scholarly journals Functional analysis of phospholipase Dδ family in tobacco pollen tubes

2019 ◽  
Author(s):  
Přemysl Pejchar ◽  
Juraj Sekereš ◽  
Ondřej Novotný ◽  
Viktor Žárský ◽  
Martin Potocký
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Catalytic Domain ◽  
Bioinformatic Analysis ◽  
Pollen Tubes ◽  
Lipid Binding ◽  
Functional Roles ◽  
Knock Down ◽  
Tobacco Pollen

SummaryPhosphatidic acid (PA), important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD) but the isoforms responsible for production of plasma membrane PA were not identified yet and their functional roles remain unknown. Following genome-wide bioinformatic analysis of PLD family in tobacco, we focused on the pollen-overrepresented PLDδ class. Combining live-cell imaging, gene overexpression or knock-down, lipid-binding and structural bioinformatics, we characterized 5 NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Knock-down, overexpression and in vivo PA level analyses revealed isofom PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. PA promotes binding of PLDδ3 to the PM, thus creating a positive feedback loop, where PA accumulation leads to the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes, that are crucial for plant cell tip growth.

scholarly journals Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 452 ◽  
Author(s):  
Irene Stenzel ◽  
Till Ischebeck ◽  
Linh Hai Vu-Becker ◽  
Mara Riechmann ◽  
Praveen Krishnamoorthy ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pollen Tube ◽  
Tip Growth ◽  
Pollen Tubes ◽  
Dominant Negative ◽  
Membrane Domains ◽  
Tobacco Pollen ◽  
Polar Tip Growth ◽  
Spinning Disc

Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2. Here, we show that fluorescence-tagged tobacco NtPLC3 displays a subapical plasma membrane distribution which frames that of fluorescence-tagged PI4P 5-kinases, suggesting that NtPLC3 may modulate PtdIns(4,5)P2-mediated processes in pollen tubes. The expression of a dominant negative NtPLC3 variant resulted in pollen tube tip swelling, consistent with a delimiting effect on PtdIns(4,5)P2 production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P2 function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P2 antagonizes the effects of PtdIns(4,5)P2 overproduction in vivo. When analyzed by spinning disc microscopy, fluorescence-tagged NtPLC3 displayed discontinuous membrane distribution omitting punctate areas of the membrane, suggesting that NtPLC3 is involved in the spatial restriction of plasma membrane domains also at the nanodomain scale. Together, the data indicate that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P2 in the subapical plasma membrane of pollen tubes.

scholarly journals Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

2020 ◽  
Author(s):  
Marta Fratini ◽  
Praveen Krishnamoorthy ◽  
Irene Stenzel ◽  
Mara Riechmann ◽  
Kirsten Bacia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Pollen Tubes ◽  
Actin Dynamics ◽  
Tobacco Pollen ◽  
Dual Distribution ◽  
Cytoskeletal Dynamics

AbstractPollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2-effects are specified is unclear. Spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labelling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, AtPIP5K2 and NtPIP5K6 display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2 and NtPIP5K6 variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, PI4P 5-kinase variants targeted to nanodomains stabilized actin, suggesting a specific function of PtdIns(4,5)P2-nanodomains. A distinct role of nanodomain-associated AtPIP5K2 in actin regulation is further supported by proximity to and interaction with the Rho-GTPase NtRac5, and by functional interplay with elements of ROP-signalling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2-functions to coordinate cytoskeletal dynamics and secretion in pollen tubes.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Extended Synaptotagmin is a presynaptic ER Ca2+ sensor that promotes neurotransmission and synaptic growth in Drosophila

2017 ◽  
Author(s):  
Koto Kikuma ◽  
Daniel Kim ◽  
David Sutter ◽  
Xiling Li ◽  
Dion K. Dickman
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Synaptic Activity ◽  
Synaptic Growth ◽  
Synaptic Physiology ◽  
Lipid Exchange ◽  
Membrane Tethering ◽  
Conserved Gene ◽  
Neuronal Functions

ABSTRACTThe endoplasmic reticulum (ER) is an extensive presynaptic organelle, exerting important influences at synapses by responding to Ca2+ and modulating transmission, growth, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, we have generated and characterized mutations in the single Extended Synaptotagmin (Esyt) ortholog in Drosophila. Esyts are evolutionarily conserved ER proteins with Ca2+ sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. We first demonstrate that Esyt localizes to an extensive ER structure that invades presynaptic terminals at the neuromuscular junction. Next, we show that synaptic growth, structure, function, and plasticity are surprisingly unperturbed at synapses lacking Esyt expression. However, presynaptic overexpression of Esyt leads to enhanced synaptic growth, neurotransmission, and sustainment of the vesicle pool during intense levels of activity, suggesting that elevated Esyt at the ER promotes constitutive membrane trafficking or lipid exchange with the plasma membrane. Finally, we find that Esyt mutants fail to maintain basal neurotransmission and short term plasticity at elevated extracellular Ca2+, consistent with Esyt functioning as an ER Ca2+ sensor that modulates synaptic activity. Thus, we identify Esyt as a presynaptic ER Ca2+ sensor that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family.

scholarly journals Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

2019 ◽  
Vol 2 (5) ◽  
pp. e201900431 ◽  
Author(s):  
Walaa E Kattan ◽  
Wei Chen ◽  
Xiaoping Ma ◽  
Tien Hung Lan ◽  
Dharini van der Hoeven ◽  
...  
Keyword(s):  
Biological Activity ◽  
Plasma Membrane ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Membrane Anchor ◽  
Normal Cells

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

scholarly journals Microtubules play a role in trafficking prevacuolar compartments to vacuoles in tobacco pollen tubes

Open Biology ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 180078 ◽  
Author(s):  
Elisabetta Onelli ◽  
Monica Scali ◽  
Marco Caccianiga ◽  
Nadia Stroppa ◽  
Piero Morandini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pollen Tube ◽  
Biochemical Analysis ◽  
Pollen Tubes ◽  
Degradation Pathways ◽  
Transient Transformation ◽  
Secretory Pathways ◽  
Tobacco Pollen ◽  
Fine Regulation ◽  
Pollen Tube Elongation

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.

scholarly journals Apical targeting of syntaxin 3 is essential for epithelial cell polarity

2006 ◽  
Vol 173 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Nikunj Sharma ◽  
Seng Hui Low ◽  
Saurav Misra ◽  
Bhattaram Pallavi ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Membrane Trafficking ◽  
Cell Polarization ◽  
Apical Plasma Membrane ◽  
Membrane Expression ◽  
Tight Junction Formation ◽  
Necessary And Sufficient ◽  
Syntaxin 3

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

scholarly journals Sphingosine kills bacteria by binding to cardiolipin

2020 ◽  
Vol 295 (22) ◽  
pp. 7686-7696 ◽  
Author(s):  
Rabea Verhaegh ◽  
Katrin Anne Becker ◽  
Michael J. Edwards ◽  
Erich Gulbins
Keyword(s):  
Plasma Membrane ◽  
Bactericidal Activity ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Central Element ◽  
Lipid Binding ◽  
Bacterial Cells ◽  
Bacterial Killing

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively–charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo. We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

scholarly journals Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

2014 ◽  
Vol 204 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Alex J. Smith ◽  
Byung-Ju Jin ◽  
Julien Ratelade ◽  
Alan S. Verkman
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Water Channel ◽  
Aquaporin 4 ◽  
Cellular Functions ◽  
Aggregation State ◽  
Functional Roles ◽  
State Dependent ◽  
And Function

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.

scholarly journals EHD Proteins Associate with Syndapin I and II and Such Interactions Play a Crucial Role in Endosomal Recycling

2005 ◽  
Vol 16 (8) ◽  
pp. 3642-3658 ◽  
Author(s):  
Anne Braun ◽  
Roser Pinyol ◽  
Regina Dahlhaus ◽  
Dennis Koch ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Crucial Role ◽  
Mutational Analysis ◽  
Protein Complexes ◽  
Binding Interface ◽  
Endosomal Recycling ◽  
Eh Domain ◽  
Ehd Proteins

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin–EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.

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