Modulate Microtubule Dynamics Research Articles

The microtubule-severing enzyme spastin regulates spindle dynamics to promote chromosome segregation in Trypanosoma brucei.

Microtubule-severing enzymes play essential roles in regulating diverse cellular processes, including mitosis and cytokinesis, by modulating microtubule dynamics. In the early branching protozoan parasite Trypanosoma brucei , microtubule-severing enzymes are involved in cytokinesis and flagellum length control during different life cycle stages, but none of them have been found to regulate mitosis in any life cycle form. Here, we report the biochemical and functional characterization of the microtubule-severing enzyme spastin in the procyclic form of T. brucei . We demonstrate that spastin catalyzes microtubule severing in vitro and ectopic overexpression of spastin disrupts spindle microtubules in vivo in trypanosome cells, leading to defective chromosome segregation. Knockdown of spastin impairs spindle integrity and disrupts chromosome alignment in metaphase and chromosome segregation in anaphase. We further show that the function of spastin requires the catalytic AAA-ATPase domain, the microtubule-binding domain, and the microtubule interacting and trafficking domain, and that the association of spastin with spindle depends on the microtubule-binding domain. Together, these results uncover an essential role for spastin in chromosome segregation by regulating spindle dynamics in this unicellular eukaryote.

BEACH domain-containing protein SPIRRIG facilitates microtubule cytoskeleton-associated trichome morphogenesis in Arabidopsis.

Our studies reveal the involvement of SPI in cytoskeleton-associated trichome morphogenesis, expanding the roles of SPI in regulating plant epidermal celldevelopment. Acquisition of distinct shapes is crucial for cells to perform their biological functions in multicellular organisms. Trichomes are specialized epidermal cells of plant aerial parts, offering an excellent paradigm for dissecting the underlying regulatory mechanism of plant cell shape development at the single-cell level. SPIRRIG (SPI) that encodes a BEACH domain-containing protein was initially identified to regulate trichome branch extension, but the possible pathway(s) through which SPI regulates trichome morphogenesis remain unclear. Here, we report that SPI facilitates microtubule-associated regulation on trichome branching in Arabidopsis. Functional loss of SPI results in trichome morphogenesis hyper-sensitive to the microtubule-disrupting drug oryzalin, implying SPI may mediate microtubule stability during trichome development. Accordingly, spi mutant has less-branched trichomes. Detailed live-cell imaging showed that the spatio-temporal microtubule organization during trichome morphogenesis is aberrant in spi mutants. Further genetic investigation indicated that SPI may cooperate with ZWICHEL (ZWI) to modulate microtubule dynamics during trichome morphogenesis. ZWI encodes a kinesin-like calmodulin-binding protein (KCBP), whose distribution is necessary for the proper microtubule organization in trichomes, and zwi mutants produce less-branched trichomes as well. Trichome branching is further inhibited in spi-3 zwi-101 double mutants compared to either of the single mutant. Moreover, we found SPI could co-localize with the MYTH4 domain of ZWI. Taken together, our results expand the role of SPI in regulating trichome morphogenesis and also reveal a molecular and genetic pathway in plant cell shape formation control.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-10.

Transmembrane formins as active cargoes of membrane trafficking.

Formins are a large, evolutionarily old family of cytoskeletal regulators whose roles include actin capping and nucleation, as well as modulation of microtubule dynamics. The plant class I formin clade is characterized by a unique domain organization, as most of its members are transmembrane proteins with possible cell wall-binding motifs exposed to the extracytoplasmic space-a structure that appears to be a synapomorphy of the plant kingdom. While such transmembrane formins are traditionally considered mainly as plasmalemma-localized proteins contributing to the organization of the cell cortex, we review, from a cell biology perspective, the growing evidence that they can also, at least temporarily, reside (and in some cases also function) in endomembranes including secretory and endocytotic pathway compartments, the endoplasmic reticulum, the nuclear envelope, and the tonoplast. Based on this evidence, we propose that class I formins may thus serve as 'active cargoes' of membrane trafficking-membrane-embedded proteins that modulate the fate of endo- or exocytotic compartments while being transported by them.

The interplay between Wnt signaling pathways and microtubule dynamics.

Wnt signaling pathways represent an evolutionarily highly conserved, intricate network of molecular interactions that regulates various aspects of cellular behavior, including embryonic development and tissue homeostasis. Wnt signaling pathways share the β-catenin-dependent (canonical) and the multiple β-catenin-independent (non-canonical) pathways. These pathways collectively orchestrate a wide range of cellular processes through distinct mechanisms of action. Both the β-catenin-dependent and β-catenin-independent pathways are closely intertwined with microtubule dynamics, underscoring the complex crosstalk between Wnt signaling and the cellular cytoskeleton. This interplay involves several mechanisms, including how the components of Wnt signaling can influence the stability, organization, and distribution of microtubules. The modulation of microtubule dynamics by Wnt signaling plays a crucial role in coordinating cellular behaviors and responses to external signals. In this comprehensive review, we discussed the current understanding of how Wnt signaling and microtubule dynamics intersect in various aspects of cellular behavior. This study provides insights into our understanding of these crucial cellular processes.

Integrated Transcriptome Analysis Reveals Molecular Subtypes and ceRNA Networks in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system (CNS). While extensively studied, its molecular subtypes and mechanisms remain poorly understood, hindering the identification of effective therapeutic targets. We used ConsensusClusterPlus to analyze transcriptome data from 215 MS patient samples, identifying distinct molecular subtypes. Differential expression analysis and variability assessments were conducted to further characterize these subtypes. Additionally, circular RNAs (circRNAs) and microRNAs (miRNAs) were screened for potential ceRNA interactions. Three molecular subtypes were identified: MS-FCRL1 (C1), MS-BTG1 (C2), and MS-RPL38 (C3). Each subtype was involved in key MS-related pathways (as annotated by KEGG), but the core genes regulating these pathways differed significantly among the subtypes. Subtype C3 exhibited neurodegenerative pathway enrichment, increased immune activity, and immune cell infiltration, suggesting a more severe disease course. Further analysis revealed 18 differentially expressed circRNAs and 22 miRNAs, with EEF1D and TUBA1A as hub targets in C3. Differential activation of immune pathways across MS subtypes suggests specific gene expression drives disease heterogeneity. We propose a circ_0045537/miR-196a-5p/TUBA1A axis in subtype C3, modulating microtubule dynamics and worsening MS severity.

Microtubules as a signal hub for axon growth in response to mechanical force.

Microtubules are highly polar structures and arecharacterized by high anisotropy and stiffness. In neurons, they play a key role in the directional transport of vesicles and organelles. In the neuronal projections called axons, they form parallel bundles, mostly oriented with theplus-end towards the axonal termination. Their physico-chemical properties have recently attracted attention as a potential candidate in sensing, processing and transducing physical signals generated by mechanical forces. Here, wediscuss the main evidence supporting the role of microtubules as a signal hub for axon growth in response to a traction force. Applying a tension to the axon appears to stabilize the microtubules, which, in turn, coordinate a modulation of axonal transport, local translation and their cross-talk. We speculate on the possible mechanisms modulating microtubule dynamics under tension, based on evidence collected in neuronal and non-neuronal cell types. However, the fundamental question of the causal relationship between these mechanisms is still elusive because the mechano-sensitive element in this chain has not yet been identified.

OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer.

Oxidative stress induced growth inhibitor 1 (OSGIN1) regulates cell death. The role and underlying molecular mechanism of OSGIN1 in non-small cell lung cancer (NSCLC) are uncharacterized. OSGIN1 expression in NSCLC samples was detected using immunohistochemistry and Western blotting. Growth of NSCLC cells and gefitinib-resistant cells expressing OSGIN1 or TUBB3 knockdown was determined by MTT, soft agar, and foci formation assays. The effect of OSGIN1 knockdown on in vivo tumor growth was assessed using NSCLC patient-derived xenograft models and gefitinib-resistant patient-derived xenograft models. Potentially interacting protein partners of OSGIN1 were identified using IP-MS/MS, immunoprecipitation, PLA, and Western blottingassays. Microtubule dynamics were explored by tubulin polymerization assay and immunofluorescence. Differential expression of signaling molecules in OSGIN1 knockdown cells was investigated using phospho-proteomics, KEGG analysis, and Western blotting. We found thatOSGIN1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and tumor size in lung cancer patients. OSGIN1 knockdown inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Knockdown of OSGIN1 strongly increased tubulin polymerization and re-established gefitinib sensitivity in vitro and in vivo. Additionally, knockdown of TUBB3 strongly inhibited NSCLC cell proliferation. Mechanistically, we found that OSGIN1 enhances DYRK1A-mediated TUBB3 phosphorylation, which is critical for inducing tubulin depolymerization. The results of phospho-proteomics and ontology analysis indicated that knockdown of OSGIN1 led to reduced propagation of the MKK3/6-p38 signaling axis. We propose that OSGIN1 modulates microtubule dynamics by enhancing DYRK1A-mediated phosphorylation of TUBB3 at serine 172. Moreover, elevated OSGIN1 expression promotes NSCLC tumor growth and gefitinib resistance through the MKK3/6-p38 signaling pathway. Our findings unveil a new mechanism of OSGIN1 and provide a promising therapeutic target for NSCLC treatment in the clinic.

Microtubule‐targeting agents for cancer treatment: Seven binding sites and three strategies

AbstractMicrotubules are pivotal in diverse cellular functions encompassing cell signaling, morphology, intracellular trafficking, and cell mitosis/division. They are validated targets for disease treatment, notably hematological cancers and solid tumors. Microtubule‐targeting agents (MTAs) exert their effects by modulating microtubule dynamics, impeding cell proliferation, and promoting cell death. Recent advances in structural biology have unveiled novel perspectives for investigating multiple binding sites and mechanisms of action used by MTAs. In this review, we first provide an overview of the intricate structure and dynamics of microtubules. Then we explore the seven binding sites and the three primary strategies (stabilization, destabilization, and degradation) harnessed by MTAs. Furthermore, we introduce the emerging domain of microtubule‐targeting degraders, exemplified by PROteolysis TArgeting Chimeras and small‐molecule degraders, which enable precise degradation of specific microtubule‐associated proteins implicated in cancer pathogenesis. Additionally, we discuss the promising realm of precision‐targeted approaches, including antibody–drug conjugates and the utilization of photopharmacology in MTAs. Lastly, we provide a comprehensive overview of the clinical applications of microtubule‐targeting therapies, assessing their efficacy and current challenges. We aim to provide a global picture of MTAs development as well as insights into the microtubule‐targeting drug discovery for cancer treatment.

Cystathionine β-synthase affects organization of cytoskeleton and modulates carcinogenesis in colorectal carcinoma cells.

Cystathionine β-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins β-actin and β-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal β-tubulin and, to a lesser extent, also to β-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

Maize tubulin folding cofactor B is required for cell division and cell growth through modulating microtubule homeostasis.

Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.

Intermittent Fasting Activates AMP-Kinase to Restructure Right Ventricular Lipid Metabolism and Microtubules

Intermittent fasting (IF) extends life span via pleotropic mechanisms, but one important molecular mediator is adenosine monophosphate-activated protein kinase (AMPK). AMPK enhances lipid metabolism and modulates microtubule dynamics. Dysregulation of these molecular pathways causes right ventricular (RV) failure in patients with pulmonary arterial hypertension. In rodent pulmonary arterial hypertension, IF activates RV AMPK, which restores mitochondrial and peroxisomal morphology and restructures mitochondrial and peroxisomal lipid metabolism protein regulation. In addition, IF increases electron transport chain protein abundance and activity in the right ventricle. Echocardiographic and hemodynamic measures of RV function are positively associated with fatty acid oxidation and electron transport chain protein levels. IF also combats heightened microtubule density, which normalizes transverse tubule structure.

Divalent metal ion mediated structural transitions in microtubule bundles in dissipative reaction mixtures revealed by synchrotron SAXS and plastic-embedded TEM.

Tau is an intrinsically disordered neuronal protein known to modulate microtubule dynamics through the suppression of dynamic instability. Recent studies have shown that Tau mediates dynamical linear microtubule bundles similar to microtubule fascicles observed in vivo, which are a cardinal feature of the axon initial segment (P. J. Chung, C. Song, et al. Nature Communications 2016, 7, 12278. DOI: 10.1038/ncomms12278 and ACS Macro Lett. 2018, 7, 228-232. DOI: 10.1021/acsmacrolett.7b00937). In order to better understand tau's role in the physiologic bundling of MTs, quantitative techniques of synchrotron small-angle x-ray scattering data—combined with plastic-embedded TEM, and other analytical techniques—have been utilized. Here we show that Ca2+ and Mg2+ in the mM concentration range (corresponding to the average concentration in cells for Mg2+) can destabilize steady-state, bundled microtubules (in the presence of GTP at 37 °C), inducing a novel bundle-to-bundle transition prior to the tau-coated tubulin ring state. This transitory bundled phase suggests that tubulin oligomers also play a role in stabilizing MT bundles.

Evolutionary perspective of Big tau structure: 4a exon variants of MAPT.

The MAPT gene encoding the microtubule-associated protein tau can generate multiple isoforms by alternative splicing giving rise to proteins which are differentially expressed in specific areas of the nervous system and at different developmental stages. Tau plays important roles in modulating microtubule dynamics, axonal transport, synaptic plasticity, and DNA repair, and has also been associated with neurodegenerative diseases (tauopathies) including Alzheimer's disease and frontotemporal dementia. A unique high-molecular-weight isoform of tau, originally found to be expressed in the peripheral nervous system and projecting neurons, has been termed Big tau and has been shown to uniquely contain the large exon 4a that significantly increases the size and 3D structure of tau. With little progress since the original discovery of Big tau, more than 25 years ago, we have now completed a comprehensive comparative study to analyze the structure of the MAPT gene against available databases with respect to the composition of the tau exons as they evolved from early vertebrates to primates and human. We focused the analysis on the evolution of the 4a exon variants and their homology relative to humans. We discovered that the 4a exon defining Big tau appears to be present early in vertebrate evolution as a large insert that dramatically changed the size of the tau protein with low sequence conservation despite a stable size range of about 250aa, and in some species a larger 4a-L exon of 355aa. We suggest that 4a exon variants evolved independently in different species by an exonization process using new alternative splicing to address the growing complexities of the evolving nervous systems. Thus, the appearance of a significantly larger isoform of tau independently repeated itself multiple times during evolution, accentuating the need across vertebrate species for an elongated domain that likely endows Big tau with novel physiological functions as well as properties related to neurodegeneration.

Analysis of Tau/Nucleoporin Interactions by Surface Plasmon Resonance Spectroscopy.

Tau, a soluble and predominantly neuronal protein, is best known for its microtubule (MT)-binding function in the cytosol, where it decisively contributes to stability as well as modulation of MT dynamics. In Alzheimer's disease and other tauopathies, Tau is altered into forming intracellular neurofibrillary tangles; additionally, also a mislocalization from the cytosol to the nucleus has been observed where interactions of Tau with the nucleus become possible. Using surface plasmon resonance (SPR), it was recently shown that Tau can directly interact with certain nucleoporins (e.g., Nup98), components of the nuclear pore complex (NPC). The NPC constitutes large regulated pores in the nuclear envelope that facilitate the bidirectional exchange of proteins, nucleic acids, and other biomolecules between the inner section of the nucleus and the cytosol, the nucleocytoplasmic transport. The mechanism of Tau/Nup interactions is as yet unknown, and a systematic interaction analysis of Tau with different Nups can be of high value to decipher the molecular binding mechanism of Tau to Nups. SPR is a useful tool to analyze binding affinities and kinetic parameters in a label-free environment. While one interaction partner is immobilized on a sensor chip, the second is supplied within a constant flow of buffer. Binding of mobile molecules to immobilized ones changes the refractive index of the medium close to the sensor surface with the signal being proportional to the bound mass. In this chapter, we describe the application of the SPR technique for the investigation of Tau binding to nucleoporins.

Stathmin expression alters the antiproliferative effect of eribulin in leiomyosarcoma cells

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ β-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

Centrosome-dependent microtubule modifications set the conditions for axon formation.

Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.

CK1 Is a Druggable Regulator of Microtubule Dynamics and Microtubule-Associated Processes.

Simple SummaryThe Casein Kinase 1 (CK1) family of serine-threonine specific protein kinases regulates the activity of key regulatory proteins and signaling pathways being involved in embryonic development but also in the adult organism. Furthermore, it plays an important role in the regulation of proliferation, differentiation, apoptotic processes, circadian rhythm, chromosome segregation, and other microtubule-associated processes. Deregulation of CK1 expression and activity, as well as mutations in the coding region, contribute to the development of many human pathologies, including cancer. Alternations in the site-specific phosphorylation of α/β-tubulin and microtubule-associated proteins affect microtubule stability, finally resulting in mitotic defects and genomic instability. Here we review our knowledge about CK1 functions in general and especially in chromosome segregation. Furthermore, an update in modulating CK1 activity by small molecule inhibitors and peptides specifically inhibiting CK1 protein interactions as new therapy concepts for the treatment of cancer will be discussed.Protein kinases of the Casein Kinase 1 family play a vital role in the regulation of numerous cellular processes. Apart from functions associated with regulation of proliferation, differentiation, or apoptosis, localization of several Casein Kinase 1 isoforms to the centrosome and microtubule asters also implicates regulatory functions in microtubule dynamic processes. Being localized to the spindle apparatus during mitosis Casein Kinase 1 directly modulates microtubule dynamics by phosphorylation of tubulin isoforms. Additionally, site-specific phosphorylation of microtubule-associated proteins can be related to the maintenance of genomic stability but also microtubule stabilization/destabilization, e.g., by hyper-phosphorylation of microtubule-associated protein 1A and RITA. Consequently, approaches interfering with Casein Kinase 1-mediated microtubule-specific functions might be exploited as therapeutic strategies for the treatment of cancer. Currently pursued strategies include the development of Casein Kinase 1 isoform-specific small molecule inhibitors and therapeutically useful peptides specifically inhibiting kinase-substrate interactions.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Photoswitchable Epothilone-Based Microtubule Stabilisers Allow GFP-Imaging-Compatible, Optical Control over the Microtubule Cytoskeleton.

Optical methods to modulate microtubule dynamics show promise for reaching the micron‐ and millisecond‐scale resolution needed to decrypt the roles of the cytoskeleton in biology. However, optical microtubule stabilisers are under‐developed. We introduce “STEpos” as GFP‐orthogonal, light‐responsive epothilone‐based microtubule stabilisers. They use a novel styrylthiazole photoswitch in a design to modulate hydrogen‐bonding and steric effects that control epothilone potency. STEpos photocontrol microtubule dynamics and cell division with micron‐ and second‐scale spatiotemporal precision. They substantially improve potency, solubility, and ease‐of‐use compared to previous optical microtubule stabilisers, and the structure‐photoswitching‐activity relationship insights in this work will guide future optimisations. The STEpo reagents can contribute greatly to high‐precision research in cytoskeleton biophysics, cargo transport, cell motility, cell division, development, and neuroscience.