Cytoskelet a endocytóza;dynamický systém pro lokalizaci transferů auxinu z buněk

Thanks to its sessile life strategy, the polarity of plant body reflects the polarity of single cells. The polarity is maintained by asymmetric distribution of various molecules downstream from extra and intracellular signals. Directional transport of auxin plays an important role in the pattern formation, morphogenesis, and directional growth responses. The positioning of PIN auxin efflux transporters has been shown to be crucial in the setting of auxin gradients. It is dependent on the plasma membrane deposition of membrane vesicles and their constitutive cycling between plasma membrane and endosomal space. Although some evidences support the idea of differential actin and microtubular cytoskeleton dependence of PIN protein trafficking, there is a significant lack of the information on the role of cytoskeleton in this process. In this paper we use combination of live cell imaging and immunofluorescence techniques to search for the molecular players of actin filaments (AFs) and proteins proteins associated with AFs in the mechanisms of endocytosis and directed PIN1 protein targeting. Seedlings of Arabidopsis thaliana carrying mutations in actin genes (ACT1, ACT2, ACT7, ACT11), Arp 2/3 complex genes (ARP2, ARP3, ARPC2, ARPC5), WAVE complex components genes (BRK1, NAP1, SRA1) and actin monomer...Hlavním cílem této disertační práce bylo popsat roli cytoskeletu a vesikulárního transportu v procesech směrovaného ukládání přenašečů auxin ven z buněk do místa jejich působení, tj. do plazmatické membrány. Propojení mezi transportem auxinu, transportem vesikulů (proteinů) a aktinovým cytoskeletem bylo již v minulosti naznačeno. V naší předchozí práci bylo zjištěno, že v buňkách tabákové kultury BY-2 inhibitor anterográdního transportu brefeldin A (BFA) zpomaluje tok auxinu z buněk a způsobuje přestavbu aktinových filament. Tento účinek na stav aktinového cytoskeletu vyvolal řadu otázek: Jak souvisí stav aktinu s auxinovým transportem? Zdal-li funkční propojení aktinu s endomembránovým systémem je nezbytné pro cyklování auxinových přenašečů a tím pro jejich funkci. Výsledky prezentované v této disertační práci byly získány s použitím modelu buněčné linie tabáku BY-2 a semenáčků rostlin Arabidopsis thaliana. S využitím farmakologického přístupu a in vivo pozorování endocytózy a lokalizace proteinů PIN1 v tabákových buňkách BY-2 bylo poprvé navrženo, že endocytóza barvy FM 4-64 zprostředkovaná klatrinem je závislá jak na aktinové, tak i na mikrotubulární síti. Dále bylo ukázáno, že shlukování aktinu stimulované působením BFA pozorované v naší předchozí práci je zřejmě aktivní proces, neboť...Department of Experimental Plant BiologyKatedra experimentální biologie rostlinFaculty of SciencePřírodovědecká fakult

Cytoskeleton and endocytosis, a dynamic system for the localization of auxin efflux carriers

Thanks to its sessile life strategy, the polarity of plant body reflects the polarity of single cells. The polarity is maintained by asymmetric distribution of various molecules downstream from extra and intracellular signals. Directional transport of auxin plays an important role in the pattern formation, morphogenesis, and directional growth responses. The positioning of PIN auxin efflux transporters has been shown to be crucial in the setting of auxin gradients. It is dependent on the plasma membrane deposition of membrane vesicles and their constitutive cycling between plasma membrane and endosomal space. Although some evidences support the idea of differential actin and microtubular cytoskeleton dependence of PIN protein trafficking, there is a significant lack of the information on the role of cytoskeleton in this process. In this paper we use combination of live cell imaging and immunofluorescence techniques to search for the molecular players of actin filaments (AFs) and proteins proteins associated with AFs in the mechanisms of endocytosis and directed PIN1 protein targeting. Seedlings of Arabidopsis thaliana carrying mutations in actin genes (ACT1, ACT2, ACT7, ACT11), Arp 2/3 complex genes (ARP2, ARP3, ARPC2, ARPC5), WAVE complex components genes (BRK1, NAP1, SRA1) and actin monomer..

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12 janvier 19381938/01/12 (A67).Appartient à l’ensemble documentaire : PoitouCh

Additional file 2: Figure S3. of NtGNL1a ARF-GEF acts in endocytosis in tobacco cells

Description of method used for the evaluation of PM associated endosomes. Endosomes close to PM were magnified (green box) and the probe (represented by red line) was applied through the vesicle and the adjacent PM. The pool of PM-associated endosomes was characterized by histogram values that did not decrease below 60 % of PM (endosome) gray values in the region between PM and endosome. The evaluation was performed using ImageJ (National Institutes of Health). (PDF 116 kb

An Arabidopsis mutant deficient in phosphatidylinositol-4-phosphate kinases ß1 and ß2 displays altered auxin-related responses in roots

International audiencePhosphatidylinositol 4-kinases (PI4Ks) are the first enzymes that commit phosphatidylinositol into the phosphoinositide pathway. Here, we show that Arabidopsis thaliana seedlings deficient in PI4Kβ1 and β2 have several developmental defects including shorter roots and unfinished cytokinesis. The pi4kβ1β2 double mutant was insensitive to exogenous auxin concerning inhibition of root length and cell elongation; it also responded more slowly to gravistimulation. The pi4kß1ß2 root transcriptome displayed some similarities to a wild type plant response to auxin. Yet, not all the genes displayed such a constitutive auxin-like response. Besides, most assessed genes did not respond to exogenous auxin. This is consistent with data with the transcriptional reporter DR5-GUS. The content of bioactive auxin in the pi4kß1ß2 roots was similar to that in wild-type ones. Yet, an enhanced auxin-conjugating activity was detected and the auxin level reporter DII-VENUS did not respond to exogenous auxin in pi4kß1ß2 mutant. The mutant exhibited altered subcellular trafficking behavior including the trapping of PIN-FORMED 2 protein in rapidly moving vesicles. Bigger and less fragmented vacuoles were observed in pi4kß1ß2 roots when compared to the wild type. Furthermore, the actin filament web of the pi4kß1ß2 double mutant was less dense than in wild-type seedling roots, and less prone to rebuilding after treatment with latrunculin B. A mechanistic model is proposed in which an altered PI4K activity leads to actin filament disorganization, changes in vesicle trafficking, and altered auxin homeostasis and response resulting in a pleiotropic root phenotypes