Biology subjects

Mavrakis, M.

Publications and source records attributed to Mavrakis, M..

3 recordsLinked to original sources

bioRxiv2021

4polar-STORM polarized super-resolution imaging of actin filament organization in cells

Advances in single-molecule localization microscopy are providing unprecedented insights into the nanometer-scale organization of protein assemblies in cells and thus a powerful means for interrogating biological function. However, localization imaging alone does not contain information on protein conformation and orientation, which constitute additional key signatures of protein function. Here, we present a new microscopy method which combines for the first time Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging with single molecule orientation and wobbling measurements using a four polarization-resolved image splitting scheme. This new method, called 4polar-STORM, allows us to determine both single molecule localization and orientation in 2D and to infer their 3D orientation, and is compatible with high labelling densities and thus ideally placed for the determination of the organization of dense protein assemblies in cells. We demonstrate the potential of this new method by studying the nanometer-scale organization of dense actin filament assemblies driving cell adhesion and motility, and reveal bimodal distributions of actin filament orientations in the lamellipodium, which were previously only observed in electron microscopy studies. 4polar-STORM is fully compatible with 3D localization schemes and amenable to live-cell observations, and thus promises to provide new functional readouts by enabling nanometer-scale studies of orientational dynamics in a cellular context.

biophysics↗

bioRxiv2021

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton. SummaryHuman septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

cell biology↗

bioRxiv2020

Membrane binding controls ordered self-assembly of animal septins

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here we show by cell-free reconstitution that membrane binding requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septins form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

cell biology↗