Variable angle fluorescence microscopy Dotaz Zobrazit nápovědu
We report a novel and multifaceted approach for the quick synthesis of highly stable single-stranded DNA (ssDNA) functionalized gold nanoparticles (AuNPs). The method is based on the combined effect of surface passivation by (1-mercaptoundec-11-yl)hexa(ethylene glycol) and low pH conditions, does not require any salt pretreatment or high excess of ssDNA, and can be generalized for oligonucleotides of any length or base sequence. The synthesized ssDNA-coated AuNPs conjugates are stable at salt concentrations as high as 3.0 M, and also functional and specific toward DNA-DNA hybridization, as shown from UV-vis spectrophotometry, scanning electron microscopy, gel electrophoresis, fluorescence, and small angle X-ray scattering based analyses. The method is highly flexible and shows an additional advantage of creating ssDNA-AuNP conjugates with a predefined number of ssDNA strands per particle. Its simplicity and tenability make it widely applicable to diverse biosensing applications involving ssDNA functionalized AuNPs.
- Klíčová slova
- DNA functionalization, gold nanoparticles, oligo ethylene glycol, salt-stability, specificity, surface passivation, variable DNA density,
- MeSH
- ethylenglykol chemie MeSH
- fluorescence MeSH
- hybridizace nukleových kyselin MeSH
- jednovláknová DNA chemie genetika MeSH
- kovové nanočástice chemie MeSH
- oligonukleotidy chemie genetika MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ethylenglykol MeSH
- jednovláknová DNA MeSH
- oligonukleotidy MeSH
- zlato 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
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
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
Membrane microdomains play vital roles in the process of bacterial infection. The membrane microdomain-associated protein Flot1 acts in an endocytic pathway and is required for seedling development, however, whether Flot1 is a part of host defense mechanisms remains unknown. During an analysis of callose deposition, we found that Flot1 amiRNAi mutants exhibited defects in response to flg22. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), structured illumination microscopy (SIM) and fluorescence cross spectroscopy (FCS), we determined that the dynamic behavior of GFP-Flot1 in Arabidopsis thaliana cotyledon epidermal cells changed significantly in plants treated with the elicitor flg22. Moreover, we found that Flot1 was constitutively recycled via an endocytic pathway and that flg22 could promote endocytosis. Importantly, targeting of Flot1 to the late endosome/vacuole for degradation increased in response to flg22 treatment; immunoblot analysis showed that when triggered by flg22, GFP-Flot1 was gradually degraded in a time-dependent manner. Taken together, these findings support the hypothesis that the changing of dynamics and oligomeric states can promote the endocytosis and degradation of Flot1 under flg22 treatment in plant cells.
- Klíčová slova
- Dynamics, Endocytosis, Flot1, SIM, VA-TIRFM,
- MeSH
- Arabidopsis účinky léků genetika metabolismus MeSH
- endocytóza účinky léků genetika fyziologie MeSH
- flagelin farmakologie MeSH
- fluorescenční mikroskopie MeSH
- geneticky modifikované rostliny genetika metabolismus MeSH
- kotyledon genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- flagelin MeSH
- proteiny huseníčku MeSH
In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
- Klíčová slova
- SAXS, bilayer, chlorophyll fluorescence, cryo-electron-tomography, electron microscopy, membrane energization, membrane networks, non-bilayer lipid phases, violaxanthin de-epoxidase,
- MeSH
- cirkulární dichroismus metody MeSH
- elektronová mikroskopie metody MeSH
- fotosyntéza genetika MeSH
- lipidy genetika MeSH
- magnetická rezonanční spektroskopie metody MeSH
- tylakoidy genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lipidy 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 major brassinosteroid (BR) receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRI1. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFlot1-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bri1-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.
- Klíčová slova
- BR signaling, BRI1, endocytosis, membrane microdomains, spatiotemporal dynamics,
- MeSH
- Arabidopsis cytologie metabolismus MeSH
- brassinosteroidy farmakologie MeSH
- časoprostorová analýza * MeSH
- difuze MeSH
- endocytóza účinky léků MeSH
- klathrin metabolismus MeSH
- membránové mikrodomény účinky léků metabolismus MeSH
- multimerizace proteinu účinky léků MeSH
- pohyb těles MeSH
- proteinkinasy metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- rostlinné buňky účinky léků metabolismus MeSH
- signální transdukce účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- brassinosteroidy MeSH
- BRI1 protein, Arabidopsis MeSH Prohlížeč
- klathrin MeSH
- proteinkinasy MeSH
- proteiny huseníčku 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.
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
