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.

DIACYLGLYCEROL KINASE 5 regulates polar tip growth of tobacco pollen tubes

2021 ◽  
Author(s):  
Patricia Scholz ◽  
Přemysl Pejchar ◽  
Max Fernkorn ◽  
Eliška Škrabálková ◽  
Roman Pleskot ◽  
...  
Keyword(s):  
Tip Growth ◽  
Diacylglycerol Kinase ◽  
Pollen Tubes ◽  
Tobacco Pollen ◽  
Polar Tip Growth

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 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 Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth

2021 ◽  
Vol 40 (2) ◽  
pp. 205-222
Author(s):  
Monica Scali ◽  
Alessandra Moscatelli ◽  
Luca Bini ◽  
Elisabetta Onelli ◽  
Rita Vignani ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Tip Growth ◽  
Male Gametophyte ◽  
Pollen Tubes ◽  
Structural Features ◽  
Two Dimensional Gel Electrophoresis ◽  
Depth Analysis

AbstractPollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identifyNicotiana tabacumDifferentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC–ESI–MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain

1999 ◽  
Vol 112 (12) ◽  
pp. 1957-1965 ◽  
Author(s):  
K. Venkateswarlu ◽  
F. Gunn-Moore ◽  
J.M. Tavare ◽  
P.J. Cullen
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Head Group ◽  
Nucleotide Exchange ◽  
Ph Domain

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

Morphology of Cell Injury: An Approach to the EDIT Programme by the Use of Tobacco Pollen Tubes

2002 ◽  
Vol 30 (3) ◽  
pp. 323-329 ◽  
Author(s):  
Udo Kristen ◽  
Natalie Bischoff ◽  
Saskia Lisboa ◽  
Enno Schirmer ◽  
Sören Witt ◽  
...  
Keyword(s):  
Tip Growth ◽  
Cell Injury ◽  
Cytosolic Calcium ◽  
Pollen Tubes ◽  
In Vitro Cytotoxicity ◽  
Toxic Compounds ◽  
In Vitro System ◽  
Tobacco Pollen ◽  
Different Types

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubu-lin immunofluorescence and rhodamin–phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.

scholarly journals Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1252-1262 ◽  
Author(s):  
Dale J. Powner ◽  
Matthew N. Hodgkin ◽  
Michael J.O. Wakelam
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Enzyme Activation ◽  
Dominant Negative ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals Signaling in Pollen Tube Growth: Beyond the Tip of the Polarity Iceberg

Plants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 156 ◽  
Author(s):  
Nolan Scheible ◽  
Andrew McCubbin
Keyword(s):  
Pollen Tube ◽  
Signaling Pathways ◽  
Pollen Tube Growth ◽  
Tip Growth ◽  
Holistic Approach ◽  
Pollen Tubes ◽  
Small Gtpase ◽  
Tube Growth ◽  
Concerted Action ◽  
Current State

The coordinated growth of pollen tubes through floral tissues to deliver the sperm cells to the egg and facilitate fertilization is a highly regulated process critical to the Angiosperm life cycle. Studies suggest that the concerted action of a variety of signaling pathways underlies the rapid polarized tip growth exhibited by pollen tubes. Ca2+ and small GTPase-mediated pathways have emerged as major players in the regulation of pollen tube growth. Evidence suggests that these two signaling pathways not only integrate with one another but also with a variety of other important signaling events. As we continue to elucidate the mechanisms involved in pollen tube growth, there is a growing importance in taking a holistic approach to studying these pathways in order to truly understand how tip growth in pollen tubes is orchestrated and maintained. This review considers our current state of knowledge of Ca2+-mediated and GTPase signaling pathways in pollen tubes, how they may intersect with one another, and other signaling pathways involved. There will be a particular focus on recent reports that have extended our understanding in these areas.

scholarly journals Pollen Tube Growth in Carya and Temporal Influence of Pollen Deposition on Fertilization Success in Pecan

1992 ◽  
Vol 117 (2) ◽  
pp. 328-331 ◽  
Author(s):  
Robert D. Marquard
Keyword(s):  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Pollen Tubes ◽  
Fertilization Success ◽  
Tube Growth ◽  
In Vivo Studies ◽  
Pollen Deposition ◽  
Cape Fear

In vivo pollen tube growth of pecan [Carya illinoinensis (Wangenh.) K. Koch] was estimated to be ≈ 150 μm·hour-1 from 3 to 8 hours postpollination. Pollen tubes averaged 47, 194, 405, and 946 μm after 2, 3, 4, and 8 hours postpollination, respectively. Pollen tube growth was strongly influenced by temperature, and in vitro studies demonstrated pollen germination and tube growth were optimal at 27C for `Cape Fear' pecan. In in vivo studies, tubes of cross-pollen did not grow significantly faster than tubes of self-pollen. Pollen tubes of water hickory [C. aquatica (Michx. f.) Nutt.] grew significantly faster than those of C. illinoinensis. Bitternut [C. cordiformis (Wangenh.) K. Koch] and mockernut hickory (C. tomentosa Nutt.) pollen tubes grew significantly slower on pecan stigmas than did pecan pollen. Pollen arriving first on the stigma has a decided advantage for fertilization success of pecan. The fertilization success rate of pecan pollen arriving 24 hours after first pollen arrival was <3%.

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