Biology subjects

Wainman, A.

Publications and source records attributed to Wainman, A..

10 recordsLinked to original sources

bioRxiv2025

Structural insights into mitotic-centrosome assembly

Centrosomal material assembles rapidly in mitosis. In Drosophila, the coiled-coil protein Cnn forms a scaffold that recruits PCM clients; in C.elegans, SPD-5 plays an analogous role. Here we show that full-length Cnn and SPD-5 can both form spherical condensates in vitro, but that the interactions driving their assembly into scaffolds inside cells appear to diverge. We show that the Cnn PReM adopts a helical hairpin fold that autoinhibits CM2 binding but that phosphorylation appears to increase hairpin breathing to permit CM2 engagement and robust scaffold assembly. Phospho-blocking mutations prevent PReM-CM2 interactions and scaffold formation, whereas phospho-mimetic substitutions partially restore function. The human homologue CDK5RAP2 contains a CM2 domain that can partially substitute for fly CM2 in vivo and we identify a candidate CDK5RAP2 PReM region that forms macromolecular networks with human CM2 in vitro. By contrast, the putative PReM and CM2 regions of SPD-5 cannot substitute for their equivalent fly domains and they do not interact detectably, suggesting a distinct assembly mechanism in worms despite conserved PLK1-dependent control of PCM growth.

cell biology↗

bioRxiv2024

Centrioles generate two scaffolds with distinct biophysical properties to build mitotic centrosomes

Mitotic centrosomes form when centrioles recruit large amounts of pericentriolar material (PCM) around themselves. The PCM comprises hundreds of proteins, yet it can assemble and disassemble within minutes, leading to much debate about its physical nature. Here we show that Drosophila Spd-2 fluxes out from centrioles, recruiting Polo and Aurora A to catalyse the assembly of a solid-like Cnn-scaffold and a more liquid-like TACC-scaffold, respectively. Both scaffolds can assemble and recruit PCM proteins independently, but both are required for proper centrosome assembly, with the Cnn-scaffold providing mechanical strength, and the TACC-scaffold locally concentrating centriole and centrosome proteins. Recruiting Spd-2, but not Cnn or TACC, to the surface of synthetic beads injected into early embryos reconstitutes several aspects of mitotic centrosome assembly on the bead surface, and this depends on the ability of Spd-2 to recruit Polo and AurA. Thus, Spd-2 molecules flux out from the centriole recruiting Polo and AurA to promote the assembly of two scaffolds with distinct properties to support mitotic centrosome assembly in flies.

cell biology↗

bioRxiv2023

Cell Cycle Dependent Orchestration of Surface Layer Biogenesis in Caulobacter crescentus

Surface layers (S-layers) are proteinaceous, two-dimensional crystals that constitute the outermost components of many prokaryotic cell envelopes. In this study, we investigated principles of S-layer biogenesis on the outer membrane in the bacterial model organism Caulobacter crescentus. Fluorescent microscopy revealed localised incorporation of new S-layer at the poles and mid-cell, consistent with elongation and division phases of the cell cycle. Next, light microscopy and electron cryotomography investigations of drug-treated bacteria revealed that bacterial actin homologue MreB is crucial for localised S-layer insertion. We further uncovered that S-layer biogenesis follows new peptidoglycan synthesis and localises to regions of high cell wall turnover. Finally, correlated cryo-light microscopy and electron cryotomographic analysis of regions of S-layer insertion showed the presence of gaps in the hexagonal S-layer lattice, contrasting with other S-layers completed by defined symmetric defects. Our findings provide insight into how C. crescentus cells form an ordered S-layer on their surface, providing evidence for coordination between the biogenesis of the cell envelope at multiple levels.

microbiology↗

bioRxiv2023

Cdk/Cyclin activity helps set mitotic centrosome size by influencing the centrosome growth rate and growth period

Mitotic centrosomes assemble when centrioles recruit large amounts of pericentriolar material (PCM) around themselves in preparation for cell division. How the mitotic PCM grows to the correct size is unclear. In Drosophila syncytial embryos, thousands of mitotic centrosomes assemble in a common cytoplasm as the embryo proceeds through 13 rounds of near-synchronous nuclear division. During nuclear cycles (NCs) 11-13 these divisions gradually slow, and we find that mitotic centrosomes respond by reciprocally slowing their growth rate and increasing their growth period so that they grow to a consistent size at each cycle. This size homeostasis is enforced, at least in part, by the Cdk/Cyclin cell cycle oscillator (CCO). Moderate levels of CCO activity appear to initially promote centrosome growth by stimulating Polo/PLK1 recruitment to centrosomes, while higher levels of activity subsequently inhibit centrosome growth by phosphorylating centrosome proteins to decrease their centrosomal recruitment and/or maintenance as the embryos enter mitosis. Thus, the CCO initially promotes, and subsequently restricts, mitotic centrosome growth to help ensure that centrosomes grow to a consistent size.

cell biology↗

bioRxiv2023

Mob4 is essential for spermatogenesis in Drosophila melanogaster

Gamete formation is essential for sexual reproduction in metazoans. In males, spermatogenesis gives rise to interconnected spermatids that have to differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. Here, we show that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role on female fertility. We describe the function of Mob4 during spermatid individualization, showing that Mob4 is required for proper axonemal structure and that the loss of Mob4 leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of Mob4RNAi developing spermatids revealed defects in axoneme structure and abnormal mitochondria biogenesis. Importantly, we find that male fertility is impaired upon depletion of other STRIPAK components, suggesting that Mob4 acts through STRIPAK to support spermiogenesis. As we show that expression of the human Mob4 gene effectively rescues all phenotypes of Mob4 downregulation, the gene is not only evolutionary but also functionally conserved. We propose that Mob4 plays a role in regulating the microtubule- and actin-cytoskeleton during spermatogenesis. This study advances our understanding of male infertility by uncovering Mob4 as a novel gene required for sperm individualization.

developmental biology↗

bioRxiv2021

Centriole distal-end proteins CP110 and Cep97 influence cartwheel growth at the proximal-end of centrioles

Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. We previously showed that an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. Here, we show that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, entrained by the core Cdk/Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110/Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, we find that changing the levels of CP110/Cep97 alters the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which may help to coordinate centriole growth.

cell biology↗

bioRxiv2021

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/450133v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@10f7bf1org.highwire.dtl.DTLVardef@192214dorg.highwire.dtl.DTLVardef@c84916org.highwire.dtl.DTLVardef@1366287_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain. Highlights O_LISingle molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics C_LIO_LIThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replication C_LIO_LIGenomic RNA is stable and persistent during the initial stages of infection C_LIO_LIB.1.1.7 variant replicates more slowly than the Victoria strain C_LI

microbiology↗

bioRxiv2020

Ana1 recruits PLK1 to mother centrioles to promote mitotic PCM assembly and centriole elongation

Polo kinase (PLK1) is a master cell cycle regulator that is recruited to various subcellular structures by its Polo-Box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo has multiple functions at centrioles and centrosomes, and we previously showed that phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the mitotic Pericentriolar Material (PCM) that assembles around mother centrioles. Here, we investigate whether additional proteins recruit Polo to centrioles and/or centrosomes, and find that Ana1 (Cep295 in mammals) helps recruit Polo to mother centrioles. If this function is impaired, mother centrioles can still duplicate and disengage from their daughters, but they can no longer efficiently assemble a mitotic PCM or elongate their centrioles in G2. Thus, Ana1 is part of a sequential phosphorylation cascade that recruits Polo to centrioles to drive mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication or disengagement.

cell biology↗

bioRxiv2019

A free-running oscillator times and executes centriole biogenesis

The accurate timing of organelle biogenesis and the precise regulation of organelle size are crucial for cell physiology. Centriole biogenesis initiates exclusively in S-phase, when a daughter centriole emerges from the side of a pre-existing mother and grows until it reaches its mothers size. This process is regulated by Polo-like kinase 4 (Plk4), which is recruited to centrioles in oscillatory waves in flies and human cells 1,2. The nature and function of Plk4 oscillations is, however, unknown. Here we discover that Plk4 forms an adaptive oscillator at the base of the growing centriole, whose function is to time and set the duration of centriole biogenesis in Drosophila embryos. We demonstrate that the Plk4 oscillator is free-running of, but is entrained and calibrated by, the core Cdk/Cyclin cell-cycle oscillator, explaining how centrioles can duplicate independently of the cell cycle 3-5. Mathematical modelling and further experiments indicate that the Plk4 oscillator is generated by a time-delayed negative-feedback loop in which Plk4 recruitment to, and dissociation from, the centriole is monitored via changes in the affinity-state of its centriolar receptor, Asterless. We postulate that such organelle-specific autonomous oscillators could regulate the timing and execution of organelle biogenesis more generally.

cell biology↗

bioRxiv2018

A positive feedback loop drives centrosome maturation in flies

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn form an extensive scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If Spd-2 cannot recruit Polo to the expanding scaffold, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. We conclude that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic centrosome in flies.

cell biology↗