Methods for Studying the Plant Cytoskeleton

Williamson, R. E.; Grolig, Franz; Hurley, Ursula A.; Jablonski, P. P.; McCurdy, David W.; Wasteneys, Geoffrey O.

doi:10.1007/978-3-642-83349-6_11

Cited by 12 publications

(3 citation statements)

References 24 publications

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“…Perfusion of internodal cells, as described by Williamson et al (1989), was used to visualize the cytoskeleton. Briefly, an internodal cell was placed on the bottom of a perfusion chamber and pressed lightly into vacuum grease lines positioned several millimeters away from the cell ends.…”

Section: Methodsmentioning

confidence: 99%

Convoluted Plasma Membrane Domains in the Green AlgaCharaare Depleted of Microtubules and Actin Filaments

et al. 2015

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Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

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Section: Methodsmentioning

confidence: 99%

Convoluted Plasma Membrane Domains in the Green AlgaCharaare Depleted of Microtubules and Actin Filaments

et al. 2015

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“…The cytoplasmic pH was measured using the dual‐excitation wavelength pH indicator BCECF conjugated to 10 kDa dextran (Molecular Probes). Very young branchlet internodal cells with lengths of up to 5 mm were injected with a few nanoliter from a 2 mM BCECF‐10kD dextran dissolved in perfusion solution (200 mM sucrose, 70 mM KCl, 4.49 mM MgCl 2 , 5 mM EGTA, 1.48 mM CaCl 2 , 10 mM pipes; pH 7.3) [Williamson et al., 1989] using a CellTram Oil manual microinjector (Eppendorf Austria GmbH). Usually only one of the two cells in the branchlet was injected.…”

Section: Methodsmentioning

confidence: 99%

Brefeldin A inhibits clathrin‐dependent endocytosis and ion transport in Chara internodal cells

Foissner

Hoeftberger

Hoepflinger

et al. 2020

Biology of the Cell

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Background. The Characeae are multicellular green algae, which are closely related to higher plants. Their internodal cells are a convenient model to study membrane transport and organelle interactions. Results. In this study, we report on the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on internodal cells of Chara australis. BFA induced the commonly observed agglomeration of Golgi bodies and trans Golgi network into 'brefeldin compartments' at concentrations between 6 and 500 μM and within 30-120 min treatment. In contrast to most other cells, however, BFA inhibited endocytosis and significantly decreased the number of clathrin-coated pits and clathrin-coated vesicles at the plasma membrane. BFA did not inhibit secretion of organelles at wounds induced by puncturing or local light damage but prevented the formation of cellulosic wound walls probably because of insufficient membrane recycling. We also found that BFA inhibited the formation of alkaline and acid regions along the cell surface ('pH banding pattern') which facilitates carbon uptake required for photosynthesis; we hypothesise that this is due to insufficient recycling of ion transporters. During long-term treatments over several days, BFA delayed the formation of complex 3D plasma membranes (charasomes). Interestingly, BFA had no detectable effect on clathrin-dependent charasome degradation. Protein sequence analysis suggests that the peculiar effects of BFA in Chara internodal cells are due to a mutation in the guanine-nucleotide exchange factor GNOM required for recruitment of membrane coats via activation of ADP-ribosylation factor proteins. Conclusions and Significance. This work provides an overview on the effects of BFA on different processes in C. australis. It revealed similarities but also distinct differences in vesicle trafficking between higher plant and algal cells. It shows that characean internodal cells are a promising model to study interactions between seemingly distant metabolic pathways.

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“…Their simple cylindrical shape and large size, as well as the excellent imaging conditions made them a useful model system for various research fields (Braun et al ., 2007). They are famous for their rapid cytoplasmic streaming and therefore attractive for studying the actin cytoskeleton (Williamson et al ., 1989; Foissner & Wasteneys, 2000a; Braun et al ., 2007). They have also been important for the elucidation of microtubule properties (Wasteneys & Williamson, 1989 and references therein).…”

Section: Introductionmentioning

confidence: 99%

The characean internodal cell as a model system for studying wound healing

Foissner

Wasteneys

2011

Journal of Microscopy

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SummaryThis work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: (1) cortical window formation characterised by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, (2) fibrillar wound walls characterised by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and (3) amorphous, callose-and membrane-containing wound walls characterised by exocytosis of vesicles and endoplasmic reticulum cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca 2+ influx, and that the secretion of Ca 2+ -loaded endoplasmic reticulum cisternae is an emergency reaction in case of severe Ca 2+ load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganisation of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally, we show that quickly growing fibrillar wound walls, even

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Methods for Studying the Plant Cytoskeleton

Cited by 12 publications

References 24 publications

Convoluted Plasma Membrane Domains in the Green AlgaCharaare Depleted of Microtubules and Actin Filaments

Convoluted Plasma Membrane Domains in the Green AlgaCharaare Depleted of Microtubules and Actin Filaments

Brefeldin A inhibits clathrin‐dependent endocytosis and ion transport in Chara internodal cells

The characean internodal cell as a model system for studying wound healing

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