The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.

• MeSH
• Arabidopsis metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• endozomy metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• Golgiho aparát metabolismus   MeSH
• konfokální mikroskopie MeSH
• listy rostlin metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny biosyntéza   genetika   MeSH
• tabák metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Specific gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy recently emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen tubes. Here, we describe the protocol for the successful employment of AODN technique in growing tobacco pollen tubes, covering AODN design, application, and analysis of the results. We also discuss the advantages and drawbacks of this method.

• MeSH
• antisense oligonukleotidy genetika   MeSH
• fenotyp MeSH
• genový knockdown * MeSH
• pylová láčka genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• tabák genetika   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Trafficking of proteins and lipids within the plant endomembrane system is essential to support cellular functions and is subject to rigorous regulation. Despite this seemingly strict regulation, endomembrane trafficking needs to be dynamically adjusted to ever-changing internal and environmental stimuli, while maintaining cellular integrity. Although often overlooked, the versatile second messenger Ca2+is intimately connected to several endomembrane-associated processes. Here, we discuss the impact of electrostatic interactions between Ca2+and anionic phospholipids on endomembrane trafficking, and illustrate the direct role of Ca2+sensing proteins in regulating endomembrane trafficking and membrane integrity preservation. Moreover, we discuss how Ca2+can control protein sorting within the plant endomembrane system. We thus highlight Ca2+signaling as a versatile mechanism by which numerous signals are integrated into plant endomembrane trafficking dynamics.

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• fosfolipidy metabolismus   MeSH
• fyziologie rostlin * MeSH
• rostlinné proteiny metabolismus   MeSH
• transport proteinů MeSH
• vápníková signalizace * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• exocytóza * MeSH
• fosfatidylinositol-4,5-difosfát chemie   metabolismus   MeSH
• kinetika MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• rho proteiny vázající GTP chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekreční dráha MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• vezikulární transportní proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Successful establishment and maintenance of cell polarity is crucial for many aspects of plant development, cellular morphogenesis, response to pathogen attack, and reproduction. Polar cell growth depends on integrating membrane and cell-wall dynamics with signal transduction pathways, changes in ion membrane transport, and regulation of vectorial vesicle trafficking and the dynamic actin cytoskeleton. In this review, we address the critical importance of protein-membrane crosstalk in the determination of plant cell polarity and summarize the role of membrane lipids, particularly minor acidic phospholipids, in regulation of the membrane traffic. We focus on the protein-membrane interface dynamics and discuss the current state of knowledge on three partially overlapping levels of descriptions. Finally, due to their multiscale and interdisciplinary nature, we stress the crucial importance of combining different strategies ranging from microscopic methods to computational modelling in protein-membrane studies.

• MeSH
• fyziologie rostlin * genetika   MeSH
• membránové lipidy metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• polarita buněk MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny metabolismus   MeSH
• signální transdukce * MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Retroviral Gag polyproteins are targeted to the inner leaflet of the plasma membrane through their N-terminal matrix (MA) domain. Because retroviruses of different morphogenetic types assemble their immature particles in distinct regions of the host cell, the mechanism of MA-mediated plasma membrane targeting differs among distinct retroviral morphogenetic types. Here, we focused on possible mechanistic differences of the MA-mediated plasma membrane targeting of the B-type mouse mammary tumor virus (MMTV) and C-type HIV-1, which assemble in the cytoplasm and at the plasma membrane, respectively. Molecular dynamics simulations, together with surface mapping, indicated that, similarly to HIV-1, MMTV uses a myristic switch to anchor the MA to the membrane and electrostatically interacts with phosphatidylinositol 4,5-bisphosphate to stabilize MA orientation. We observed that the affinity of MMTV MA to the membrane is lower than that of HIV-1 MA, possibly related to their different topologies and the number of basic residues in the highly basic MA region. The latter probably reflects the requirement of C-type retroviruses for tighter membrane binding, essential for assembly, unlike for D/B-type retroviruses, which assemble in the cytoplasm. A comparison of the membrane topology of the HIV-1 MA, using the surface-mapping method and molecular dynamics simulations, revealed that the residues at the HIV-1 MA C terminus help stabilize protein-protein interactions within the HIV-1 MA lattice at the plasma membrane. In summary, HIV-1 and MMTV share common features such as membrane binding of the MA via hydrophobic interactions and exhibit several differences, including lower membrane affinity of MMTV MA.

• MeSH
• buněčná membrána metabolismus   patologie   MeSH
• HIV infekce metabolismus   patologie   MeSH
• HIV-1 fyziologie   MeSH
• infekce onkogenními viry metabolismus   patologie   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• molekulární modely MeSH
• myši MeSH
• retrovirové infekce metabolismus   patologie   MeSH
• sestavení viru MeSH
• virus myšího tumoru prsní žlázy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Plants respond to diverse biotic and abiotic stimuli as well as to endogenous developmental cues. Many of these stimuli result in altered activity of phospholipase D (PLD), an enzyme that hydrolyzes structural phospholipids producing phosphatidic acid (PA). PA is a key signaling intermediate in animals, but its targets in plants are relatively uncharacterized. Recent studies have demonstrated that the cytoskeleton is a major target of PLD-PA signaling and identified a positive feedback loop between actin turnover and PLD activity. Moreover, two cytoskeletal proteins, capping protein and MAP65-1, have been identified as PA-binding proteins regulating actin and microtubule organization and dynamics. In this review, we highlight the role of the PLD-PA module as an important hub for housekeeping and stress-induced regulation of membrane-associated cytoskeletal dynamics.

• MeSH
• aktiny metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• fyziologický stres MeSH
• fyziologie rostlin MeSH
• kyseliny fosfatidové metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• rostliny enzymologie   MeSH
• signální transdukce MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP(2)). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP(2)/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP-membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.

• MeSH
• aktin zastřešující proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• fosfatidylinositolfosfáty chemie   metabolismus   MeSH
• fylogeneze MeSH
• hydrofobní a hydrofilní interakce MeSH
• kur domácí MeSH
• kyseliny fosfatidové chemie   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• proteiny huseníčku antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• ptačí proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vazba proteinů MeSH
• výpočetní biologie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics.

• MeSH
• biosenzitivní techniky metody   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• diglyceridy metabolismus   MeSH
• fluorescence MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• fotovybělování MeSH
• kyseliny fosfatidové analýza   metabolismus   MeSH
• počítačové zpracování obrazu MeSH
• proteiny Qb-SNARE genetika   metabolismus   MeSH
• proteiny Qc-SNARE genetika   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• tabák cytologie   metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Membrane lipids and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane-cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD-actin interactions. Inhibition of PLD by n-butanol compromises pollen tube actin, and PA rescues the detrimental effect of n-butanol on F-actin, showing clearly the importance of the PLD-PA interaction for pollen tube F-actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDbeta1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP(2)-dependent PLD (PLDbeta) is specifically enhanced by F-actin and inhibited by G-actin. We then identified the NtPLDbeta1 domain responsible for actin interactions. Using sequence- and structure-based analysis, together with site-directed mutagenesis, we identified Asn323 and Thr382 of NtPLDbeta1 as the crucial amino acids in the actin-interacting fold. The effect of antisense-mediated suppression of NtPLDbeta1 or NtPLDdelta on pollen tube F-actin dynamics shows that NtPLDbeta1 is the active partner in PLD-actin interplay. The positive feedback loop created by activation of PLDbeta by F-actin and of F-actin by PA provides an important mechanism to locally increase membrane-F-actin dynamics in the cortex of plant cells.

• MeSH
• aktiny metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• genový knockdown MeSH
• izoenzymy metabolismus   MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• pylová láčka růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• tabák enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH