Biology subjects

Woodruff, J. B.

Publications and source records attributed to Woodruff, J. B..

13 recordsLinked to original sources

bioRxiv2026

Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

Animal oocytes undergo highly asymmetric divisions to expel excess copies of their genome into compact cells called polar bodies. This requires tight clustering and cortical positioning of meiotic chromosomes, yet the mechanism remains incompletely understood. Using C. elegans oocytes, we found that the meiotic spindle pole protein ZYG-9/ch-TOG delocalizes from microtubules to spread along the surface of chromosomes and prevent their dispersal in meiotic anaphase. This effect was more pronounced in the absence of the spindle, where ZYG-9 formed into a micron-scale droplet that enveloped all chromosomes. Purified ZYG-9 was sufficient to bind DNA and coat reconstituted chromatin. Mutations that perturb ZYG-9-DNA binding impaired chromosome packaging into polar bodies, resulting in oocytes carrying extra chromosomes and reduced fertility. We propose that liquid-like assemblies of spindle pole proteins are repurposed as surface-acting glue to tightly package meiotic chromosomes into polar bodies, thus ensuring oocytes have the correct genome copy number.

cell biology↗

bioRxiv2025

TRIM37 recognizes a bipartite degron to ubiquitinate centrosome substrates

Dysregulation of the E3 ubiquitin ligase TRIM37 is associated with tumor formation and Mulibrey nanism, a recessive developmental syndrome. TRIM37 regulates steady-state levels of centrosome proteins and limits their ectopic assembly, but how it recognizes and ubiquitinates its substrates is poorly understood. We found that TRIM37 directly ubiquitinates the centrosome-forming protein Cep192 at 7 lysines clustered near its C-terminus. TRIM37 binds Cep192 at a C-terminal intrinsically disordered region followed by an ASH domain (IDR+ASH8). Mutation of the 7 lysines or the IDR+ASH8 domain increased Cep192 levels and stability in cells, indicating loss of TRIM37-based regulation. Fusing IDR+ASH8 to an unrelated protein (GFP-EB1) was sufficient to enable its degradation via TRIM37. Biochemical assays revealed that IDR+ASH8 is primarily monomeric and binds TRIM37 via two separate coiled-coil motifs with mid-nanomolar affinity. We propose that the IDR+ASH8 motif is a bipartite degron for TRIM37, enabling it to target centrosome proteins and adjust their levels.

cell biology↗

bioRxiv2025

In vitro reconstitution of minimal human centrosomes

CDK5RAP2/CEP215 is a key pericentriolar material (PCM) protein that recruits microtubule-nucleating factors at human centrosomes. Using an in vitro reconstitution system, we show that CDK5RAP2 is sufficient to form micron-scale scaffolds around a nanometer-scale nucleator in a PLK-1-regulated manner. CDK5RAP2 assemblies recruited and activated gamma tubulin ring complexes ({gamma}-TuRCs) which, in the presence of /{beta} tubulin, generated microtubule asters. We found that F75 in CDK5RAP2 is partially needed to recruit {gamma}-TuRC yet is indispensable for {gamma}-TuRC activation. Furthermore, our system recapitulated key features of centrosome-amplified cancer cells. CDK5RAP2 scaffolds selectively recruited the molecular motor KifC1/HSET, which enhanced concentration of /{beta} tubulin, microtubule polymerization, and clustering of the assemblies. Our results highlight the specificity and selectivity of in vitro generated CDK5RAP2 scaffolds and identify a minimal set of components required for human centrosome assembly and function. This minimal centrosome model offers a powerful tool for studying centrosome biology and dysfunction in human health and disease.

cell biology↗

bioRxiv2024

eIF4ET regulates meiotic proteome levels to enable oocyte formation and storage

Animals store oocytes in a dormant state for weeks to decades before ovulation. The homeostatic programs oocytes use to endure long-term storage are poorly understood. Using female nematodes as a short-lived model, we found that oocyte formation and storage required IFET-1, the conserved eIF4E-Transporter protein (eIF4ET). IFET-1 co-assembled with CAR-1 (Lsm14) to form micron-scale condensates in stored oocytes, which dissipated after oocyte activation. Depletion of IFET-1 destabilized the stored oocyte proteome, leading to lower translation, a decline in microtubule maintenance proteins, and errors in microtubule organization and meiotic spindle assembly. Deleting individual domains within IFET-1 impaired oocyte storage without affecting oocyte formation. Thus, in addition to establishing a healthy oocyte reserve in young mothers, IFET-1 ensures that correct levels of cytoskeletal proteins are maintained as oocytes age. Human eIF4ET also localized to micron-scale puncta in dormant oocytes in a reproductively healthy patient. Our results clarify the role for eIF4ET in maintaining the oocyte reserve and further support eIF4ET dysfunction as an upstream cause of embryonic aneuploidy and age-related infertility.

developmental biology↗

bioRxiv2024

Phospho-regulated tuning of viscoelastic properties balances centrosome growth and strength

Centrosomes are membranelles organelles containing centrioles encapsulated by pericentriolar material (PCM). PCM nucleates microtubules that help position and segregate chromosomes during mitosis, yet how PCM resists microtubule-mediated forces is poorly understood at the material level. Here, we show that PLK-1 phosphorylation of SPD-5 tunes the dynamics and material properties of the PCM scaffold in C. elegans embryos. Microrheology of reconstituted PCM condensates reveals that PLK-1 phosphorylation decreases SPD-5 dynamics and increases condensate viscoelasticity. Similarly, in embryos, phospho-mimetic SPD-5 is less dynamic than wild-type SPD-5, which itself is less dynamic than phospho-null SPD-5. PCM built with phospho-null SPD-5 is smaller than normal, but its assembly can be partially rescued by reducing microtubule-dependent forces. The same is true for PCM built with phospho-mimetic SPD-5, yet the underlying causes are distinct: under force, phospho-null SPD-5 fails to assemble, while phospho-mimetic SPD-5 forms hyper-stable foci that fail to cohere into a uniform, spherical mass. Both mutants have defects with chromosome segregation and viability. Thus, tuning of SPD-5 phosphorylation optimizes PCM material properties to achieve correct PCM size, integrity, and function. Our results demonstrate how regulated chemical modification of a scaffolding protein modulates the material properties and function of a membraneless organelle.

cell biology↗

bioRxiv2024

Native molecular architectures of centrosomes in C. elegans embryos

Centrosomes organize microtubules that are essential for mitotic divisions in animal cells. They consist of centrioles surrounded by Pericentriolar Material (PCM). Questions related to mechanisms of centriole assembly, PCM organization, and microtubule formation remain unanswered, in part due to limited availability of molecular-resolution structural analyses in situ. Here, we use cryo-electron tomography to visualize centrosomes across the cell cycle in cells isolated from C. elegans embryos. We describe a pseudo-timeline of centriole assembly and identify distinct structural features including a cartwheel in daughter centrioles, and incomplete microtubule doublets surrounded by a star-shaped density in mother centrioles. We find that centriole and PCM microtubules differ in protofilament number (13 versus 11) indicating distinct nucleation mechanisms. This difference could be explained by atypical {gamma}-tubulin ring complexes with 11-fold symmetry identified at the minus ends of short PCM microtubules. We further characterize a porous and disordered network that forms the interconnected PCM. Thus, our work builds a three-dimensional structural atlas that helps explain how centrosomes assemble, grow, and achieve function.

cell biology↗

bioRxiv2023

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength

During mitotic spindle assembly, microtubules generate tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. The molecular interactions that enable PCM to assemble rapidly and resist external forces are unknown. Here we use cross-linking mass spectrometry to identify interactions underlying supramolecular assembly of SPD-5, the main PCM scaffold protein in C. elegans. Crosslinks map primarily to alpha helices within the phospho-regulated region (PReM), a long C-terminal coiled-coil, and a series of four N-terminal coiled-coils. PLK-1 phosphorylation of SPD-5 creates new homotypic contacts, including two between PReM and the CM2-like domain, and eliminates numerous contacts in disordered linker regions, thus favoring coiled-coil-specific interactions. Mutations within these interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces. Thus, PCM assembly and strength are interdependent. In vitro, self-assembly of SPD-5 scales with coiled-coil content, although there is a defined hierarchy of association. We propose that multivalent interactions among coiled-coil regions of SPD-5 build the PCM scaffold and contribute sufficient strength to resist microtubule-mediated forces.

cell biology↗

bioRxiv2023

A central helical hairpin in SPD-5 enables centrosome strength and assembly

Centrosomes organize microtubules for mitotic spindle assembly and positioning. Forces mediated by these microtubules create tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. How PCM resists these stresses is unclear at the molecular level. Here, we use cross-linking mass spectrometry (XL-MS) to map interactions underlying multimerization of SPD-5, an essential PCM scaffold component in C. elegans. We identified an interaction hotspot in an alpha helical hairpin motif in SPD-5 (a.a. 541-677). XL-MS data, ab initio structural predictions, and mass photometry suggest that this region dimerizes to form a tetrameric coiled-coil. Mutating a helical section (a.a. 610-640) or a single residue (R592) inhibited PCM assembly in embryos. This phenotype was rescued by eliminating microtubule pulling forces, revealing that PCM assembly and material strength are interrelated. We propose that interactions mediated by the helical hairpin strongly bond SPD-5 molecules to each other, thus enabling PCM to assemble fully and withstand stresses generated by microtubules.

cell biology↗

bioRxiv2021

Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

The tubby family protein-TULP3 coordinates with the intraflagellar transport complex-A (IFT-A) in trafficking certain transmembrane proteins to cilia. These transmembrane cargoes have short motifs that are necessary and sufficient for TULP3-mediated trafficking. However, whether TULP3 regulates trafficking of membrane-associated proteins is not well understood. Here we show that TULP3 is required for transport of the atypical GTPase ARL13B into cilia, and for ciliary enrichment of ARL13B-dependent farnesylated and myristoylated proteins. ARL13B transport requires TULP3 binding to IFT-A core but not to phosphoinositides, unlike transmembrane cargo transport that requires binding to both by TULP3. A conserved lysine in TULP3s tubby domain mediates direct ARL13B binding and trafficking of lipidated and transmembrane cargoes. An N-terminal amphipathic helix in ARL13B flanking the palmitoylation site mediates binding to TULP3 and directs trafficking to cilia even in absence of palmitoylation and RVxP sorting motif. Therefore, TULP3 transports transmembrane proteins and ARL13B into cilia by capture of short sequences through a shared tubby domain site.

cell biology↗

bioRxiv2019

Material aging causes centrosome weakening and disassembly during mitotic exit

Centrosomes must resist microtubule-mediated forces for mitotic chromosome segregation. During mitotic exit, however, centrosomes are deformed and fractured by those same forces, which is a key step in centrosome disassembly. How the functional material properties of centrosomes change throughout the cell cycle, and how they are molecularly tuned remain unknown. Here, we used optically-induced flow perturbations to determine the molecular basis of centrosome strength and ductility in C. elegans embryos. We found that both properties declined sharply at anaphase onset, long before natural disassembly. This mechanical transition required PP2A phosphatase and correlated with inactivation of PLK-1 (Polo Kinase) and SPD-2 (Cep192). In vitro, PLK-1 and SPD-2 directly protected centrosome scaffolds from force-induced disassembly. Our results suggest that, prior to anaphase, PLK-1 and SPD-2 confer strength and ductility to the centrosome scaffold so that it can resist microtubule-pulling forces. In anaphase, centrosomes lose PLK-1 and SPD-2 and transition to a weak, brittle state that enables force-mediated centrosome disassembly.

cell biology↗

bioRxiv2018

FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing

Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-{beta} family growth factors. To overcome these limitations, we engineered \"FlexiBAC\", a system that simplifies baculovirus production and permits furin-driven proteolytic maturation of targets. This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 146 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. We demonstrate that this system can be used to produce high levels of mature, active forms of TGF-{beta} family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains.

biochemistry↗

bioRxiv2017

Phosphatase PP2A and microtubule pulling forces disassemble centrosomes during mitotic exit

Centrosomes are major microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM) from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6), together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, [~]25% of PCM persisted from one cell cycle into the next, resulting in cytokinetic furrow ingression errors, excessive centrosome accumulation, and embryonic death. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and catastrophic rupture of the PCM scaffold.

cell biology↗

bioRxiv2016

The centrosome is a selective phase that nucleates microtubules by concentrating tubulin

Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C.elegans proteins. We found that macromolecular crowding drives phase separation of the key PCM scaffold protein SPD-5 into spherical droplets that morphologically and dynamically resemble in vivo PCM. These SPD-5 droplets recruited the microtubule polymerase ZYG-9 (XMAP215 homologue) and the microtubule-stabilizing protein TPXL-1 (TPX2 homologue). Together, these three proteins concentrated tubulin ~4- fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.\n\nOne Sentence SummaryPhase separation of C. elegans centrosome proteins drive the formation of micron-sized microtubule organizing centers.

cell biology↗