 over 5 min 99% B2 for 20 min 99–2.5% over 5 min 2.5% for 30 min | 2.5% B2 over 5 min, 2.5–35% B2 over 105 min, 35–99 % B2 over 5 min 99% B2 for 20 min 99–2.5% over 5 min 2.5% for 40 min | ||
| In-source CID | off | 80 eV | off | MS1 settings |
| Use lock masses | off | on (m/z 445.12003) | ||
| Resolution at m/z 200 (FWHM) | 70,000 | |||
| Chromatographic peak width | 15 s | |||
| AGC target | 3e6 | |||
| Maximum injection time | 100 ms | 50 ms | ||
| Scan range | 600–3000 m/z | 350–1600 m/z | ||
| Mass tags | off | on | off | |
| TopN | 12 | n/a | MS2 settings | |
| Resolution at m/z 200 (FWHM) | 17,500 | 35,000 | ||
| Isolation window | 2 m/z | 2 m/z (offset 0.5 m/z) | ||
| AGC target | 1e5 | |||
| Maximum injection time | 120 ms | |||
| Normalized collision energy (NCE) | 30 | 27 | ||
| Minimum AGC target | 1.25e3 | n/a | ||
| Intensity threshold | 1e4 | n/a | ||
| Charge exclusion | unassigned,>5 | n/a | ||
| Dynamic exclusion | 15 s | n/a | ||
