Microtubule organization and the effects of GFP-tubulin expression inDictyostelium discoideum

Kimble, Mary; Kuzmiak, C.; McGovern, Kelly; Hostos, Eugenio L. de

doi:10.1002/1097-0169(200009)47:1<48::aid-cm5>3.0.co;2-q

Cited by 34 publications

(7 citation statements)

References 60 publications

(62 reference statements)

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“…It was proposed that incorporation of Nterminal-tagged α-tubulin inhibits GTP hydrolysis, thus producing polymerization-prone MTs with an extended GTP cap (Abe and Hashimoto 2005). In Dictyostelium, expression of GFP-α-tubulin has been shown to interfere with MT nucleation and/or assembly (Kimble et al 2000). As discussed by Kimble et al (2000), the addition of GFP to the amino terminus of α-tubulin could directly hinder MT assembly because the amino terminus of α-tubulin is located between adjacent tubulin dimers, a location that would be important for assembly of the MT polymer.…”

Section: Discussionmentioning

confidence: 99%

“…In Dictyostelium, expression of GFP-α-tubulin has been shown to interfere with MT nucleation and/or assembly (Kimble et al 2000). As discussed by Kimble et al (2000), the addition of GFP to the amino terminus of α-tubulin could directly hinder MT assembly because the amino terminus of α-tubulin is located between adjacent tubulin dimers, a location that would be important for assembly of the MT polymer. Therefore, the marked alteration of MT organization by expression of excess GFP-TUA6 observed in the present study may be caused by an abnormal MT dynamicity and/or assembly.…”

Section: Discussionmentioning

confidence: 99%

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Disruption of Cortical Microtubules by Overexpression of Green Fluorescent Protein‐Tagged α‐Tubulin 6 Causes a Marked Reduction in Cell Wall Synthesis

Burk

Zhong

Morrison

et al. 2006

JIPB

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It has been known that the transverse orientation of cortical microtubules (MTs) along the elongation axis is essential for normal cell morphogenesis, but whether cortical MTs are essential for normal cell wall synthesis is still not clear. In the present study, we have investigated whether cortical MTs affect cell wall synthesis by direct alteration of the cortical MT organization in Arabidopsis thaliana. Disruption of the cortical MT organization by expression of an excess amount of green fluorescent protein-tagged α α α α α-tubulin 6 (GFP-TUA6) in transgenic Arabidopsis plants was found to cause a marked reduction in cell wall thickness and a decrease in the cell wall sugars glucose and xylose. Concomitantly, the stem strength of the GFP-TUA6 overexpressors was markedly reduced compared with the wild type. In addition, expression of excess GFP-TUA6 results in an alteration in cell morphogenesis and a severe effect on plant growth and development. Together, these results suggest that the proper organization of cortical MTs is essential for the normal synthesis of plant cell walls.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Disruption of Cortical Microtubules by Overexpression of Green Fluorescent Protein‐Tagged α‐Tubulin 6 Causes a Marked Reduction in Cell Wall Synthesis

Burk

Zhong

Morrison

et al. 2006

JIPB

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“…Surprisingly, all the DdINCENP null cells expressing GFP-tubulin died shortly after transformation. Although GFP-tubulin has been widely used in Dictyostelium for live microscopy of microtubule structures (Neujahr et al, 1998), it has been shown that GFP-tubulin can slightly affect microtubule dynamics in vivo (Kimble et al, 2000). This raised the possibility that the microtubule organization in the DdINCENP mutant cells might be unstable relative to that in wild-type cells, thus making such cells hypersensitive to the destabilization of microtubules as a result of expressing GFP-tubulin.…”

Section: Ddincenp Is Important For the Stability Of The Mitotic Spindlementioning

confidence: 99%

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and Cytokinesis

Chen

Lozanne

2006

MBoC

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“…Low-level expression of fluorescent protein-tagged tubulin subunits [18–20] can be used to label microtubules, since fluorescent protein-tagged tubulin, unlike tagged actin, can co-polymerize with native monomers. However, studies in a non-plant system ( Dictyostelium ) suggest that labeled tubulin may affect microtubule dynamics [21]. …”

Section: Introductionmentioning

confidence: 99%

A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex

Cvrčková

Oulehlová

2017

Plant Methods

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BackgroundCytoskeleton can be observed in live plant cells in situ with high spatial and temporal resolution using a combination of specific fluorescent protein tag expression and advanced microscopy methods such as spinning disc confocal microscopy (SDCM) or variable angle epifluorescence microscopy (VAEM). Existing methods for quantifying cytoskeletal dynamics are often either based on laborious manual structure tracking, or depend on costly commercial software. Current automated methods also do not readily allow separate measurements of structure lifetime, lateral mobility, and spatial anisotropy of these parameters.ResultsWe developed a new freeware-based, operational system-independent semi-manual technique for analyzing VAEM or SDCM data, QuACK (Quantitative Analysis of Cytoskeletal Kymograms), and validated it on data from Arabidopsis thaliana fh1 formin mutants, previously shown by conventional methods to exhibit altered actin and microtubule dynamics compared to the wild type. Besides of confirming the published mutant phenotype, QuACK was used to characterize surprising differential effects of various fluorescent protein tags fused to the Lifeact actin probe on actin dynamics in A. thaliana cotyledon epidermis. In particular, Lifeact-YFP slowed down actin dynamics compared to Lifeact-GFP at marker expression levels causing no macroscopically noticeable phenotypic alterations, although the two fluorophores are nearly identical. We could also demonstrate the expected, but previously undocumented, anisotropy of cytoskeletal dynamics in elongated epidermal cells of A. thaliana petioles and hypocotyls.ConclusionsOur new method for evaluating plant cytoskeletal dynamics has several advantages over existing techniques. It is intuitive, rapid compared to fully manual approaches, based on the free ImageJ software (including macros we provide here for download), and allows measurement of multiple parameters. Our approach was already used to document unexpected differences in actin mobility in transgenic A. thaliana expressing Lifeact fusion proteins with different fluorophores, highlighting the need for cautious interpretation of experimental results, as well as to reveal hitherto uncharacterized anisotropy of cytoskeletal mobility in elongated plant cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0171-9) contains supplementary material, which is available to authorized users.

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Microtubule organization and the effects of GFP-tubulin expression inDictyostelium discoideum

Cited by 34 publications

References 60 publications

Disruption of Cortical Microtubules by Overexpression of Green Fluorescent Protein‐Tagged α‐Tubulin 6 Causes a Marked Reduction in Cell Wall Synthesis

Disruption of Cortical Microtubules by Overexpression of Green Fluorescent Protein‐Tagged α‐Tubulin 6 Causes a Marked Reduction in Cell Wall Synthesis

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and Cytokinesis

A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex

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