scholarly journals Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation

2021 ◽  
Vol 118 (36) ◽  
pp. e2024109118
Author(s):  
Halim Kusumaatmaja ◽  
Alexander I. May ◽  
Mistianne Feeney ◽  
Joseph F. McKenna ◽  
Noboru Mizushima ◽  
...  
Keyword(s):  
Storage Proteins ◽  
Spontaneous Curvature ◽  
Liquid Droplets ◽  
Protein Storage Vacuoles ◽  
Plant Vacuole ◽  
Dicotyledonous Plants ◽  
Membrane Budding ◽  
Membrane Wetting

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241–254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142–146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

Missing pieces in protein deposition and mobilization inside legume seed storage vacuoles: calcium and magnesium ions

2012 ◽  
Vol 22 (4) ◽  
pp. 249-258 ◽  
Author(s):  
Cláudia N. Santos ◽  
Marta M. Alves ◽  
Isabel T. Bento ◽  
Ricardo B. Ferreira
Keyword(s):  
Alkaline Earth ◽  
Storage Proteins ◽  
Lupinus Albus ◽  
Physiological Role ◽  
Experimental Conditions ◽  
Protein Storage Vacuoles ◽  
Aggregation And Disaggregation ◽  
Alkaline Earth Cations

AbstractDuring the maturation of dicotyledonous seeds, organic carbon, nitrogen and sulphur are stored in protein storage vacuoles (PSVs) as storage globulins. Several studies point to the coexistence of storage proteins with proteases responsible for their degradation inside PSVs. Different mechanisms have been proposed to explain why there is no proteolysis during this period. Protein aggregation to form large supramolecular structures resistant to proteolytic attack could be the reason. However, during germination, and particularly following its completion, the globulin aggregates must undergo disintegration to allow protease attack for protein reserve mobilization. Based on the well-described concentration-dependent ability of Ca2+ and Mg2+ to promote in vitro aggregation and disaggregation of globulins, we explored a possible role for these alkaline earth cations in globulin packaging and mobilization. Ca2+ and Mg2+ measurements in purified PSVs [6.37 μmol and 43.9 μmol g− 1 dry weight (DW) of cotyledons, respectively] showed the presence of these two alkaline earth cations within this compartment. To our knowledge, this is the first time that Ca2+ and Mg2+ have been quantified in purified PSVs from Lupinus albus seeds. Considering the importance of these two alkaline earth cations inside PSVs, which represent 14.6% and 60.7% of the total seed Mg2+and Ca2+, respectively, globulin aggregation and disaggregation profiles were assayed using experimental conditions closer to those that are physiologically present (proportion of Ca2+ and Mg2+, and acidic pH). Based on: (1) the high in vivo abundance of Ca2+ and Mg2+ inside PSVs; and (2) globulin aggregation and disaggregation profiles, together with structural and physiological evidence already reported in the literature, an important physiological role for Ca2+ and Mg2+ in globulin packaging and mobilization inside PSVs is suggested.

scholarly journals Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry

2018 ◽  
Vol 115 (6) ◽  
pp. E1127-E1136 ◽  
Author(s):  
Katharina B. Beer ◽  
Jennifer Rivas-Castillo ◽  
Kenneth Kuhn ◽  
Gholamreza Fazeli ◽  
Birgit Karmann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Vesicle ◽  
Endosomal Trafficking ◽  
Vesicle Budding ◽  
Sorting Nexins ◽  
Dnaj Protein ◽  
Membrane Budding ◽  
Pleiotropic Functions

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans. However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

scholarly journals Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads

2021 ◽  
Author(s):  
Alexandre SOUCHAUD ◽  
Arthur BOUTILLON ◽  
Gaëlle CHARRON ◽  
Atef ASNACIOS ◽  
Camille NOÛS ◽  
...  
Keyword(s):  
Shear Stress ◽  
Covalent Binding ◽  
Three Dimensional ◽  
Contour Method ◽  
Shear Stresses ◽  
Liquid Droplets ◽  
Mechanical Constraints

To investigate the role of mechanical constraints in morphogenesis and development, we develop a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane (PDMS) mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate and submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the Zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue, and its time evolution.

scholarly journals Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

2018 ◽  
Author(s):  
Ruchika Sachdev ◽  
Maria Hondele ◽  
Miriam Linsenmeier ◽  
Pascal Vallotton ◽  
Christopher F. Mugler ◽  
...  
Keyword(s):  
Phase Separation ◽  
Direct Interaction ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
The Dead ◽  
Processing Body ◽  
Liquid Liquid Phase Separation

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

Comparative biochemical and morphological changes in imbibed cotton seed hypocotyls and radicles in situ and in vitro – Protein breakdown and elongation growth

1995 ◽  
Vol 5 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Eugene L. Vigil ◽  
Tung K. Fang
Keyword(s):  
Matrix Protein ◽  
Storage Proteins ◽  
Morphological Changes ◽  
Fold Increase ◽  
Hypocotyl Length ◽  
Cortical Cells ◽  
Protein Storage Vacuoles ◽  
Sds Page ◽  
Cotton Seeds

AbstractAxes, hypocotyls and radicles excised from dry cotton seeds (Gossypium hirsutum L. cv. M-8, a double haploid) were imbibed for 24 h and compared with axial segments (excised sections of embryos below the cotyledons) of imbibed, intact seeds. Radicles of excised axes had a 7.4-fold increase in length compared with only 5.2- and 5.7-fold increases, respectively, in radicles of intact seeds and in those isolated when dry. Change in hypocotyl length was not as extensive. EM data for hypocotyl and radicle cortical cells from dry and imbibed seeds revealed a major reduction in matrix protein in protein storage vacuoles along with significant organelle development at 24 h from the start of imbibition. This occurred in parallel with a reduction in salt-extracted proteins and an increase in 2% SDS-extractable proteins. SDS-PAGE of protein from low (0.2 M NaCI) and high (1.0 M NaCI) salt extracts showed a reduction in amount of the major storage proteins (53 and 48 kDa), these bands being almost totally absent in gels of protein extracts from imbibed radicles and significantly reduced in hypocotyls, within 24 h from the start of imbibition. These results indicate that initial elongation of hypocotyls and radicles in intact seeds or of excised axes, after 24 h imbibition, involves breakdown of storage proteins in these axial parts to supply nutrients for growth, with very limited contribution from the cotyledons.

scholarly journals Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin.

1982 ◽  
Vol 93 (3) ◽  
pp. 638-647 ◽  
Author(s):  
H Tamir ◽  
T C Theoharides ◽  
M D Gershon ◽  
P W Askenase
Keyword(s):  
Mast Cells ◽  
Storage Proteins ◽  
Binding Protein ◽  
Protein S ◽  
Intracellular Distribution ◽  
Con A ◽  
Intracellular Storage ◽  
Rbl Cells

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.

scholarly journals Paraspeckles: Paragons of functional aggregation

2015 ◽  
Vol 210 (4) ◽  
pp. 527-528 ◽  
Author(s):  
Edward Courchaine ◽  
Karla M. Neugebauer
Keyword(s):  
Gene Expression ◽  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Body ◽  
Liquid Droplets ◽  
Cell Biol

Low-complexity proteins undergo phase separation in vitro, forming hydrogels or liquid droplets. Whether these form in vivo, and under what conditions, is still unclear. In this issue, Hennig et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201504117) show that formation of the paraspeckle, a nuclear body that regulates gene expression, requires low-complexity prion-like domains (PLDs) within paraspeckle proteins. The same proteins were shown to form hydrogels, shedding light on the role of “functional aggregation” in nuclear substructure.

scholarly journals Curling of epithelial monolayers reveals coupling between active bending and tissue tension

2019 ◽  
Author(s):  
Jonathan Fouchard ◽  
Tom Wyatt ◽  
Amsha Proag ◽  
Ana Lisica ◽  
Nargess Khalilgharibi ◽  
...  
Keyword(s):  
Free Edge ◽  
The Body ◽  
Spontaneous Curvature ◽  
Cell Sheets ◽  
Mechanical Coupling ◽  
Epithelial Monolayers ◽  
Bending Modulus ◽  
Out Of Plane

Epithelial monolayers are two-dimensional cell sheets which compartmentalise the body and organs of multi-cellular organisms. Their morphogenesis during development or pathology results from patterned endogenous and exogenous forces and their interplay with tissue mechanical properties. In particular, bending of epithelia is thought to results from active torques generated by the polarization of myosin motors along their apico-basal axis. However, the contribution of these out-of-plane forces to morphogenesis remains challenging to evaluate because of the lack of direct mechanical measurement. Here, we use epithelial curling to characterize the out-of-plane mechan ics of epithelial monolayers. We find that curls of high curvature form spontaneously at the free edge of epithelial monolayers devoid of substrate in vivo and in vitro. Curling originates from an enrichment of myosin in the basal domain that generates an active spontaneous curvature. By measuring the force necessary to flatten curls, we can then estimate the active torques and the bending modulus of the tissue. Finally, we show that the extent of curling is controlled by the interplay between in-plane and out-of-plane stresses in the monolayer. Such mechanical coupling implies an unexpected role for in-plane stresses in shaping epithelia during morphogenesis.

scholarly journals Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ruchika Sachdev ◽  
Maria Hondele ◽  
Miriam Linsenmeier ◽  
Pascal Vallotton ◽  
Christopher F Mugler ◽  
...  
Keyword(s):  
Phase Separation ◽  
Direct Interaction ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
The Dead ◽  
Processing Body ◽  
Liquid Liquid Phase Separation

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

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