Plant cell morphogenesis involves concerted rearrangements of microtubules and actin microfilaments. We previously reported that FH1, the main Arabidopsis thaliana housekeeping Class I membrane-anchored formin, contributes to actin dynamics and microtubule stability in rhizodermis cells. Here we examine the effects of mutations affecting FH1 (At3g25500) on cell morphogenesis and above-ground organ development in seedlings, as well as on cytoskeletal organization and dynamics, using a combination of confocal and variable angle epifluorescence microscopy with a pharmacological approach. Homozygous fh1 mutants exhibited cotyledon epinasty and had larger cotyledon pavement cells with more pronounced lobes than the wild type. The pavement cell shape alterations were enhanced by expression of the fluorescent microtubule marker GFP-microtubule-associated protein 4 (MAP4). Mutant cotyledon pavement cells exhibited reduced density and increased stability of microfilament bundles, as well as enhanced dynamics of microtubules. Analogous results were also obtained upon treatments with the formin inhibitor SMIFH2 (small molecule inhibitor of formin homology 2 domains). Pavement cell shape in wild-type (wt) and fh1 plants in some situations exhibited a differential response towards anti-cytoskeletal drugs, especially the microtubule disruptor oryzalin. Our observations indicate that FH1 participates in the control of microtubule dynamics, possibly via its effects on actin, subsequently influencing cell morphogenesis and macroscopic organ development.

• Klíčová slova
• Arabidopsis thaliana, Confocal microscopy, Cotyledon pavement cells, Cytoskeleton, Formin, Variable angle epifluorescence microscopy,
• MeSH
• aktiny metabolismus   MeSH
• Arabidopsis cytologie   účinky léků   metabolismus   MeSH
• biologické markery metabolismus   MeSH
• biologické modely MeSH
• cytoskelet účinky léků   metabolismus   MeSH
• fluorescence MeSH
• forminy MeSH
• klathrin metabolismus   MeSH
• kotyledon účinky léků   metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mikrofilamenta účinky léků   metabolismus   MeSH
• mikrotubuly účinky léků   metabolismus   MeSH
• mutace genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• semenáček účinky léků   růst a vývoj   metabolismus   MeSH
• thioketony farmakologie   MeSH
• tvar buňky * účinky léků   MeSH
• uracil analogy a deriváty   farmakologie   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AFH1 protein, Arabidopsis MeSH Prohlížeč
• aktiny MeSH
• biologické markery MeSH
• forminy MeSH
• klathrin MeSH
• membránové proteiny MeSH
• proteiny huseníčku MeSH
• SMIFH2 compound MeSH Prohlížeč
• thioketony MeSH
• uracil MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: Cytoskeleton 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. RESULTS: We 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. CONCLUSIONS: Our 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.

• Klíčová slova
• Actin, Anisotropy, FH1 (At3g25500), Kymogram, Lateral mobility, Lifeact, Microtubules, Spinning disc confocal microscopy, Structure stability, Variable angle fluorescence microscopy,
• Publikační typ
• časopisecké články MeSH

The cortical microtubule and actin meshworks play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM), spinning disk confocal microscopy (SDCM), and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of free computing tools based on the publicly available ImageJ software package, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

• Klíčová slova
• Actin, CLSM, Fluorescent proteins, Image analysis, ImageJ, Microtubules, SDCM, VAEM,
• MeSH
• Arabidopsis ultrastruktura   MeSH
• cytoskelet ultrastruktura   MeSH
• fluorescenční mikroskopie metody   MeSH
• konfokální mikroskopie metody   MeSH
• mikrotubuly ultrastruktura   MeSH
• počítačové zpracování obrazu metody   MeSH
• rostlinné buňky ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

• MeSH
• Arabidopsis genetika   metabolismus   ultrastruktura   MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• cytoplazma metabolismus   ultrastruktura   MeSH
• cytoskelet metabolismus   ultrastruktura   MeSH
• epidermis rostlin genetika   metabolismus   ultrastruktura   MeSH
• exocytóza MeSH
• exprese genu MeSH
• fluorescenční mikroskopie MeSH
• kořeny rostlin genetika   metabolismus   ultrastruktura   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny SNARE genetika   metabolismus   MeSH
• Rab proteiny vázající GTP genetika   metabolismus   MeSH
• sekreční vezikuly metabolismus   ultrastruktura   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku MeSH
• proteiny SNARE MeSH
• Rab proteiny vázající GTP MeSH

The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of suitable computing techniques, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

• MeSH
• aktiny chemie   MeSH
• Arabidopsis anatomie a histologie   chemie   MeSH
• cytoskelet chemie   MeSH
• laserová skenovací cytometrie metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH

Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin-microtubule cross-talk.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• forminy MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mikrofilamenta genetika   metabolismus   MeSH
• mikrotubuly genetika   metabolismus   MeSH
• mutace * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AFH1 protein, Arabidopsis MeSH Prohlížeč
• forminy MeSH
• membránové proteiny MeSH
• proteiny huseníčku MeSH