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

© The Authors, 2019. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 4.0 International License. The definitive version was published in Journal of Cell Biology (2019), doi:10.1083/jcb.201807211.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.We thank the Gladfelter laboratory and Danny Lew for useful discussions, Matthias Garten for ideas in setting up the rod assay, and the University of North Carolina EM facility (Victoria Madden and Kristen White) for support with scanning electron microscope. This work was supported by a Howard Hughes Medical Institute Faculty Scholars award to A.S. Gladfelter, and K.S. Cannon was supported in part by a grant from the National Institute of General Medical Sciences under award T32 GM119999.2019-07-0

The hierarchical assembly of septins revealed by high-speed AFM

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Jiao, F., Cannon, K. S., Lin, Y. C., Gladfelter, A. S., & Scheuring, S. The hierarchical assembly of septins revealed by high-speed AFM. Nature Communications, 11(1), (2020): 5062, doi:10.1038/s41467-020-18778-x.Septins are GTP-binding proteins involved in diverse cellular processes including division and membrane remodeling. Septins form linear, palindromic heteromeric complexes that can assemble in filaments and higher-order structures. Structural studies revealed various septin architectures, but questions concerning assembly-dynamics and -pathways persist. Here we used high-speed atomic force microscopy (HS-AFM) and kinetic modeling which allowed us to determine that septin filament assembly was a diffusion-driven process, while formation of higher-order structures was complex and involved self-templating. Slightly acidic pH and increased monovalent ion concentrations favor filament-assembly, -alignment and -pairing. Filament-alignment and -pairing further favored diffusion-driven assembly. Pairing is mediated by the septin N-termini face, and may occur symmetrically or staggered, likely important for the formation of higher-order structures of different shapes. Multilayered structures are templated by the morphology of the underlying layers. The septin C-termini face, namely the C-terminal extension of Cdc12, may be involved in membrane binding.We thank J. Thorner for the generous gift of the CTE mutant plasmids. K.S.C. was supported in part by a grant from NIGMS under award T32 GM119999 and A.S.G., F.J. and S.S. were supported by NIH RO1 GM130934

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

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Woods, B. L., Cannon, K. S., Vogt, E. J. D., Crutchley, J. M., & Gladfelter, A. S. Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling. Molecular Biology of the Cell, 32(20), (2021): mbcE20050303, https://doi.org/10.1091/mbc.E20-05-0303.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 nonessential 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.This work was supported by National Institutes of Health (NIH) Grant no. R01GM-130934 to A.S.G. B.L.W. was supported by the NIH Training Grant no. 2T32AI052080-16. K.S.C. and E.J.D.V. were supported in part by a grant from the National Institute of General Medical Sciences under award T32 GM119999

Multi-monoubiquitylation controls VASP-mediated actin dynamics Icon for The Forest of Biologists

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics

Multimodal x-ray and electron microscopy of the Allende meteorite.

Multimodal microscopy that combines complementary nanoscale imaging techniques is critical for extracting comprehensive chemical, structural, and functional information, particularly for heterogeneous samples. X-ray microscopy can achieve high-resolution imaging of bulk materials with chemical, magnetic, electronic, and bond orientation contrast, while electron microscopy provides atomic-scale spatial resolution with quantitative elemental composition. Here, we combine x-ray ptychography and scanning transmission x-ray spectromicroscopy with three-dimensional energy-dispersive spectroscopy and electron tomography to perform structural and chemical mapping of an Allende meteorite particle with 15-nm spatial resolution. We use textural and quantitative elemental information to infer the mineral composition and discuss potential processes that occurred before or after accretion. We anticipate that correlative x-ray and electron microscopy overcome the limitations of individual imaging modalities and open up a route to future multiscale nondestructive microscopies of complex functional materials and biological systems

Emerging Infectious Disease leads to Rapid Population Decline of Common British Birds

Emerging infectious diseases are increasingly cited as threats to wildlife, livestock and humans alike. They can threaten geographically isolated or critically endangered wildlife populations; however, relatively few studies have clearly demonstrated the extent to which emerging diseases can impact populations of common wildlife species. Here, we report the impact of an emerging protozoal disease on British populations of greenfinch Carduelis chloris and chaffinch Fringilla coelebs, two of the most common birds in Britain. Morphological and molecular analyses showed this to be due to Trichomonas gallinae. Trichomonosis emerged as a novel fatal disease of finches in Britain in 2005 and rapidly became epidemic within greenfinch, and to a lesser extent chaffinch, populations in 2006. By 2007, breeding populations of greenfinches and chaffinches in the geographic region of highest disease incidence had decreased by 35% and 21% respectively, representing mortality in excess of half a million birds. In contrast, declines were less pronounced or absent in these species in regions where the disease was found in intermediate or low incidence. Also, populations of dunnock Prunella modularis, which similarly feeds in gardens, but in which T. gallinae was rarely recorded, did not decline. This is the first trichomonosis epidemic reported in the scientific literature to negatively impact populations of free-ranging non-columbiform species, and such levels of mortality and decline due to an emerging infectious disease are unprecedented in British wild bird populations. This disease emergence event demonstrates the potential for a protozoan parasite to jump avian host taxonomic groups with dramatic effect over a short time period