Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel

Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9 fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6

Santiago Harald Andany, Niccolo Banterle, Georg Fantner, Pierre Gönczy, Adrian Pascal Nievergelt

The self-assembly of protein complexes is at the core of many fundamental biological processes1, ranging from the polymerization of cytoskeletal elements, such as microtubules2, to viral capsid formation and organelle assembly3. To reach a comprehensive understanding of the underlying mechanisms of self-assembly, high spatial and temporal resolutions must be attained. This is complicated by the need to not interfere with the reaction during the measurement. As self-assemblies are often governed by weak interactions, they are especially difficult to monitor with high-speed atomic force microscopy (HS-AFM) due to the non-negligible tip–sample interaction forces involved in current methods. We have developed a HS-AFM technique, photothermal off-resonance tapping (PORT), which is gentle enough to monitor self-assembly reactions driven by weak interactions. We apply PORT to dissect the self-assembly reaction of SAS-6 proteins, which form a nine-fold radially symmetric ring-containing structure that seeds the formation of the centriole organelle. Our analysis reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical structure.

2018

Analysis of Fluid-Structure Interaction by Means of Dynamic Unstructured Meshes

Pénélope Leyland

This paper presents a computational analysis on forced vibration fluid-structure interaction in compressible flow regimes. A so-called staggered approach is pursued where the fluid and structure are integrated in time by distinct solvers. Their interaction is then taken into account by a coupling algorithm. The unsteady fluid motion is simulated by means of an explicit time-accurate solver. For the fluid-structure interaction problems which are considered here the effects due to the viscosity can be neglected. The fluid is hence modeled by the Euler equations for compressible inviscid flow. Unstructured grids are used to discretise the fluid domain. These grids are particularly suited to simulate unsteady flows over complex geometries by their capacity of being dynamically refined and unrefined. Dynamic mesh adaptation is used to enhance the computational precision with minimal CPU and memory constraints. Fluid-structure interaction involves moving boundaries. Therefore the Arbitrary Lagrange Euler method (ALE-method) is adopted to solve the Euler equations on a moving domain. The deformation of the mesh is controlled by means of a spring analogy in conjunction with a boundary correction to circumvent the principle of Saint Venant. To take advantage of the differences between fluid and structure time scales, the fluid calculation is subcycled within the structural time step. Numerical results are presented for large rotation, pitching oscillation and aeroelastic motion of the NACA0012 airfoil. The boundary deformation is validated by comparing the numerical solution for a flat plate under supersonic flow with the analytical solution.

1998

Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans

Pierre Gönczy

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Galpha(i1)(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD

2008