Biology subjects

Billault-Chaumartin, I.

Publications and source records attributed to Billault-Chaumartin, I..

7 recordsLinked to original sources

bioRxiv2025

Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts

Actin is an essential cytoskeletal protein that is extremely well conserved across the tree of life. Similarities and differences in the way actin from different species self-assembles into filaments inform our understanding of its evolution. However, this basic knowledge is largely incomplete. Here, we address this issue by systematically characterizing assembly kinetics for actin from two yeast species that are five hundred million years apart in evolution, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and compare them to the well-studied actin from rabbit skeletal muscle from which they diverged a billion years ago. By monitoring individual actin filaments under controlled conditions, we accurately quantified their barbed end elongation from ATP-actin monomers, their disassembly in the ADP{middle dot}Pi and ADP states, as well as the release of inorganic phosphate, which marks their aging. We find that, in the ATP state, both yeast actins behave strikingly like mammalian actin at filament barbed ends. In contrast, yeast actin filaments in both the ADP{middle dot}Pi and the ADP states depolymerize several-fold faster than their mammalian counterparts, and they release inorganic phosphate over 20-fold faster. We show that the absence of methylation on histidine 73 largely accounts for this faster aging of yeast actin filaments. We also reveal differences between the actins of the two yeasts. In particular, ADP-actin filaments depolymerize faster and are mechanically more flexible in Saccharomyces cerevisiae compared to Schizosaccharomyces pombe. Our findings suggest that actins expressed across species possess more diverse and specialized biochemical characteristics than previously recognized.

biochemistry↗

bioRxiv2022

mNeonGreen-tagged fusion proteins and nanobodies reveal localization of tropomyosin to patches, cables, and contractile actomyosin rings in live yeast cells

Tropomyosins are structurally conserved -helical coiled-coil dimeric proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments in non-muscle cells and are essential for calcium regulation of myosin II contractility in the muscle. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live cell imaging tools currently available in many organisms. Here, we report mNeon-Green (mNG) tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin localization and dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8), and Saccharomyces cerevisiae (Tpm1 and Tpm2), in vivo and in isolated S. pombe cell division apparatuses. We extended this approach to also visualize the mammalian TPM2 isoform. Finally, we generated a camelid-nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo without significantly influencing cell growth and dynamics of actin cytoskeleton. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring, and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and actin cables in vivo.

cell biology↗

bioRxiv2022

Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion

Spatial accumulation of secretory vesicles underlies various cellular processes, such as neurotransmitter release at neuronal synapses [1], hyphal steering in filamentous fungi [2, 3], and local cell wall digestion preceding the fusion of yeast gametes [4]. Secretory vesicles transported on actin filaments by myosin V motors form clusters that serve as pool for local content release. During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes [5-7]. Focus position and coalescence are controlled by local signalling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis [6, 8-10], but the mechanisms of focusing have been elusive. Here, we show that the regulatory N-terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude other macromolecules. Fus1 lacking its IDR fails to condense in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, replacement of Fus1 IDR with a heterologous low-complexity region that forms liquid condensates fully restores Fus1 condensation and function. By contrast, replacement of Fus1 IDR with a domain that forms more stable oligomers restores condensation but poorly supports cell fusion, suggesting that condensation is tuned to yield a structure selectively permeable for secretory vesicles. We propose that condensation of actin structures by an intrinsically disordered region may be a general mechanism for actin network organisation and the selective local concentration of secretory vesicles.

cell biology↗

bioRxiv2022

The actin assembly requirements of the formin Fus1 to build the fusion focus

Formins form the largest family of actin filament nucleators and elongators, involved in the assembly of diverse actin structures. Actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, common to all formins. However, the rate of these reactions varies between formins by at least 20-fold. Typically, each cell expresses several distinct formins, each contributing to the assembly of one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in the fission yeast Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates for function in vivo. Fus1 assembles the actin fusion focus, an aster-like structure of actin filaments at the contact site between gametes, necessary for the process of cell fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on the architecture of the fusion focus, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in the Fus1 FH2 domain that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional property of the FH2 domain. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

cell biology↗

bioRxiv2021

Genomic contacts reveal the control of sister chromosome decatenation in E. coli

In bacteria, chromosome segregation occurs progressively, from the origin to the terminus, a few minutes after the replication of each locus. In-between replication and segregation, sister loci are maintained in an apparent cohesive state by topological links. Whereas topoisomerase IV (Topo IV), the main bacteria decatenase, controls segregation, little is known regarding the influence of the cohesion step on chromosome folding. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, a massive chromosome reorganization takes place, associated with increases in trans-contacts between catenated sister chromatids and in long-range cis-contacts between the terminus and distant loci on the genome. A genetic analysis of these signals allowed us to decipher specific roles for Topo IV and Topo III, an accessory decatenase. Moreover we revealed the role of MatP, the terminus macrodomain organizing system and MukB, the E. coli SMC in organizing sister chromatids tied by persistent catenation links. We propose that large-scale conformation changes observed in these conditions reveal a defective decatenation hub located in the terminus area. Altogether, our findings support a model of spatial and temporal partition of the tasks required for sister chromosome segregation.

molecular biology↗

bioRxiv2019

A toolbox of Stable Integration Vectors (SIV) in the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe is a widely used model organism that resembles higher eukaryotes in many aspects of cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.

cell biology↗

bioRxiv2019

Capping protein insulates Arp2/3-assembled actin patches from formins

How actin structures of distinct identities and functions co-exist within the same environment is a critical self-organization question. Fission yeast cells have a simple actin cytoskeleton made of four structures: Arp2/3 assembles actin patches around endocytic pits; the formins For3, Cdc12 and Fus1 assemble actin cables, the cytokinetic ring during division, and the fusion focus during sexual reproduction, respectively. The focus concentrates the delivery of hydrolases by myosin V to digest the cell wall for cell fusion. We discovered that cells lacking capping protein (CP), a heterodimer that blocks barbed-end dynamics and associates with actin patches, exhibit a delay in fusion. Consistent with CP-formin competition for barbed-end binding, Fus1, F-actin and the linear filament marker tropomyosin hyper-accumulate at the fusion focus in absence of CP. However, myosin V and exocytic cargoes are diverted to ectopic foci and reduced at the fusion focus, which underlies the fusion defect. Remarkably, ectopic foci coincide with actin patches, which now contain low levels of Fus1. During mitotic growth, actin patches lacking CP similarly display a dual identity, as they accumulate the formins For3 and Cdc12 and are co-decorated by tropomyosin and the patch marker fimbrin. Thus, CP serves to protect Arp2/3-nucleated structures from formin activity.

cell biology↗