Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the non-essential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain ( cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled. [Media: see text]

Download Full-text

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

10.1101/2020.05.12.090787 ◽

2020 ◽

Author(s):

Benjamin L. Woods ◽

Kevin S. Cannon ◽

Amy S. Gladfelter

Keyword(s):

Cell Shape ◽

Binding Proteins ◽

Membrane Curvature ◽

Amphipathic Helix ◽

Multiple Functions ◽

Gtp Binding Proteins ◽

Amphipathic Helices ◽

Gtp Binding ◽

Necessary And Sufficient ◽

Sensing Membrane

AbstractThe curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the non-essential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In mutants lacking a fully functional Cdc12 AH domain, the Shs1 AH domain becomes essential. Moreover, we find that the Cdc12 AH domain is also important for septin bundling, suggesting multiple functions for septin AH domains.

Download Full-text

An amphipathic helix enables septins to sense micrometer-scale membrane curvature

The Journal of Cell Biology ◽

10.1083/jcb.201807211 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1128-1137 ◽

Cited By ~ 26

Author(s):

Kevin S. Cannon ◽

Benjamin L. Woods ◽

John M. Crutchley ◽

Amy S. Gladfelter

Keyword(s):

Membrane Curvature ◽

Amphipathic Helix ◽

Curvature Sensing ◽

C Terminus ◽

Number Of Binding Sites ◽

Curved Membranes ◽

Necessary And Sufficient ◽

Micrometer Scale

Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

Download Full-text

The long and short of membrane curvature sensing by septins

The Journal of Cell Biology ◽

10.1083/jcb.201903045 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1083-1085 ◽

Cited By ~ 2

Author(s):

Michael A. McMurray

Keyword(s):

Membrane Curvature ◽

Amphipathic Helix ◽

Curvature Sensing ◽

Cell Biol ◽

Necessary And Sufficient

Septin proteins form hetero-oligomers that associate with membranes of specific curvatures, but the mechanism is unknown. In this issue, Cannon et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807211) identify a single amphipathic helix that is necessary and sufficient for membrane curvature sensing by septins.

Download Full-text

An amphipathic helix enables septins to sense micron-scale membrane curvature

10.1101/379982 ◽

2018 ◽

Cited By ~ 2

Author(s):

Kevin S. Cannon ◽

Benjamin L. Woods ◽

John M. Crutchley ◽

Amy S. Gladfelter

Keyword(s):

Membrane Curvature ◽

Amphipathic Helix ◽

Curvature Sensing ◽

C Terminus ◽

Number Of Binding Sites ◽

Curved Membranes ◽

Necessary And Sufficient ◽

Micrometer Scale

AbstractThe geometry of cells is well described by membrane curvature. Septins are filament forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

Download Full-text

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614349 ◽

1999 ◽

Vol 82 (09) ◽

pp. 1177-1181 ◽

Cited By ~ 27

Author(s):

Hubert de Leeuw ◽

Pauline Wijers-Koster ◽

Jan van Mourik ◽

Jan Voorberg

Keyword(s):

Endothelial Cells ◽

Binding Proteins ◽

Binding Protein ◽

Control Release ◽

Small Gtpase ◽

Gtp Binding Protein ◽

Two Dimensional Gel Electrophoresis ◽

Gtp Binding Proteins ◽

Dense Granules ◽

Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Download Full-text

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615073 ◽

1998 ◽

Vol 79 (04) ◽

pp. 832-836 ◽

Cited By ~ 3

Author(s):

Thomas Fischer ◽

Christina Duffy ◽

Gilbert White

Keyword(s):

Molecular Weight ◽

Divalent Cation ◽

Binding Proteins ◽

Binding Protein ◽

Platelet Membrane ◽

Low Molecular Weight ◽

Membrane Glycoproteins ◽

Gtp Binding Protein ◽

Gtp Binding Proteins ◽

Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

Download Full-text

Glucose stimulates islet beta-cell mitogenesis through GTP-binding proteins and by protein kinase C-dependent mechanisms

Diabetes ◽

10.2337/diabetes.46.7.1141 ◽

1997 ◽

Vol 46 (7) ◽

pp. 1141-1147 ◽

Cited By ~ 2

Author(s):

A. Sjoholm

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Beta Cell ◽

Binding Proteins ◽

Islet Beta Cell ◽

Gtp Binding Proteins ◽

Kinase C ◽

Gtp Binding

Download Full-text

Spotting Differences in Molecular Dynamic Simulations of Influenza A M2 Protein-Ligand Complexes by Varying M2 construct, Lipid Bilayer and Force Field

10.26434/chemrxiv.11365943.v1 ◽

2019 ◽

Author(s):

Dimitrios Kolokouris ◽

Iris Kalenderoglou ◽

Panagiotis Lagarias ◽

Antonios Kolocouris

Keyword(s):

Molecular Dynamic ◽

Lipid Bilayer ◽

Influenza A ◽

Md Simulations ◽

Membrane Curvature ◽

Buffer Size ◽

M2 Protein ◽

Dynamic Simulations ◽

Amphipathic Helices ◽

Drug Protein Binding

We studied by molecular dynamic (MD) simulations systems including the inwardclosed state of influenza A M2 protein in complex with aminoadamantane drugs in membrane bilayers. We varied the M2 construct and performed MD simulations in M2TM or M2TM with amphipathic helices (M2AH). We also varied the lipid bilayer by changing either the lipid, DMPC or POPC, POPE or POPC/cholesterol (chol), or the lipids buffer size, 10x10 Å2 or 20x20 Å2. We aimed to suggest optimal system conditions for the computational description of this ion channel and related systems. Measures performed include quantities that are available experimentally and include: (a) the position of ligand, waters and chlorine anion inside the M2 pore, (b) the passage of waters from the outward Val27 gate of M2 S31N in complex with an aminoadamantane-aryl head blocker, (c) M2 orientation, (d) the AHs conformation and structure which is affected from interactions with lipids and chol and is important for membrane curvature and virus budding. In several cases we tested OPLS2005, which is routinely applied to describe drug-protein binding, and CHARMM36 which describes reliably protein conformation. We found that for the description of the ligands position inside the M2 pore, a 10x10 Å2 lipids buffer in DMPC is needed when M2TM is used but 20x20 Å2 lipids buffer of the softer POPC; when M2AH is used all 10x10 Å2 lipid buffers with any of the tested lipids can be used. For the passage of waters at least M2AH with a 10x10 Å2 lipid buffer is needed. The folding conformation of AHs which is defined from hydrogen bonding interactions with the bilayer and the complex with chol is described well with a 10x10 Å2 lipids buffer and CHARMM36.

Download Full-text