Biology subjects

Habenstein, B.

Publications and source records attributed to Habenstein, B..

6 recordsLinked to original sources

bioRxiv2026

ATP-driven membrane binding and polymerization of bacterial actin MreB promotes local membrane fluidization

The bacterial actin homologue MreB plays a key role in rod cell shape determination. We recently showed that MreB from the Gram-positive bacterium Geobacillus stearothermophilus (MreBGs) polymerizes into straight pairs of protofilaments in the presence of both ATP and a lipid surface. Membrane interaction is thought to be mediated by electrostatic interactions with anionic lipids, with final anchoring relying on two spatially close hydrophobic motifs that protrude from the MreBGs monomers, forming a putative membrane-insertion domain. Here, we determined the binding properties of ATP and ADP to MreBGs using fluorescence anisotropy, and monitored ATP-mediated binding and polymer formation on lipid bilayers using liposome binding assays and AFM, respectively. Finally, we used solid-state NMR to visualize the interaction between the membrane and MreBGs at the atomic level. Our findings reveal that MreBGs has similar affinity for both ATP and ADP, unlike eukaryotic actin. We also show that monomeric MreBGs establishes peripheral contacts with the membrane likely through electrostatic interactions, while ATP-induced MreBGs filaments insert into the lipid bilayer without interfering with the membrane lamellar phase and have a significant local fluidifying effect. Statement of significanceBacteria rely on the actin-like protein MreB to determine and maintain their cell shape, like actin does in eukaryotic cells. To perform its tightly regulated morphogenetic function, MreB forms membrane-associated nanofilaments in vivo, which control the cell wall biosynthetic machinery. We recently demonstrated that, in vitro, MreB from the Gram-positive bacterium Geobacillus stearothermophilus requires both ATP and lipids to polymerize into pairs of filaments. Here, we show that in the presence of ATP, Geobacillus MreB forms membrane-bound filaments that directly impact local membrane fluidity, which could translate into a regulatory effect on cell wall synthetic enzymes. We further reveal that MreB binds both ATP and ADP with similar affinity, unlike eukaryotic actin, which preferentially binds ATP over ADP, and speculate that this could be a mechanism modulating the pool of polymerization-competent MreB in bacteria.

biophysics↗

bioRxiv2023

The actin depolymerizing factor StADF2 alters StREM1.3 plasma membrane nanodomains to inhibit the Potato Virus X

The dynamic regulation of the plasma membrane (PM) organization at the nanoscale emerged as a key element shaping the outcome of host-microbe interactions. Protein organization into nanodomains (ND) is often assumed to be linked to the activation of cellular processes. In contrast, we have previously shown that the phosphorylation of the Solanum tuberosum REM1.3 (StREM1.3) N-terminal domain disperses its native ND organization and promotes its inhibitory effect on Potato Virus X (PVX) cell-to-cell movement. Here, we show that the phosphorylation of StREM1.3 modify the chemical environment of numerous residues in its intrinsically-disordered N-terminal domain. We leveraged exploratory screens to identify potential phosphorylation-dependent interactors of StREM1.3. Herewith, we uncovered uncharacterized regulators of PVX cell-to-cell movement, linking StREM1.3 to autophagy, water channels and the actin cytoskeleton. We show that the Solanum tuberosum actin depolymerizing factors 2 (StADF2) alters StREM1.3 NDs and limits PVX cell-to-cell movement in a REMORIN-dependent manner. Mutating a conserved single residue reported to affect ADFs affinity to actin inhibits StADF2 effect on StREM1.3 ND organization and PVX cell-to-cell movement. These observations provide functional links between the organization of plant PM and the actin cytoskeleton and suggests that the alteration of StREM1.3 ND organization promotes plant anti-viral responses. We envision that analogous PM re-organization applies for additional signaling pathways in plants and in other organisms.

cell biology↗

bioRxiv2022

Driving forces behind remorin nanodomain formation in anionic lipid membranes

Remorins are a family of multigenic phosphoproteins of the plasma membrane, involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nano-sized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains, however the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated PhosphatidylCholine (PC), PhosphatidylInositol Phosphates (PIPs), and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nano-sized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces. Summary headingUsing reconstituted membranes, we demonstrated the clustering of the plant protein remorins StREM1.3 to the lipid bilayer external leaflet and monitor the formation of nanodomains of the protein.

plant biology↗

bioRxiv2021

Protein resonance assignment by solid-state NMR based on 1H-detected 13C-based double-quantum spectroscopy at fast MAS

Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (1H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of 1H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) 13C spectroscopy, detected on 1H, is introduced at fast MAS (70 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional 1H detected experiments correlating a 13C DQ dimension respectively to its intra-residue and sequential 15N-1H pairs, a sequential walk through DQ (C+CO) resonance is obtained. Our approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process.

biophysics↗

bioRxiv2020

Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behaviour and distinct patho-phenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and shed new light on key molecular features concerning an important class of pathogenic agents.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Emergence of stealth polymorphs that escape α-synuclein amyloid monitoring, take over and acutely spread in neurons

The conformational strain diversity characterizing -synuclein (-syn) amyloid fibrils is possibly at the origin of the different clinical presentations of synucleinopathies. Experimentally, various -syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe Thioflavin T (ThT). We report here that besides classical ThT positive products, fibrillization in saline simultaneously gives rise to competing fibril polymorphs that are invisible to ThT (stealth polymorphs), and that can take over. Due to competition, spontaneous generation of such stealth polymorphs bears on the apparent fibrillization kinetics and on the final plateau values. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. Compared to their ThT-positive counterparts, and as judged from their chemical shift resonances fingerprint, these new stealth polymorphs present a yet undescribed atomic organization and show an exacerbated propensity (approx. 20-fold) towards self-replication in cortical neurons. They also trigger a long distance synucleinopathic spread along nigro-striatal projections in vivo. In order to rapidly screen fibrillization products for the presence of such stealth polymorphs, we designed a simple multiplexed assay that can be easily and rapidly operated. This assay allows us to demonstrate the sustainability of the conformational replication of these novel and particularly invasive strains. It should also be of help to avoid erroneous upstream interpretations of fibrillization rates based on sole ThT, and to expedite further structural and functional characterization of stealth amyloid assemblies. One Sentence Summarystealth -synuclein fibrils take over

neuroscience↗