Erratum to: Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

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.

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

Open Biology ◽

10.1098/rsob.180078 ◽

2018 ◽

Vol 8 (10) ◽

pp. 180078 ◽

Cited By ~ 1

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.

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

Plants ◽

10.3390/plants9040452 ◽

2020 ◽

Vol 9 (4) ◽

pp. 452 ◽

Cited By ~ 2

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.

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Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

Journal of Virology ◽

10.1128/jvi.00469-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8162-8181 ◽

Cited By ~ 17

Author(s):

Audrey Thomas ◽

Charlotte Mariani-Floderer ◽

Maria Rosa López-Huertas ◽

Nathalie Gros ◽

Elise Hamard-Péron ◽

...

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Signaling Pathway ◽

Rho Gtpases ◽

Particle Formation ◽

Actin Dynamics ◽

Membrane Localization ◽

Cortical Actin ◽

Particle Release ◽

Hiv 1

ABSTRACTDuring HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes.IMPORTANCEDuring HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.

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Actin dynamics in cell migration

Essays in Biochemistry ◽

10.1042/ebc20190015 ◽

2019 ◽

Vol 63 (5) ◽

pp. 483-495 ◽

Cited By ~ 22

Author(s):

Matthias Schaks ◽

Grégory Giannone ◽

Klemens Rottner

Keyword(s):

Plasma Membrane ◽

Cell Migration ◽

Actin Filament ◽

Rho Gtpases ◽

Rho Gtpase ◽

Actin Dynamics ◽

Regulatory Complex ◽

Membrane Protrusions ◽

Filament Networks ◽

Contractile Forces

Abstract Cell migration is an essential process, both in unicellular organisms such as amoeba and as individual or collective motility in highly developed multicellular organisms like mammals. It is controlled by a variety of activities combining protrusive and contractile forces, normally generated by actin filaments. Here, we summarize actin filament assembly and turnover processes, and how respective biochemical activities translate into different protrusion types engaged in migration. These actin-based plasma membrane protrusions include actin-related protein 2/3 complex-dependent structures such as lamellipodia and membrane ruffles, filopodia as well as plasma membrane blebs. We also address observed antagonisms between these protrusion types, and propose a model – also inspired by previous literature – in which a complex balance between specific Rho GTPase signaling pathways dictates the protrusion mechanism employed by cells. Furthermore, we revisit published work regarding the fascinating antagonism between Rac and Rho GTPases, and how this intricate signaling network can define cell behavior and modes of migration. Finally, we discuss how the assembly of actin filament networks can feed back onto their regulators, as exemplified for the lamellipodial factor WAVE regulatory complex, tightly controlling accumulation of this complex at specific subcellular locations as well as its turnover.

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Functional analysis of phospholipase Dδ family in tobacco pollen tubes

10.1101/807719 ◽

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.

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A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

Science Advances ◽

10.1126/sciadv.abc6345 ◽

2021 ◽

Vol 7 (13) ◽

pp. eabc6345

Author(s):

Shrawan Kumar Mageswaran ◽

Wei Yuan Yang ◽

Yogaditya Chakrabarty ◽

Catherine M. Oikonomou ◽

Grant J. Jensen

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Electron Tomography ◽

Membrane Damage ◽

Light And Electron Microscopy ◽

Actin Dynamics ◽

Endosomal Sorting ◽

Plasma Membrane Damage ◽

Cryo Electron Tomography ◽

Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

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