The kinesin motor Kif9 regulates centriolar satellite positioning and mitotic progression.

Centrosomes are the principal microtubule-organizing centers of the cell and play an essential role in mitotic spindle function. Centrosome biogenesis is achieved by strict control of protein acquisition and phosphorylation prior to mitosis. Defects in this process promote fragmentation of pericentriolar material culminating in multipolar spindles and chromosome missegregation. Centriolar satellites, membrane-less aggrupations of proteins involved in the trafficking of proteins toward and away from the centrosome, are thought to contribute to centrosome biogenesis. Here we show that the microtubule plus-end directed kinesin motor Kif9 localizes to centriolar satellites and regulates their pericentrosomal localization during interphase. Lack of Kif9 leads to aggregation of satellites closer to the centrosome and increased centrosomal protein degradation that disrupts centrosome maturation and results in chromosome congression and segregation defects during mitosis. Our data reveal roles for Kif9 and centriolar satellites in the regulation of cellular proteostasis and mitosis.

KIF2C/MCAK a prognostic biomarker and its oncogenic potential in malignant progression, and prognosis of cancer patients: a systematic review and meta-analysis as biomarker.

KIF2C/MCAK (KIF2C) is the most well-characterized member of the kinesin-13 family, which is critical in the regulation of microtubule (MT) dynamics during mitosis, as well as interphase. This systematic review briefly describes the important structural elements of KIF2C, its regulation by multiple molecular mechanisms, and its broad cellular functions. Furthermore, it systematically summarizes its oncogenic potential in malignant progression and performs a meta-analysis of its prognostic value in cancer patients. KIF2C was shown to be involved in multiple crucial cellular processes including cell migration and invasion, DNA repair, senescence induction and immune modulation, which are all known to be critical during the development of malignant tumors. Indeed, an increasing number of publications indicate that KIF2C is aberrantly expressed in multiple cancer entities. Consequently, we have highlighted its involvement in at least five hallmarks of cancer, namely: genome instability, resisting cell death, activating invasion and metastasis, avoiding immune destruction and cellular senescence. This was followed by a systematic search of KIF2C/MCAK's expression in various malignant tumor entities and its correlation with clinicopathologic features. Available data were pooled into multiple weighted meta-analyses for the correlation between KIF2Chigh protein or gene expression and the overall survival in breast cancer, non-small cell lung cancer and hepatocellular carcinoma patients. Furthermore, high expression of KIF2C was correlated to disease-free survival of hepatocellular carcinoma. All meta-analyses showed poor prognosis for cancer patients with KIF2Chigh expression, associated with a decreased overall survival and reduced disease-free survival, indicating KIF2C's oncogenic potential in malignant progression and as a prognostic marker. This work delineated the promising research perspective of KIF2C with modern in vivo and in vitro technologies to further decipher the function of KIF2C in malignant tumor development and progression. This might help to establish KIF2C as a biomarker for the diagnosis or evaluation of at least three cancer entities.

Production of CRISPR-Cas9 Transgenic Cell Lines for Knocksideways Studies.

Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin-relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off-target effects are minimized by testing for the most efficient target site using a split-GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split-GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners.

Microtubule Targeting Agents in Disease: Classic Drugs, Novel Roles.

Microtubule-targeting agents (MTAs) represent one of the most successful first-line therapies prescribed for cancer treatment. They interfere with microtubule (MT) dynamics by either stabilizing or destabilizing MTs, and in culture, they are believed to kill cells via apoptosis after eliciting mitotic arrest, among other mechanisms. This classical view of MTA therapies persisted for many years. However, the limited success of drugs specifically targeting mitotic proteins, and the slow growing rate of most human tumors forces a reevaluation of the mechanism of action of MTAs. Studies from the last decade suggest that the killing efficiency of MTAs arises from a combination of interphase and mitotic effects. Moreover, MTs have also been implicated in other therapeutically relevant activities, such as decreasing angiogenesis, blocking cell migration, reducing metastasis, and activating innate immunity to promote proinflammatory responses. Two key problems associated with MTA therapy are acquired drug resistance and systemic toxicity. Accordingly, novel and effective MTAs are being designed with an eye toward reducing toxicity without compromising efficacy or promoting resistance. Here, we will review the mechanism of action of MTAs, the signaling pathways they affect, their impact on cancer and other illnesses, and the promising new therapeutic applications of these classic drugs.

Kinase-anchoring proteins in ciliary signal transduction.

Historically, the diffusion of chemical signals through the cell was thought to occur within a cytoplasmic soup bounded by the plasma membrane. This theory was predicated on the notion that all regulatory enzymes are soluble and moved with a Brownian motion. Although enzyme compartmentalization was initially rebuffed by biochemists as a 'last refuge of a scoundrel', signal relay through macromolecular complexes is now accepted as a fundamental tenet of the burgeoning field of spatial biology. A-Kinase anchoring proteins (AKAPs) are prototypic enzyme-organizing elements that position clusters of regulatory proteins at defined subcellular locations. In parallel, the primary cilium has gained recognition as a subcellular mechanosensory organelle that amplifies second messenger signals pertaining to metazoan development. This article highlights advances in our understanding of AKAP signaling within the primary cilium and how defective ciliary function contributes to an increasing number of diseases known as ciliopathies.

A brain-penetrant microtubule-targeting agent that disrupts hallmarks of glioma tumorigenesis.

BACKGROUND: Glioma is sensitive to microtubule-targeting agents (MTAs), but most MTAs do not cross the blood brain barrier (BBB). To address this limitation, we developed the new chemical entity, ST-401, a brain-penetrant MTA. METHODS: Synthesis of ST-401. Measures of MT assembly and dynamics. Cell proliferation and viability of patient-derived (PD) glioma in culture. Measure of tumor microtube (TM) parameters using immunofluorescence analysis and machine learning-based workflow. Pharmacokinetics (PK) and experimental toxicity in mice. In vivo antitumor activity in the RCAS/tv-a PDGFB-driven glioma (PDGFB-glioma) mouse model. RESULTS: We discovered that ST-401 disrupts microtubule (MT) function through gentle and reverisible reduction in MT assembly that triggers mitotic delay and cell death in interphase. ST-401 inhibits the formation of TMs, MT-rich structures that connect glioma to a network that promotes resistance to DNA damage. PK analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft flank tumor mouse model demonstrates significant antitumor activity and no over toxicity in mice. In the PDGFB-glioma mouse model, ST-401 enhances the therapeutic efficacies of temozolomide (TMZ) and radiation therapy (RT). CONCLUSION: Our study identifies hallmarks of glioma tumorigenesis that are sensitive to MTAs and reports ST-401 as a promising chemical scaffold to develop brain-penetrant MTAs.

Phosphorylation of NMDA receptors by cyclin B/CDK1 modulates calcium dynamics and mitosis.

N-methyl-D-aspartate receptors (NMDAR) are glutamate-gated calcium channels named after their artificial agonist. NMDAR are implicated in cell proliferation under normal and pathophysiological conditions. However, the role of NMDAR during mitosis has not yet been explored in individual cells. We found that neurotransmitter-evoked calcium entry via endogenous NMDAR in cortical astrocytes was transient during mitosis. The same occurred in HEK293 cells transfected with the NR1/NR2A subunits of NMDAR. This transient calcium entry during mitosis was due to phosphorylation of the first intracellular loop of NMDAR (S584 of NR1 and S580 of NR2A) by cyclin B/CDK1. Expression of phosphomimetic mutants resulted in transient calcium influx and enhanced NMDAR inactivation independent of the cell cycle phase. Phosphomimetic mutants increased entry of calcium in interphase and generated several alterations during mitosis: increased mitotic index, increased number of cells with lagging chromosomes and fragmentation of pericentriolar material. In summary, by controlling cytosolic calcium, NMDAR modulate mitosis and probably cell differentiation/proliferation. Our results suggest that phosphorylation of NMDAR by cyclin B/CDK1 during mitosis is required to preserve mitotic fidelity. Altering the modulation of the NMDAR by cyclin B/CDK1 may conduct to aneuploidy and cancer.

Cell Biology: Social Distancing of Microtubule Ends Increases Their Assembly Rates.

Microtubules assembled from artificial centrosomes in microfluidic chambers of defined size are amenable to high resolution live imaging of their dynamics and space-filling properties. By using this experimental regime in conjunction with cytoplasmic extract, a new study finds that microtubule end density negatively influences their assembly rates.

Gravin-associated kinase signaling networks coordinate γ-tubulin organization at mitotic spindle poles.

Mitogenic signals that regulate cell division often proceed through multienzyme assemblies within defined intracellular compartments. The anchoring protein Gravin restricts the action of mitotic kinases and cell-cycle effectors to defined mitotic structures. In this report we discover that genetic deletion of Gravin disrupts proper accumulation and asymmetric distribution of γ-tubulin during mitosis. We utilize a new precision pharmacology tool, Local Kinase Inhibition, to inhibit the Gravin binding partner polo-like kinase 1 at spindle poles. Using a combination of gene-editing approaches, quantitative imaging, and biochemical assays, we provide evidence that disruption of local polo-like kinase 1 signaling underlies the γ-tubulin distribution defects observed with Gravin loss. Our study uncovers a new role for Gravin in coordinating γ-tubulin recruitment during mitosis and illuminates the mechanism by which signaling enzymes regulate this process at a distinct subcellular location.

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons.

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.

Functional characterization of MCAK/Kif2C cancer mutations using high-throughput microscopic analysis.

The microtubule (MT)-depolymerizing activity of MCAK/Kif2C can be quantified by expressing the motor in cultured cells and measuring tubulin fluorescence levels after enough hours have passed to allow tubulin autoregulation to proceed. This method allows us to score the impact of point mutations within the motor domain. We found that, despite their distinctly different activities, many mutations that impact transport kinesins also impair MCAK/Kif2C's depolymerizing activity. We improved our workflow using CellProfiler to significantly speed up the imaging and analysis of transfected cells. This allowed us to rapidly interrogate a number of MCAK/Kif2C motor domain mutations documented in the cancer database cBioPortal. We found that a large proportion of these mutations adversely impact the motor. Using green fluorescent protein-FKBP-MCAK CRISPR cells we found that one deleterious hot-spot mutation increased chromosome instability in a wild-type (WT) background, suggesting that such mutants have the potential to promote tumor karyotype evolution. We also found that increasing WT MCAK/Kif2C protein levels over that of endogenous MCAK/Kif2C similarly increased chromosome instability. Thus, endogenous MCAK/Kif2C activity in normal cells is tuned to a mean level to achieve maximal suppression of chromosome instability.

Subcellular drug targeting illuminates local kinase action.

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

GTP-tubulin loves microtubule plus ends but marries the minus ends.

Microtubule minus ends are inherently more stable than plus ends despite the fact that free tubulin associates more avidly to the plus end. In this issue, Strothman et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201905019) measure, for the first time, the off-rate for GTP-tubulin and find that it is different for the two ends, suggesting that this parameter may control the transition to disassembly at microtubule ends.

GPR124 regulates microtubule assembly, mitotic progression, and glioblastoma cell proliferation.

GPR124 is involved in embryonic development and remains expressed by select organs. The importance of GPR124 during development suggests that its aberrant expression might participate in tumor growth. Here we show that both increases and decreases in GPR124 expression in glioblastoma cells reduce cell proliferation by differentially altering the duration mitotic progression. Using mass spectrometry-based proteomics, we discovered that GPR124 interacts with ch-TOG, a known regulator of both microtubule (MT)-plus-end assembly and mitotic progression. Accordingly, changes in GPR124 expression and ch-TOG similarly affect MT assembly measured by real-time microscopy in cells. Our study describes a novel molecular interaction involving GPR124 and ch-TOG at the plasma membrane that controls glioblastoma cell proliferation by modifying MT assembly rates and controlling the progression of distinct phases of mitosis.

The quantification and regulation of microtubule dynamics in the mitotic spindle.

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.

Arf GAPs and molecular motors.

Arf GTPase-activating proteins (Arf GAPs) were first identified as regulators of the small GTP-binding proteins ADP-ribosylation factors (Arfs). The Arf GAPs are a large family of proteins in metazoans, outnumbering the Arfs that they regulate. The members of the Arf GAP family have complex domain structures and some have been implicated in particular cellular functions, such as cell migration, or with particular pathologies, such as tumor invasion and metastasis. The specific effects of Arfs sometimes depend on the Arf GAP involved in their regulation. These observations have led to speculation that the Arf GAPs themselves may affect cellular activities in capacities beyond the regulation of Arfs. Recently, 2 Arf GAPs, ASAP1 and AGAP1, have been found to bind directly to and influence the activity of myosins and kinesins, motor proteins associated with filamentous actin and microtubules, respectively. The Arf GAP-motor protein interaction is critical for cellular behaviors involving the actin cytoskeleton and microtubules, such as cell migration and other cell movements. Arfs, then, may function with molecular motors through Arf GAPs to regulate microtubule and actin remodeling.

De novo design of self-assembling helical protein filaments.

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo-electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

Modified carbazoles destabilize microtubules and kill glioblastoma multiform cells.

Small molecules that target microtubules (MTs) represent promising therapeutics to treat certain types of cancer, including glioblastoma multiform (GBM). We synthesized modified carbazoles and evaluated their antitumor activity in GBM cells in culture. Modified carbazoles with an ethyl moiety linked to the nitrogen of the carbazole and a carbonyl moiety linked to distinct biaromatic rings exhibited remarkably different killing activities in human GBM cell lines and patient-derived GBM cells, with IC50 values from 67 to >10,000 nM. Measures of the activity of modified carbazoles with tubulin and microtubules coupled to molecular docking studies show that these compounds bind to the colchicine site of tubulin in a unique low interaction space that inhibits tubulin assembly. The modified carbazoles reported here represent novel chemical tools to better understand how small molecules disrupt MT functions and kill devastating cancers such as GBM.

ß-tubulin carboxy-terminal tails exhibit isotype-specific effects on microtubule dynamics in human gene-edited cells.

Microtubules are highly dynamic structures that play an integral role in fundamental cellular functions. Different α- and ß-tubulin isotypes are thought to confer unique dynamic properties to microtubules. The tubulin isotypes have highly conserved structures, differing mainly in their C-terminal tail sequences. However, little is known about the importance of the C-terminal tail in regulating and co-ordinating microtubule dynamics. We developed syngeneic human cell models using gene-editing to precisely modify the ß-tubulin C-terminal tail region while preserving the endogenous microtubule network. Fluorescent microscopy of live cells, coupled with advanced image analysis revealed that the ß-tubulin C-terminal tails differentially co-ordinate the collective and individual dynamic behaviour of microtubules by affecting microtubule growth rates and explorative microtubule assembly in an isotype-specific manner. Furthermore, ßI- and ßIII-tubulin C-terminal tails differentially regulate the sensitivity of microtubules to tubulin-binding agents and the microtubule depolymerising protein MCAK. The sequence of the ß-tubulin tail encodes regulatory information that instructs and co-ordinates microtubule dynamics, thereby fine-tuning microtubule dynamics to support cellular functions.