Centrifuge Microscopy to Analyze the Sedimentary Movements of Amyloplasts

Toyota, Masatsugu; Ikeda, Norifumi; Tasaka, Masao; Morita, Miyo Terao

doi:10.21769/bioprotoc.1229

Cited by 4 publications

(6 citation statements)

References 3 publications

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“…To address this question, we utilized hypergravity conditions to increase sedimentary movement and analysed amyloplast behavior using a centrifuge microscope (Toyota et al , 2013 a ,b). Two types of gravistimulation were used at 10 g , one applied longitudinally (along the shoot–root longitudinal axis) and a second applied perpendicularly, similar to a 90° reorientation assay.…”

Section: Resultsmentioning

confidence: 99%

“…A centrifuge microscope was used for hypergravity experiments as previously described (Toyota et al , 2013 a ,b). Four-day-old etiolated seedlings were mounted in a chamber to fix the specimen on the centrifuge microscope before rotation; 10 g was applied after 10–20s of imaging and held continuously for 60s.…”

Section: Methodsmentioning

confidence: 99%

“…Tracking of amyloplasts during hypergravity experiments was carried out with G-Track spot-tracking software (G-Angstrom, Japan) as described (Toyota et al , 2013 a ). …”

Section: Methodsmentioning

confidence: 99%

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Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsiszigzag1hypocotyls

Alvarez

Han

Toyota

et al. 2016

EXBOTJ

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsiszigzag1hypocotyls

Alvarez

Han

Toyota

et al. 2016

EXBOTJ

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“…Centrifugal acceleration creates a hypergravity condition in which sedimentary movement of highdensity amyloplasts is more dominant than random diffusive movements, forcing all amyloplasts to the bottom. This makes centrifugation a useful technique to investigate the relationship between amyloplast dynamics and gravity sensing (Toyota et al 2014). Indeed, the reduced gravitropic sensitivities in pgm and sgr mutants were restored by centrifugal hypergravity (Fitzelle and Kiss 2001;Mori et al 2016;, which correlated with the resumption of amyloplast sedimentation (Fitzelle and Kiss 2001;Toyota et al 2013b).…”

Section: Introductionmentioning

confidence: 99%

Micromanipulation of amyloplasts with optical tweezers in <i>Arabidopsis</i> stems

Abe

Meguriya

Matsuzaki

et al. 2020

Plant Biotechnology

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“…Centrifugal acceleration creates a hypergravity condition in which sedimentary movement of high-density amyloplasts is more dominant than random diffusive movements, forcing all amyloplasts to the bottom. This makes centrifugation a useful technique to investigate the relationship between amyloplast dynamics and gravity sensing (Toyota et al 2014). Indeed, the reduced gravitropic sensitivities in pgm and sgr mutants were restored by centrifugal hypergravity (Fitzelle and Kiss 2001; Toyota et al 2014; Mori et al 2016), which correlated with the resumption of amyloplast sedimentation (Fitzelle and Kiss 2001; Toyota et al 2013b).…”

Section: Introductionmentioning

confidence: 99%

Micromanipulation of amyloplasts with optical tweezers inArabidopsisstems

Abe

Meguriya

Matsuzaki

et al. 2020

Preprint

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Intracellular sedimentation of highly dense, starch-filled amyloplasts toward the gravity vector is likely a key initial step for gravity sensing in plants. However, recent live-cell imaging technology revealed that most amyloplasts continuously exhibit dynamic, saltatory movements in the endodermal cells of Arabidopsis stems. These complicated movements led to questions about what type of amyloplast movement triggers gravity sensing. Here we show that a confocal microscope equipped with optical tweezers can be a powerful tool to trap and manipulate amyloplasts noninvasively, while simultaneously observing cellular responses such as vacuolar dynamics in living cells. A near-infrared (λ = 1064 nm) laser that was focused into the endodermal cells at 1 mW of laser power attracted and captured amyloplasts at the laser focus. The optical force exerted on the amyloplasts was theoretically estimated to be up to 1 pN. Interestingly, endosomes and trans-Golgi networks were trapped at 30 mW but not at 1 mW, which is probably due to lower refractive indices of these organelles than that of the amyloplasts. Because amyloplasts are in close proximity to vacuolar membranes in endodermal cells, their physical interaction could be visualized in real time. The vacuolar membranes drastically stretched and deformed in response to the manipulated movements of amyloplasts by optical tweezers. Our new method provides deep insights into the biophysical properties of plant organelles in vivo and opens a new avenue for studying gravity-sensing mechanisms in plants.

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Centrifuge Microscopy to Analyze the Sedimentary Movements of Amyloplasts

Cited by 4 publications

References 3 publications

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsiszigzag1hypocotyls

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsiszigzag1hypocotyls

Micromanipulation of amyloplasts with optical tweezers in <i>Arabidopsis</i> stems

Micromanipulation of amyloplasts with optical tweezers inArabidopsisstems

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