Isolation of <i>Chlamydomonas</i> Flagella

Craige, Branch; Brown, Jason; Witman, George B.

doi:10.1002/0471143030.cb0341s59

Cited by 57 publications

(44 citation statements)

References 27 publications

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“…Except where noted, flagella were isolated using the dibucaine method [40]. Treatment with 1% NP40 in HMEK followed by centrifugation for 20 min at 16,000 rpm in an SS34 rotor (30,590 RCF; Sorvall) fractionated flagella into M+M and axonemal fractions.…”

Section: Methodsmentioning

confidence: 99%

Assembly of IFT Trains at the Ciliary Base Depends on IFT74

et al. 2015

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SUMMARY Intraflagellar transport (IFT) moves IFT trains carrying cargoes from the cell body into the flagellum and from the flagellum back to the cell body. IFT trains are composed of complexes IFT-A and IFT-B and cargo adapters such as the BBSome. The IFT-B core proteins IFT74 and IFT81 interact directly through central and C-terminal coiled-coil domains, and recently it was shown that the N-termini of these proteins form a tubulin-binding module important for ciliogenesis. To investigate the function of IFT74 and its domains in vivo, we have utilized Chlamydomonas reinhardtii ift74 mutants. In a null mutant, lack of IFT74 destabilized IFT-B, leading to flagella assembly failure. In this null background, expression of IFT74 lacking 130 aa of the charged N terminus stabilized IFT-B and promoted slow assembly of nearly full-length flagella. A further truncation (lacking aa 1-196 including part of coiled-coil 1) also stabilized IFT-B, but failure in IFT-A / IFT-B interaction within the pool at the base of the flagellum prevented entry of IFT-A into the flagellum and led to severely decreased IFT injection frequency and flagellar-assembly defects. Decreased IFT-A in these short flagella resulted in aggregates of stalled IFT-B in the flagella. We conclude that IFT74 is required to stabilize IFT-B; aa 197-641 are sufficient for this function in vivo. The N terminus of IFT74 may be involved in but is not required for tubulin entry into flagella. It is required for association of IFT-A and IFT-B at the base of the flagellum and flagellar import of IFT-A.

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Section: Methodsmentioning

confidence: 99%

Assembly of IFT Trains at the Ciliary Base Depends on IFT74

et al. 2015

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“…2 L cultures inoculated either into TAP 48h before experiment and grown at 50 μEm-2s-1 (a), or in Minimal Medium 72h before experiment and grown aerated under at 200 μEm-2s-1 (b), were kept in darkness the night preceding the experiment. Cells were washed twice in 20 mM HEPES-KOH pH 7.2 (a), or washed once in 10 mM HEPES-KOH pH 7.2 and then subjected to an acid shock to remove flagella as described in Craige et al (2013) (1min at pH 4.5 by adding 1M acetic acid, then neutralising back to pH 7.2 with 1M KOH) (b). Working in a cold room, cells were then resuspended in 50 mL ice cold breaking buffer (300 mM Sorbitol, 3 mM MgCl2, 5 mM EDTA, 0.5% Polyvinylpyrrolidone PVP40, in 67 mM HEPES-KOH pH 7.2) and disrupted by nebulization with Argon using either one pass at 40 psi (a), or four passes at 80 psi (b), after which an aliquot was set aside.…”

Section: Biochemistrymentioning

confidence: 99%

Evidence supporting an antimicrobial origin of targeting peptides to endosymbiotic organelles

Garrido

Caspari

Choquet

et al. 2020

Preprint

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Mitochondria and chloroplasts emerged from primary endosymbiosis. Most endosymbiont proteins have subsequently been expressed in the nucleo-cytosol of the host and organelle-targeted via the acquisition of N-terminal presequences, whose evolutionary origin remains enigmatic. Using a quantitative assessment of their physico-chemical properties, we show that organelle targeting peptides, which are distinct from signaling peptides targeting other subcellular compartments, group with a subset of antimicrobial peptides. We demonstrate that extant antimicrobial peptides target a fluorescent reporter to either the mitochondria or the chloroplast in the green alga Chlamydomonas reinhardtii and, conversely, that extant targeting peptides display antimicrobial activity. Thus, we provide conclusive computational and functional evidence for an evolutionary link between organelle-targeting and antimicrobial peptides. Our results support the view that resistance of bacterial progenitors of organelles to the attack of host antimicrobial peptides, has been instrumental in eukaryogenesis and in the subsequent emergence of photosynthetic eukaryotes.

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“…Wild-type Chlamydomonas assemble two cilia that are 10 to 12 µm long (22). Cilia of Chlamydomonas can experimentally be removed from the cell body using a variety of methods including mechanical shear or chemical stress (23,24). The cells immediately begin to grow new cilia as the stress ends (25,26).…”

Section: Introductionmentioning

confidence: 99%

How does cilium length affect beating?

Bottier

Thomas

Dutcher

et al. 2018

Preprint

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The effects of cilium length on the dynamics of cilia motion were investigated by high-speed video microscopy of uniciliate mutants of the swimming alga, Chlamydomonas reinhardtii. Cells with short cilia were obtained by deciliating cells via pH shock and allowing cilia to reassemble for limited times.The frequency of cilia beating was estimated from motion of the cell body and of the cilium. Key features of the ciliary waveform were quantified from polynomial curves fitted to the cilium in each image frame. Most notably, periodic beating did not emerge until the cilium reached a critical length between 2-4 µm. Surprisingly, in cells that exhibited periodic beating, the frequency of beating was similar for all lengths with only a slight decrease in frequency as length increased from 4 µm to the normal length of 10-12 µm. The waveform average curvature (rad/µm) was also conserved as the cilium grew. The mechanical metrics of ciliary propulsion: force, torque, and power all increased in proportion to length. Mechanical efficiency of beating appeared to be maximal at the normal wild-type length of 10-12 μm. These quantitative features of ciliary behavior illuminate the biophysics of cilia motion and, in future studies, may help distinguish competing hypotheses of the underlying mechanism of oscillation.

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Isolation of Chlamydomonas Flagella

Cited by 57 publications

References 27 publications

Assembly of IFT Trains at the Ciliary Base Depends on IFT74

Assembly of IFT Trains at the Ciliary Base Depends on IFT74

Evidence supporting an antimicrobial origin of targeting peptides to endosymbiotic organelles

How does cilium length affect beating?

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