Team Publications

Summary

Summary

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.

Summary

Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays a central role in structural biology, with high impact on research fields such as cell and developmental biology, bioinformatics, cell physics and applied mathematics. It allows the determination of structures of purified proteins within cells. This review describes the main recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and the avenues for future development.

Summary

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 binding to flat lipid membranes 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 septin hexamers 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.

Summary

Protein enrichment at specific membrane locations in cells is crucial for many cellular functions. It is well-recognized that the ability of some proteins to sense membrane curvature contributes partly to their enrichment in highly curved cellular membranes. In the past, different theoretical models have been developed to reveal the physical mechanisms underlying curvature-driven protein sorting. This review aims to provide a detailed discussion of the two continuous models that are based on the Helfrich elasticity energy, (1) the spontaneous curvature model and (2) the curvature mismatch model. These two models are commonly applied to describe experimental observations of protein sorting. We discuss how they can be used to explain the curvature-induced sorting data of two BAR proteins, amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently the membrane curvature generated by BAR proteins, could influence protein organization on the curved membranes. Finally, we address future directions in extending these models to describe some cellular phenomena involving protein sorting.

Summary

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either use as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor

Summary

Abstract
Motile and morphological cellular processes require a spatially and temporally
coordinated branched actin network that is controlled by the activity of various regulatory
proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously
reported that myosin 1b regulates the density of the actin network in the growth cone. Using
in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we
show in this report that this molecular motor flattens the Arp2/3-dependent actin branches
up to breaking them and reduces the probability to form new branches. This experiment
reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated
actin junction. Together with the former in vivo studies, this work emphasizes the essential
role played by myosins in the architecture and in the dynamics of actin networks in different
cellular regions.

Summary

Budding yeast septins are essential for cell division and polarity. Septins assemble as
palindromic linear octameric complexes. The function and ultra-structural organization of
septins are finely governed by their molecular polymorphism. In particular, in budding
yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for
the first time, the behavior of the Shs1 protomer bound to membranes at nanometer
resolution, in complex with the other septins. Using electron microscopy, we have shown
that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or twodimensional
gauzes. Using a set of specific mutants we have demonstrated a synergistic
role of both nucleotides and lipids for the organization and oligomerization of budding
yeast septins. Besides, cryo-electron tomography assays show that vesicles are
deformed by the interaction between Shs1 oligomers and lipids. The Shs1–Shs1 interface
is stabilized by the presence of phosphoinositides, allowing the visualization of micrometric
long filaments formed by Shs1 protomers. In addition, molecular modeling experiments
have revealed a potential molecular mechanism regarding the selectivity of septin
subunits for phosphoinositide lipids.

Summary

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90–Arp2/3 nucleated filaments and formation of a ternary SPIN90–Arp2/3–mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90–Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.

Summary

Giant vesicles are widely used as a model membrane system, both for basic biological systems and for their promising applications in the development of smart materials and cell mimetics, as well as in driving new technologies in synthetic biology and for the cosmetics and pharmaceutical industry. The reader is guided to use giant vesicles, from the formation of simple membrane platforms to advanced membrane and cell system models. It also includes fundamentals for understanding lipid or polymer membrane structure, properties and behavior. Every chapter includes ideas for further applications and discussions on the implications of the observed phenomena towards understanding membrane-related processes. The Giant Vesicle Book is meant to be a road companion, a trusted guide for those making their first steps in this field as well as a source of information required by experts. Key Features • A complete summary of the field, covering fundamental concepts, practical methods, core theory, and the most promising applications • A start-up package of theoretical and experimental information for newcomers in the field • Extensive protocols for establishing the required preparations and assays • Tips and instructions for carefully performing and interpreting measurements with giant vesicles or for observing them, including pitfalls • Approaches developed for investigating giant vesicles as well as brief overviews of previous studies implementing the described techniques • Handy tables with data and structures for ready reference