The microtubule targeting agent ST-401 triggers cell death in interphase and prevents the formation of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

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.

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.

Mitosis exit followed by death in interphase prevents the development of polyploid giant cancer cells.

Microtubule targeting agents ( MTAs ) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells can escape death in mitosis and exit mitosis, and become malignant polyploid giant cancer cells ( PGCC ). Considering the low number of malignant cells undergoing mitosis in tumor tissue, killing these cells in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule assembly, preferentially kills cancer cells in interphase as opposed to mitosis, and avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces an integrated stress response and promotes mitochondria fission accompanied by a reduction in energy metabolism. This cell response may underly death in interphase and avoid the development of PGCC.

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.

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.

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.

A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays.

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.

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.

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.

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.

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems.

ST-11: A New Brain-Penetrant Microtubule-Destabilizing Agent with Therapeutic Potential for Glioblastoma Multiforme.

Glioblastoma multiforme is a devastating and intractable type of cancer. Current antineoplastic drugs do not improve the median survival of patients diagnosed with glioblastoma multiforme beyond 14 to 15 months, in part because the blood-brain barrier is generally impermeable to many therapeutic agents. Drugs that target microtubules (MT) have shown remarkable efficacy in a variety of cancers, yet their use as glioblastoma multiforme treatments has also been hindered by the scarcity of brain-penetrant MT-targeting compounds. We have discovered a new alkylindole compound, ST-11, that acts directly on MTs and rapidly attenuates their rate of assembly. Accordingly, ST-11 arrests glioblastoma multiforme cells in prometaphase and triggers apoptosis. In vivo analyses reveal that unlike current antitubulin agents, ST-11 readily crosses the blood-brain barrier. Further investigation in a syngeneic orthotopic mouse model of glioblastoma multiforme shows that ST-11 activates caspase-3 in tumors to reduce tumor volume without overt toxicity. Thus, ST-11 represents the first member of a new class of brain-penetrant antitubulin therapeutic agents. Mol Cancer Ther; 15(9); 2018-29. ©2016 AACR.

Divergent microtubule assembly rates after short- versus long-term loss of end-modulating kinesins.

Depletion of microtubule (MT) regulators can initiate stable alterations in MT assembly rates that affect chromosome instability and mitotic spindle function, but the manner by which cellular MT assembly rates can stably increase or decrease is not understood. To investigate this phenomenon, we measured the response of microtubule assembly to both rapid and long-term loss of MT regulators MCAK/Kif2C and Kif18A. Depletion of MCAK/Kif2C by siRNA stably decreases MT assembly rates in mitotic spindles, whereas depletion of Kif18A stably increases rates of assembly. Surprisingly, this is not phenocopied by rapid rapamycin-dependent relocalization of MCAK/Kif2C and Kif18A to the plasma membrane. Instead, this treatment yields opposite affects on MT assembly. Rapidly increased MT assembly rates are balanced by a decrease in nucleated microtubules, whereas nucleation appears to be maximal and limiting for decreased MT assembly rates and also for long-term treatments. We measured amplified tubulin synthesis during long-term depletion of MT regulators and hypothesize that this is the basis for different phenotypes arising from long-term versus rapid depletion of MT regulators.

Mad2, Bub3, and Mps1 regulate chromosome segregation and mitotic synchrony in Giardia intestinalis, a binucleate protist lacking an anaphase-promoting complex.

The binucleate pathogen Giardia intestinalis is a highly divergent eukaryote with a semiopen mitosis, lacking an anaphase-promoting complex/cyclosome (APC/C) and many of the mitotic checkpoint complex (MCC) proteins. However, Giardia has some MCC components (Bub3, Mad2, and Mps1) and proteins from the cohesin system (Smc1 and Smc3). Mad2 localizes to the cytoplasm, but Bub3 and Mps1 are either located on chromosomes or in the cytoplasm, depending on the cell cycle stage. Depletion of Bub3, Mad2, or Mps1 resulted in a lowered mitotic index, errors in chromosome segregation (including lagging chromosomes), and abnormalities in spindle morphology. During interphase, MCC knockdown cells have an abnormal number of nuclei, either one nucleus usually on the left-hand side of the cell or two nuclei with one mislocalized. These results suggest that the minimal set of MCC proteins in Giardia play a major role in regulating many aspects of mitosis, including chromosome segregation, coordination of mitosis between the two nuclei, and subsequent nuclear positioning. The critical importance of MCC proteins in an organism that lacks their canonical target, the APC/C, suggests a broader role for these proteins and hints at new pathways to be discovered.