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The Journal of Cell Biology Apr 2023Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins...
Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.
Topics: Centrioles; Centrosome; Cytoskeleton; Microtubule-Associated Proteins; Microtubules; Cell Cycle Proteins; Humans
PubMed: 36752787
DOI: 10.1083/jcb.202208062 -
Cells Jun 2020Distinctly organized microtubule networks contribute to the function of differentiated cell types such as neurons, epithelial cells, skeletal myotubes, and... (Review)
Review
Distinctly organized microtubule networks contribute to the function of differentiated cell types such as neurons, epithelial cells, skeletal myotubes, and cardiomyocytes. In striated (i.e. skeletal and cardiac) muscle cells, the nuclear envelope acts as the dominant microtubule-organizing center (MTOC) and the function of the centrosome-the canonical MTOC of mammalian cells-is attenuated, a common feature of differentiated cell types. We summarize the mechanisms known to underlie MTOC formation at the nuclear envelope, discuss the significance of the nuclear envelope MTOC for muscle function and cell cycle progression, and outline potential mechanisms of centrosome attenuation.
Topics: Animals; Cell Cycle; Centrosome; Humans; Microtubule-Organizing Center; Microtubules; Muscle Cells; Muscle, Striated
PubMed: 32503326
DOI: 10.3390/cells9061395 -
Cells Aug 2021This essay focuses on the role of plectin and its various isoforms in mediating intermediate filament (IF) network functions. It is based on previous studies that... (Review)
Review
This essay focuses on the role of plectin and its various isoforms in mediating intermediate filament (IF) network functions. It is based on previous studies that provided comprehensive evidence for a concept where plectin acts as an IF recruiter, and plectin-mediated IF networking and anchoring are key elements in IF function execution. Here, plectin's global role as modulator of IF functionality is viewed from different perspectives, including the mechanical stabilization of IF networks and their docking platforms, contribution to cellular viscoelasticity and mechanotransduction, compartmentalization and control of the actomyosin machinery, connections to the microtubule system, and mechanisms and specificity of isoform targeting. Arguments for IF networks and plectin acting as mutually dependent partners are also given. Lastly, a working model is presented that describes a unifying mechanism underlying how plectin-IF networks mechanically control and propagate actomyosin-generated forces, affect microtubule dynamics, and contribute to mechanotransduction.
Topics: Actomyosin; Animals; Humans; Intermediate Filaments; Microtubules; Plectin; Protein Isoforms
PubMed: 34440923
DOI: 10.3390/cells10082154 -
Current Opinion in Neurobiology Aug 2019Neurons are exquisitely polarized cells whose structure and function relies on microtubules. Microtubules in signal-receiving dendrites and signal-sending axons differ... (Review)
Review
Neurons are exquisitely polarized cells whose structure and function relies on microtubules. Microtubules in signal-receiving dendrites and signal-sending axons differ in their organization and microtubule-associated proteins. These differences, coupled with microtubule post-translational modifications, combine to locally regulate intracellular transport, morphology, and function. Recent discoveries provide new insight into the regulation of non-centrosomal microtubule arrays in neurons, the relationship between microtubule acetylation and mechanosensation, and the spatial patterning of microtubules that regulates motor activity and cargo delivery in axons and dendrites. Together, these new studies bring us closer to understanding how microtubule function is locally tuned to match the specialized tasks associated with signal reception and transmission.
Topics: Microtubule-Associated Proteins; Microtubules; Neurons
PubMed: 30738328
DOI: 10.1016/j.conb.2019.01.003 -
Cell Oct 2019The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme...
The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
Topics: Axoneme; Cell Movement; Cilia; Ciliopathies; Cryoelectron Microscopy; Humans; Microtubule Proteins; Microtubules; Stress, Mechanical
PubMed: 31668805
DOI: 10.1016/j.cell.2019.09.030 -
Molecules (Basel, Switzerland) Aug 2020Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for... (Review)
Review
Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule-MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the consequences of such a phenomenon, including drug resistance development.
Topics: Animals; Cell Division; Humans; Microtubule-Associated Proteins; Microtubules; Neoplasms
PubMed: 32823874
DOI: 10.3390/molecules25163705 -
Cells Nov 2021The primary cilium, an antenna-like structure that protrudes out from the cell surface, is present in most cell types. It is a microtubule-based organelle that serves as... (Review)
Review
The primary cilium, an antenna-like structure that protrudes out from the cell surface, is present in most cell types. It is a microtubule-based organelle that serves as a mega-signaling center and is important for sensing biochemical and mechanical signals to carry out various cellular processes such as proliferation, migration, differentiation, and many others. At any given time, cilia length is determined by a dynamic balance of cilia assembly and disassembly processes. Abnormally short or long cilia can cause a plethora of human diseases commonly referred to as ciliopathies, including, but not limited to, skeletal malformations, obesity, autosomal dominant polycystic kidney disease, retinal degeneration, and bardet-biedl syndrome. While the process of cilia assembly is studied extensively, the process of cilia disassembly and its biological role(s) are less well understood. This review discusses current knowledge on ciliary disassembly and how different cellular processes and molecular signals converge to carry out this process. This information will help us understand how the process of ciliary disassembly is regulated, identify the key steps that need further investigation, and possibly design therapeutic targets for a subset of ciliopathies that are causally linked to defective ciliary disassembly.
Topics: Animals; Cilia; Humans; Microtubules; Models, Biological; Polymerization; Signal Transduction
PubMed: 34831200
DOI: 10.3390/cells10112977 -
Molecular Biology of the Cell Jun 2023Microtubules are noncovalent polymers built from αβ-tubulin dimers. The disordered C-terminal tubulin tails are functionalized with multiple glutamate chains of...
Microtubules are noncovalent polymers built from αβ-tubulin dimers. The disordered C-terminal tubulin tails are functionalized with multiple glutamate chains of variable lengths added and removed by tubulin tyrosine ligases (TTLLs) and carboxypeptidases (CCPs). Glutamylation is abundant on stable microtubule arrays such as in axonemes and axons, and its dysregulation leads to human pathologies. Despite this, the effects of glutamylation on intrinsic microtubule dynamics are unclear. Here we generate tubulin with short and long glutamate chains and show that glutamylation slows the rate of microtubule growth and increases catastrophes as a function of glutamylation levels. This implies that the higher stability of glutamylated microtubules in cells is due to effectors. Interestingly, EB1 is minimally affected by glutamylation and thus can report on the growth rates of both unmodified and glutamylated microtubules. Finally, we show that glutamate removal by CCP1 and 5 is synergistic and occurs preferentially on soluble tubulin, unlike TTLL enzymes that prefer microtubules. This substrate preference establishes an asymmetry whereby once the microtubule depolymerizes, the released tubulin is reset to a less-modified state, while polymerized tubulin accumulates the glutamylation mark. Our work shows that a modification on the disordered tubulin tails can directly affect microtubule dynamics and furthers our understanding of the mechanistic underpinnings of the tubulin code.
Topics: Humans; Tubulin; Microtubules; Glutamic Acid; Axoneme; Protein Processing, Post-Translational
PubMed: 37074962
DOI: 10.1091/mbc.E23-01-0030 -
Proceedings of the National Academy of... Aug 2023Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here,...
Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here, we show that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid networks. When CLIP-170 and EB3 act together, the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells, these liquid networks can concentrate tubulin. In vitro, in the presence of microtubules, phase separation of EB3/CLIP-170 can enrich tubulin all along the microtubule. In this condition, microtubule growth speed increases up to twofold and the frequency of depolymerization events are strongly reduced compared to conditions in which there is no phase separation. Our data show that phase separation of EB3/CLIP-170 adds an additional layer of regulation to the control of microtubule growth dynamics.
Topics: Tubulin; Microtubules
PubMed: 37603768
DOI: 10.1073/pnas.2301457120 -
Current Opinion in Cell Biology Oct 2019Microtubules play essential roles in cellular organization, cargo transport, and chromosome segregation during cell division. During mitosis microtubules form a... (Review)
Review
Microtubules play essential roles in cellular organization, cargo transport, and chromosome segregation during cell division. During mitosis microtubules form a macromolecular structure known as the mitotic spindle that is responsible for the accurate segregation of chromosomes between the two daughter cells. This is accomplished thanks to finely tuned control of microtubule dynamics. Even small changes in microtubule dynamics during spindle formation and/or operation may lead to chromosome mis-segregation, chromosome instability and aneuploidy. These three events are directly correlated with human diseases like cancer and developmental defects. Precise measurements of microtubule dynamics in the spindle will allow us to discover new molecules involved in regulating microtubule dynamics and enable a deeper understanding of the mechanisms that underlie mitosis and cancer emergence and development. Moreover, many chemotherapeutic agents for cancer treatment are targeted to microtubules, so continued investigation of their dynamics with utmost precision will facilitate the development of new drugs. Measuring microtubule dynamics in the spindle has been a difficult task until recently. With the development of new and gentler microscopic techniques, and new computer programs, we can perform better and more accurate measurements of microtubule dynamics during mitosis.
Topics: Animals; Chromosomal Instability; Chromosome Segregation; Humans; Microtubules; Mitosis; Models, Biological; Spindle Apparatus
PubMed: 31108428
DOI: 10.1016/j.ceb.2019.03.017