Membrane curvature sensing of the lipid-anchored K-Ras small GTPase

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-RasG12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-RasG12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-RasG12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.

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Nanoscale manipulation of membrane curvature for probing endocytosis in live cells

10.1101/122275 ◽

2017 ◽

Author(s):

Wenting Zhao ◽

Lindsey Hanson ◽

Hsin-Ya Lou ◽

Matthew Akamatsu ◽

Praveen D. Chowdary ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Curvature ◽

Live Cells ◽

Radius Of Curvature ◽

Reciprocal Regulation ◽

Associated Proteins ◽

Curved Membranes ◽

Related Proteins ◽

Curved Membrane ◽

Positively Curved

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species1,2. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature1,3,4. However, the reciprocal regulation of how plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions5-8. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference toward positive membrane curvatures with a radius < 200 nm. Of ten CME related proteins we examined, all show preferences to positively curved membrane. By contrast, other membrane-associated proteins and non-CME endocytic protein, caveolin1, show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

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Rho1 regulates apoptosis via activation of the JNK signaling pathway at the plasma membrane

The Journal of Cell Biology ◽

10.1083/jcb.200912010 ◽

2010 ◽

Vol 189 (2) ◽

pp. 311-323 ◽

Cited By ~ 49

Author(s):

Amanda L. Neisch ◽

Olga Speck ◽

Beth Stronach ◽

Richard G. Fehon

Keyword(s):

Plasma Membrane ◽

Cell Survival ◽

Cell Shape ◽

Rho Kinase ◽

Small Gtpase ◽

Cell Cortex ◽

Jnk Pathway ◽

Jnk Signaling ◽

Upstream Regulator ◽

Jnk Signaling Pathway

Precisely controlled growth and morphogenesis of developing epithelial tissues require coordination of multiple factors, including proliferation, adhesion, cell shape, and apoptosis. RhoA, a small GTPase, is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton. In this study, we examine a less well-characterized RhoA function in cell survival. We demonstrate that the Drosophila melanogaster RhoA, Rho1, promotes apoptosis independently of Rho kinase through its effects on c-Jun NH2-terminal kinase (JNK) signaling. In addition, Rho1 forms a complex with Slipper (Slpr), an upstream activator of the JNK pathway. Loss of Moesin (Moe), an upstream regulator of Rho1 activity, results in increased levels of Rho1 at the plasma membrane and cortical accumulation of Slpr. Together, these results suggest that Rho1 functions at the cell cortex to regulate JNK activity and implicate Rho1 and Moe in epithelial cell survival.

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Aurora-A Phosphorylates, Activates, and Relocalizes the Small GTPase RalA

Molecular and Cellular Biology ◽

10.1128/mcb.00916-08 ◽

2009 ◽

Vol 30 (2) ◽

pp. 508-523 ◽

Cited By ~ 76

Author(s):

Kian-Huat Lim ◽

Donita C. Brady ◽

David F. Kashatus ◽

Brooke B. Ancrile ◽

Channing J. Der ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Growth ◽

Phosphorylation Site ◽

Small Gtpase ◽

Effector Protein ◽

Ras Signaling ◽

Aurora A ◽

Transformed Cell ◽

Oncogenic Ras ◽

Extracellular Signals

ABSTRACT The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA.

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C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609917113 ◽

2016 ◽

Vol 113 (47) ◽

pp. E7590-E7599 ◽

Cited By ~ 29

Author(s):

Jihong Gong ◽

Ying Lai ◽

Xiaohong Li ◽

Mengxian Wang ◽

Jeremy Leitz ◽

...

Keyword(s):

Plasma Membrane ◽

Neurotransmitter Release ◽

Membrane Curvature ◽

Neuronal Cultures ◽

Spontaneous Release ◽

Vesicle Membrane ◽

Curvature Sensing ◽

Postsynaptic Currents ◽

Terminal Domain

In presynaptic nerve terminals, complexin regulates spontaneous “mini” neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

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Annexin A4 trimers are recruited by high membrane curvatures in Giant Plasma Membrane Vesicles

10.1101/2020.02.20.957183 ◽

2020 ◽

Cited By ~ 1

Author(s):

Christoffer Florentsen ◽

Alexander Kamp-Sonne ◽

Guillermo Moreno-Pescador ◽

Weria Pezeshkian ◽

Ali Asghar Hakami Zanjani ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Membrane Curvature ◽

Biological Functions ◽

Membrane Repair ◽

Plasma Membrane Vesicles ◽

Curvature Sensing ◽

Curved Structures ◽

Annexin A4 ◽

Membrane Budding

AbstractThe plasma membrane of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the plasma membrane (PM) is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including plasma membrane repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant plasma membrane vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

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Endophilin-A controls recruitment, priming and fusion of neurosecretory vesicles

10.1101/540864 ◽

2019 ◽

Author(s):

Sindhuja Gowrisankaran ◽

Vicky Steubler ◽

Sébastien Houy ◽

Johanna G. Peña del Castillo ◽

Monika Gelker ◽

...

Keyword(s):

Plasma Membrane ◽

Chromaffin Cells ◽

Sh3 Domain ◽

Membrane Curvature ◽

List Type ◽

Secretory Vesicles ◽

Curvature Sensing ◽

Vesicle Pools ◽

Endocytic Traffic ◽

Efficient Distribution

SUMMARYEndophilins-A are conserved endocytic adaptors with membrane curvature-sensing and - inducing properties. We show here that, independently of their role in endocytosis, endophilin-A1 and endophilin-A2 regulate exocytosis of neurosecretory vesicles. The number of neurosecretory vesicles was not altered in chromaffin cells without endophilin, yet fast capacitance and amperometry measurements revealed reduced exocytosis, smaller vesicle pools and changed fusion kinetics. Both endophilin-A1 (brain-enriched) and A2 (ubiquitous) rescued exocytic defects, but endophilin-A2 was more efficient. Distribution of neurosecretory vesicles was altered in the plasma membrane proximity, but levels and distributions of main exocytic and endocytic factors were unchanged, and slow compensatory endocytosis was not robustly affected. Endophilin’s role in exocytosis is mediated through its SH3-domain and, at least in part, interaction with intersectin, a coordinator of exocytic and endocytic traffic. Altogether, we report that endophilins-A, key endocytic proteins linked to neurodegeneration, directly regulate exocytosis by controlling vesicle recruitment, priming and fusion.Abstract FigureRecruitment, priming and fusion of secretory vesicles is controlled by endophilinLack of endophilins alters the distribution of secretory vesicles near the PMEndophilin’s role in exocytosis is mediated through its SH3-domainEndophilin regulates intersectin localization by keeping it away from the PM

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Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002440 ◽

1980 ◽

Vol 38 ◽

pp. 552-553

Author(s):

K.I. Pagh ◽

M.R. Adelman

Keyword(s):

Cell Shape ◽

Physarum Polycephalum ◽

Slime Mold ◽

Live Cells ◽

Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

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