Michal Wieczorek

Last Name

Wieczorek

First Name

Michal

ORCID

0000-0003-3067-7314

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09763 - Wieczorek, Michal / Wieczorek, Michal

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Publications 1 - 10 of 20

Conformational Regulation of Vertebrate γ‐Tubulin Ring Complexes by CM1 Proteins
Wieczorek, Michal (2024)
Cytoskeleton
Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development
Kowanda, Michelle; Bergalet, Julie; Wieczorek, Michal; et al. (2016)
Biology Open
The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3′UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in midoogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila.
Macromolecular Crowding Pushes Catalyzed Microtubule Growth to Near the Theoretical Limit
Wieczorek, Michal; Chaaban, Sami; Brouhard, Gary J. (2013)
Cellular and Molecular Bioengineering
Purification of Affinity Tag-free Recombinant Tubulin from Insect Cells
Ti, Shih-Chieh; Wieczorek, Michal; Kapoor, Tarun M. (2020)
STAR Protocols
α/β-tubulin heterodimers, which can harbor diverse isotypes and post-translational modifications, polymerize into microtubules that are fundamental to many cellular processes. Due to long-standing challenges in generating recombinant tubulin, however, it has been difficult to examine the properties of specific tubulin isotypes. Here, we provide a protocol for purifying milligrams of affinity tag-free, isotypically pure recombinant tubulin. Our method can be applicable to any tubulin of interest, opening the door to dissecting how tubulin diversity regulates microtubule function. For complete details on the use and execution of this protocol, please see Ti et al. (2018).
Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality
Noujaim, Michael; Bechstedt, Susanne; Wieczorek, Michal; et al. (2014)
PLoS ONE
Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a ‘‘reduction in dimensionality.’’ We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubuleassociated substrates.
Variations on the Single-Molecule Assay for Microtubule-Associated Proteins and Kinesins
Bechstedt, Susanne; Wieczorek, Michal; Noujaim, Michael; et al. (2011)
Methods in Molecular Biology ~ Microtubule Dynamics
CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC
Rai, Dipti; Song, Yinlong; Hua, Shasha; et al. (2024)
Nature Cell Biology
γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.
Microtubules in Asgard archaea
Wollweber, Florian; Xu, Jingwei; Ponce-Toledo, Rafael I.; et al. (2025)
Cell
Microtubules are a hallmark of eukaryotes. Archaeal and bacterial homologs of tubulins typically form homopolymers and non-tubular superstructures. The origin of heterodimeric tubulins assembling into microtubules remains unclear. Here, we report the discovery of microtubule-forming tubulins in Asgard archaea, the closest known relatives of eukaryotes. These Asgard tubulins (AtubA/B) are closely related to eukaryotic α/β-tubulins and the enigmatic bacterial tubulins BtubA/B. Proteomics of Candidatus Lokiarchaeum ossiferum showed that AtubA/B were highly expressed. Cryoelectron microscopy structures demonstrate that AtubA/B form eukaryote-like heterodimers, which assembled into 5-protofilament bona fide microtubules in vitro. The additional paralog AtubB2 lacks a nucleotide-binding site and competitively displaced AtubB. These AtubA/B2 heterodimers polymerized into 7-protofilament non-canonical microtubules. In a sub-population of Ca. Lokiarchaeum ossiferum cells, cryo-tomography revealed tubular structures, while expansion microscopy identified AtubA/B cytoskeletal assemblies. Our findings suggest a pre-eukaryotic origin of microtubules and provide a framework for understanding the fundamental principles of microtubule assembly.
Structure and assembly of the A-C linker connecting microtubule triplets in centrioles
Tsai, Bin; Xu, Jingwei; Collet, Erik H.; et al. (2025)
Science Advances
Centriole assembly involves the coordination of centriolar modules. One module is the A-C linker, an enigmatic protein assembly connecting the A-microtubule of one microtubule triplet to the C-microtubule of the neighboring triplet. Here, we integrated biochemistry, multiscale cryo–electron microscopy, and AlphaFold modeling to investigate the architecture of the centriole. Using an improved centriole isolation method, we determined the structure of the A-C linker bound to microtubule triplets, which revealed how the A-C linker cross-links microtubules and integrates with the B-C junction. We found marked changes in the structure and composition of the A-C linker that correlate with the presence of other centriolar modules, including the pinhead, cartwheel, and inner scaffold. Our findings show that the A-C linker is a highly integrated component of the centriole whose polymorphism may orchestrate the assembly of spatially distinct centriolar modules, and provide a framework for dissecting the biology of centrioles.
Improved Helix and Kink Characterization in Membrane Proteins Allows Evaluation of Kink Sequence Predictors
Langelaan, David N.; Wieczorek, Michal; Blouin, Christian; et al. (2010)
Journal of Chemical Information and Modeling

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Michal Wieczorek

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