Biology subjects

Gönczy, P.

Publications and source records attributed to Gönczy, P..

9 recordsLinked to original sources

bioRxiv2025

C. elegans SAS-1 ensures centriole integrity and ciliary function, and operates with SSNA-1

Centrioles are microtubule-based organelles critical for signaling, motility and division. The microtubule-binding protein SAS-1 is homologous to the human ciliopathy component C2CD3 and contributes to centriole integrity in C. elegans, but how this function is exerted is incompletely understood. Here, through the generation of a null allele and analysis with U-Ex-STED, we establish that SAS-1 is dispensable for the onset of centriole assembly, but essential for organelle integrity during oogenesis, spermatogenesis and in the early embryo. Additionally, we uncover that SAS-1 is present at the transition zone of sensory neurons, where it contributes in a partially redundant manner to ciliary function. Furthermore, we investigate the relationship between SAS-1 and the C. elegans Sjogren Syndrome Nuclear Antigen 1 protein SSNA-1, establishing that SSNA-1 localizes next to the SAS-1 C-terminus in the centriole architecture. Moreover, through molecular epistasis experiments with null alleles of both components, we reveal that SAS-1 is essential for SSNA-1 localization to centrioles during oogenesis and to the transition zone during ciliogenesis. Moreover, using a heterologous human cell assay, we establish that SAS-1 recruits SSNA-1 to microtubules. Overall, our findings help clarify how SAS-1, together with SSNA-1, ensures centriole integrity and reveals that it contributes to cilium function.

cell biology↗

bioRxiv2023

Centriole elimination during C. elegans oogenesis initiates with loss of the central tube protein SAS-1

Centrioles are lost during oogenesis in most metazoans, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in C. elegans. Centriole elimination begins with loss of the so-called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule- and dynein-dependent manner to the plasma membrane. Moreover, we find that neither Polo-like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila, do so in C. elegans. Furthermore, we demonstrate that the central tube protein SAS-1 normally departs first from the organelle, which loses integrity earlier in sas-1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination.

cell biology↗

bioRxiv2022

Grand canonical Brownian dynamics simulations of adsorption and self-assembly of SAS-6 rings on a surface

The protein SAS-6 forms dimers, which then self-assemble into rings that are critical for the nine-fold symmetry of the centriole organelle. It has recently been shown experimentally that the self-assembly of SAS-6 rings is strongly facilitated on a surface, shifting the reaction equilibrium by four orders of magnitude compared to the bulk. Moreover, a fraction of non-canonical symmetries (i.e., different from nine) was observed. In order to understand which aspects of the system are relevant to ensure efficient self-assembly and selection of the nine-fold symmetry, we have performed Brownian dynamics computer simulation with patchy particles and then compared our results with experimental ones. Adsorption onto the surface was simulated by a Grand Canonical Monte Carlo procedure and Random Sequential Adsorption kinetics. Furthermore, self-assembly was described by Langevin equations with hydrodynamic mobility matrices. We find that as long as the interaction energies are weak, the assembly kinetics can be described well by the coagulation-fragmentation equations in the reaction-limited approximation. By contrast, larger interaction energies lead to kinetic trapping and diffusion-limited assembly. We find that selection of nine-fold symmetry requires a small value for the angular interaction range. These predictions are confirmed by the experimentally observed reaction constant and angle fluctuations. Overall, our simulations suggest that the SAS-6 system works at the crossover between a relatively weak binding energy that avoids kinetic trapping and a small angular range that favors the nine-fold symmetry.

biophysics↗

bioRxiv2022

Molecular architecture of the C. elegans centriole

Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. C. elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion microscopy (U-Ex-STED), as well as electron microscopy (EM) and tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of [~]14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We uncovered that the procentriole assembles from a location on the centriole margin characterized by SPD-2 and ZYG-1 accumulation. Moreover, we found that SAS-6 and SAS-5 are present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we establish that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.

cell biology↗

bioRxiv2022

Atypical and distinct microtubule radial symmetries in the centriole and the axoneme of Lecudina tuzetae.

The centriole is a minute cylindrical organelle present in a wide range of eukaryotic species. Most centrioles have a signature 9-fold radial symmetry of microtubules that is imparted onto the axoneme of the cilia and flagella they template, with 9 centriolar microtubule doublets growing into 9 axonemal microtubule doublets. There are exceptions to the 9-fold symmetrical arrangement of axonemal microtubules, with lower or higher fold symmetries in some species. In the few cases where this has been examined, such alterations in axonemal symmetries are grounded in likewise alterations in centriolar symmetries. Here, we examine the question of microtubule number continuity between centriole and axoneme in flagellated gametes of the gregarine Lecudina tuzetae, which have been reported to exhibit a 6-fold radial symmetry of axonemal microtubules. We used time-lapse differential interference microscopy to identify the stage at which flagellated gametes are present. Thereafter, using electron microscopy and ultrastructure-expansion microscopy coupled to STimulated Emission Depletion (STED) super-resolution imaging, we uncover that a 6- or 5-fold radial symmetry in the axoneme is accompanied by an 8-fold radial symmetry in the centriole. We conclude that there can be plasticity in the transition between centriolar and axonemal microtubules.

cell biology↗

bioRxiv2021

Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis

Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of C. elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.

cell biology↗

bioRxiv2020

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin stability

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease characterized by impaired growth and increased tumorigenesis, whose cellular etiology is poorly understood. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterized these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We established that an atypical de novo assembly pathway is notably involved in forming Cenpas, which can nevertheless trigger further centriole assembly and act as MTOCs. We found also that Cenpas are present and act similarly in Mulibrey patient cells. Through correlative light electron microscopy, we uncovered that Cenpas correspond to centriole related structures and elongated electron-dense structures with stripes. Importantly, we established that TRIM37 regulates the stability and solubility of the centriolar protein Centrobin. Our findings suggest that elongated Centrobin assemblies are a major constituent of the striped electron dense structures. Furthermore, we established that Cenpas formation upon TRIM37 depletion requires PLK4 activity, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents the formation of Cenpas that would otherwise threaten genome integrity, including possibly in Mulibrey patients.

cell biology↗

bioRxiv2020

Surface-catalyzed SAS-6 self-assembly directs centriole formation through kinetic and structural mechanisms

Discovering the physical principles directing organelle assembly is a fundamental pursuit in biology. Centrioles are evolutionarily conserved organelles with a 9-fold rotational symmetry of chiral microtubules imparted onto the cilia they template1. Centriole assemble from likewise symmetrical ring polymers of SAS-6 proteins, orthogonal to a toroidal surface surrounding the resident centriole2-4. How surface properties ensure ring assembly with proper symmetry and orthogonal arrangement is not known. Here, we deployed photothermally-actuated off-resonance tapping high-speed atomic force microscopy (PORT-HS-AFM) to decipher physical principles of surface-guided SAS-6 self-assembly. Using machine learning to quantify the polymerization reaction and developing a coagulation-fragmentation model, we discovered that the surface shifts the reaction equilibrium by [~]104 compared to the solution situation, explaining orthogonal organelle emergence. Moreover, molecular dynamics and PORT-HS-AFM revealed that the surface converts helical SAS-6 polymers into 9-fold ring polymers with residual asymmetry, which may impart chiral features to centrioles and cilia. Overall, we discovered two fundamental physical principles directing robust centriole organelle assembly.

biophysics↗

bioRxiv2020

Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead and A-C-linker. Moreover, we report [~]25 [A] resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicates that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.

cell biology↗