Macromolecular Crowding Tailors the Microtubule Cytoskeleton Through Tubulin Modifications and Microtubule-Associated Proteins

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Июнь 14, 2023

Cells remodel their cytoskeletal networks to adapt environment. Here, we analyze the mechanisms utilized by cell tailor its microtubule landscape in response changes osmolarity that alter macromolecular crowding. By integrating live imaging,

Язык: Английский

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Doublecortin restricts neuronal branching by regulating tubulin polyglutamylation

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Июнь 3, 2023

Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) that binds directly to microtubules via two (DC) domains. The DC domains sense the nucleotide state, longitudinal curvature, and protofilament number of microtubule lattice, indicating role in regulation structure neurons. Mutations DCX cause lissencephaly subcortical band heterotopia (also known as double-cortex syndrome) due impaired migration. To better understand migration, we developed model system based on induced pluripotent stem cells (iPSCs). We used CRISPR/Cas9 knock-out Dcx gene iPSCs differentiated into cortical Compared control neurons, DCX-KO neurons showed reduced velocities nuclear movements. coincided with an increase neurites early development, consistent migration phenotype previous findings mouse model. Neurite branching regulated by host MAPs other factors, well polymerization dynamics. However, EB comet dynamics were unchanged similar growth rates, lifetimes, numbers. Rather, observed significant reduction α-tubulin polyglutamylation Polyglutamylation levels rescued expression or TTLL11, glutamylase. Using U2OS orthogonal system, show TTLL11 act synergistically promote polyglutamylation. regulates numerous MAPs, severing enzymes, molecular motors. Consistently, observe lysosomes their processivity. propose acts positive regulator restricts neurite branching. Our results indicate unexpected for homeostasis tubulin code.

Язык: Английский

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Differential modification of the C-terminal tails of different α-tubulins and their importance for microtubule function in vivo

eLife, Год журнала: 2023, Номер 12

Опубликована: Июнь 22, 2023

Microtubules (MTs) are built from α-/β-tubulin dimers and used as tracks by kinesin dynein motors to transport a variety of cargos, such mRNAs, proteins, organelles, within the cell. Tubulins subjected several post-translational modifications (PTMs). Glutamylation is one them, it responsible for adding or more glutamic acid residues branched peptide chains C-terminal tails both α- β-tubulin. However, very little known about specific found on different tubulin isotypes in vivo role these PTMs MT other cellular processes vivo. In this study, we that Drosophila ovaries, glutamylation α-tubulin occurred clearly ends αTub84B αTub84D (αTub84B/D). contrast, ovarian α-tubulin, αTub67C, not glutamylated. The αTub84B/D glutamylated at glutamyl sidechains various combinations. TTLL5 required mono- poly-glutamylation with proper localization microtubules. Similarly, normal distribution kinesin-1 germline relies . Next, two kinesin-1-dependent processes, precise Staufen fast, bidirectional ooplasmic streaming, depend , too, suggesting causative pathway. nervous system, mutation inactivates its enzymatic activity decreases pausing anterograde axonal mitochondria. Our results demonstrate roles differential α-tubulins point importance functions involving microtubule transport.

Язык: Английский

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A Platform for Medium‐Throughput Cell‐Free Analyses of Microtubule‐Interacting Proteins Using Mammalian Cell Lysates

Current Protocols, Год журнала: 2024, Номер 4(6)

Опубликована: Июнь 1, 2024

Abstract The microtubule (MT) cytoskeleton performs a variety of functions in cell division, architecture, neuronal differentiation, and ciliary beating. These are controlled by proteins that directly interact with MTs, commonly referred to as microtubule‐associated (MAPs). Out the many reported only some have been biochemically functionally characterized so far. One limitations classical vitro assays single‐MT reconstitution approaches is they typically performed purified proteins. As purification can be difficult time‐consuming, previous studies focused on few proteins, while systematic analyses different were not possible. Here we present detailed protocol using lysates mammalian cells instead overcomes this limitation. Those contain all molecular components required for MT including endogenous tubulin recombinant MAPs, which form assemblies upon injection into microscopy chamber. This allows observe dynamic behavior growing well fluorescently labeled associated total internal reflection fluorescence (TIRF) microscopy. Strikingly, tested far functional our approach, thus providing possibility test virtually any protein interest. also opens screen impact patient mutations binding MAPs medium‐throughput manner. In addition, lysate approach easily adapted other applications predominantly far, such investigating cytoskeletal systems crosstalk, or study structures bound MTs cryo‐electron Our versatile, expandable, easy‐to‐use method characterize broad spectrum function. © 2024 Authors. Current Protocols published Wiley Periodicals LLC. Basic Protocol 1 : Preparation human TIRF 2 Quantification GFP‐tagged MAP concentration Support Purification KIF5B(N555/T92A) (dead kinesin) GMPCPP seeds 3 TIRF‐based MT‐MAP

Язык: Английский

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Selective regulation of kinesin-5 function by β-tubulin carboxy-terminal tails

The Journal of Cell Biology, Год журнала: 2024, Номер 224(3)

Опубликована: Дек. 17, 2024

The tubulin code hypothesis predicts that tails create programs for selective regulation of microtubule-binding proteins, including kinesin motors. However, the molecular mechanisms determine and their relevance in cells are poorly understood. We report budding yeast kinesin-5 motors by β-tubulin tail. Cin8, but not Kip1, requires tail recruitment to mitotic spindle, creating a balance both spindle efficient progression. identify negatively charged patch mediates interaction with Cin8. Using vitro reconstitution genetically modified tubulin, we demonstrate increases Cin8 plus-end-directed velocity processivity. Finally, positively amino-terminal extension coordinates interactions Our work identifies mechanism underlying closely related how this promotes proper function spindle.

Язык: Английский

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