Plus-end Binding Protein Research Articles

Force-transducing molecular ensembles at growing microtubule tips control mitotic spindle size.

Correct mitotic spindle size is required for accurate chromosome segregation during cell division. It is controlled by mechanical forces generated by molecular motors and non-motor proteins acting on spindle microtubules. However, how forces generated by individual proteins enable bipolar spindle organization is not well understood. Here, we develop tools to measure contributions of individual molecules to this force balance. We show that microtubule plus-end binding proteins act at microtubule tips synergistically with minus-end directed motors to produce a system that can generate both pushing and pulling forces. To generate pushing force, the system harnesses forces generated by the growing tips of microtubules providing unique contribution to the force balance distinct from all other motors that act in the mitotic spindle. Our results reveal that microtubules are essential force generators for establishing spindle size and pave the way for understanding how mechanical forces can be fine-tuned to control the fidelity of chromosome segregation.

Endomembrane trafficking driven by microtubule growth regulates stomatal movement in Arabidopsis

Microtubule-based vesicle trafficking usually relies upon kinesin and dynein motors and few reports describe microtubule polymerisation driving directional vesicle trafficking. Here we show that Arabidopsis END BINDING1b (EB1b), a microtubule plus-end binding protein, directly interacts with SYP121, a SNARE protein that mediates the trafficking of the K+ channel KAT1 and its distribution to the plasma membrane (PM) in Arabidopsis guard cells. Knockout of AtEB1b and its homologous proteins results in a modest but significant change in the distribution of KAT1 and SYP121 in guard cells and consequently delays light-induced stomatal opening. Live-cell imaging reveals that a portion of SYP121-associated endomembrane compartments co-localise with AtEB1b at the growing ends of microtubules, trafficking along with the growth of microtubules for targeting to the PM. Our study reveals a mechanism of vesicle trafficking driven by microtubule growth, which is involved in the redistribution of PM proteins to modulate guard cell movement.

Abstract 2009: Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance

Abstract Taxanes are microtubule (MT)-stabilizing drugs widely used for the treatment of solid tumors, including the highly prevalent cancers of the breast, the prostate and gastric cancer. However, despite their initial activity, taxane resistance invariably occurs, leaving patients with limited therapeutic options. Currently, the molecular determinants of clinical taxane resistance remain poorly understood. We recently identified CLIP-170S, a novel truncated variant of the MT plus-end binding protein CLIP-170, that confers taxane resistance in vitro and is associated with clinical taxane resistance in patients with gastric cancer. We further showed that CLIP-170S is aberrantly localized along the MT lattice, thus impairing taxane access to its binding site in the MT lumen. However, the mechanism underlying the resistance-inducing extensive lattice localization of CLIP-170S is unknown. The activity and MT binding properties of canonical CLIP-170 are regulated by conformational changes mediated by the interaction of its N- and C-terminal domains. In its inactive, “closed” conformation, CLIP-170 is soluble as the N- and C-terminal domains interact with each other. In the active “open” conformation, CLIP-170’s N- and C-terminal domains are apart, thus exposing its MT-binding domain and allowing MT plus-end binding. As CLIP-170S is missing part of its N-terminal domain, we hypothesized that CLIP-170S is constitutively displayed in an open conformation that promotes its extensive and aberrant localization to the MT lattice. To prove this hypothesis, we used the FKBP-rapalog-FRB system to “force” CLIP-170S to assume a “closed” conformation. We generated a fusion CLIP-170S protein tagged with FKBP and FRB domains in its N- and C-terminal, respectively. The addition of rapalog induced FKBP (N-ter) and FRB (C-ter) domains to interact, promoting CLIP-170S to adopt a “closed” conformation. CLIP-170S in this “closed” conformation resulted in its release from the MT-lattice and restored taxane binding to MTs. We next extended our search to other CLIP-170 clinically prevalent variants with sequence alteration predicted to bind to the MT-lattice and potentially interfere with taxane sensitivity. This search identified CLIP-170IR (an intron retention variant) and several other truncation mutants prevalent in gastric, breast and lung cancer cell lines. Experimentally, we showed that these variants not only phenocopy CLIP-170S binding to the MT- lattice but also confer taxane-resistance. Together, these data demonstrate a novel mechanism of taxane resistance mediated by CLIP-170 mutations that induce CLIP-170 to adopt a constitutively “open” MT-lattice binding conformation, which renders them susceptible to causing taxane resistance. Citation Format: Urko del Castillo, Kiran Kumari Sahu, William G. Stone, Manish A. Shah, Paraskevi Giannakakou. Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2009.

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and β-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and β-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Evidence for microtubule nucleation at the Golgi in breast cancer cells.

Golgi-derived microtubule (MT) arrays are essential to directionally persistent cell migration and vesicle transport. In this study, we have examined MT nucleation sites in two breast cancer cell lines, MDA-MB-231 and MCF-7, with the hypothesis that only the migratory invasive MDA-MB-231 cells exhibit MTs originating from the Golgi. MTs were disassembled and allowed to slightly regrow so individual nucleation sites could then be observed via fluorescently tagged antibodies (α-tubulin, cis-Golgi marker GM130, and EB1-a MT plus-end binding protein) and confocal microscopy. To determine if MT nucleation at the Golgi is more apparent during active migration compared to when cells are stationary, cells were treated with the chemoattractant epidermal growth factor (EGF) and examined for colocalizations between the Golgi, α-tubulin, and γ-tubulin. Images were analyzed qualitatively for color overlap, and quantitatively using Manders Colocalization Coefficients. Differences between groups were tested for significance using one-way analysis of variances and Tukey's post hoc test. Significantly higher colocalization values (coloc) in the highly invasive MDA-MB-231 cells (α-tubulin coloc GM130 = 0.39, GM130 coloc α-tubulin = 0.82, GM130 coloc EB1 = 0.24, and EB1 coloc GM130 = 0.38) compared to the weakly invasive MCF-7 cells (0.15, 0.08, 0.02, and 0.16, respectively) were observed. EGF-treated cells exhibited higher colocalization values than control cells for three of the four protein combinations tested, but EGF-treated MDA-MB-231 cells exhibited significantly higher values (α-tubulin coloc GM130 = 0.20, GM130 coloc α-tubulin = 0.89, and γ-tubulin coloc GM130 = 0.47) than both control groups as well as the EGF-treated MCF-7 cells. Results support the hypothesis that MT nucleation at the Golgi occurs more frequently in the invasive MDA-MB-231 cell line compared to the weakly invasive MCF-7 cells. The presence or absence of Golgi-derived MTs may help to explain the difference in migratory potential commonly exhibited by these two cell lines.

EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment in Plasmodium male gametogenesis

Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium undergoes endomitosis in this multinucleated cell division, which is highly reliant on proper spindle-kinetochore attachment. However, the mechanisms underlying the spindle-kinetochore attachment remain elusive. End-binding proteins (EBs) are conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here, we report that the Plasmodium EB1 is an orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays reveal that the Plasmodium EB1 losses MT plus-end tracking but possesses MT-lattice affinity. This MT-binding feature of Plasmodium EB1 is contributed by both CH domain and linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission. EB1 is localized at the nucleoplasm of male gametocytes. During the gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment is observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice binding affinity fulfills the spindle-kinetochore lateral attachment in male gametogenesis.

Abstract 1742: Cediranib reverses taxane resistance in CLIP-170S expressing gastric cancer

Abstract Taxane-based chemotherapy is a first-line treatment for patients with Gastric Cancer (GC), however, many will not respond due to mechanisms of resistance. Recently, we reported the discovery of a novel truncated variant of the microtubule plus-end binding protein CLIP-170, hereafter CLIP-170S, whose expression is enriched in taxane-resistant cell lines and in 65% of patients with GC, non-responsive to Cabazitaxel monotherapy. CLIP-170S knockdown reversed taxane resistance and its ectopic expression led to resistance, suggesting causation. Computational analysis of RNA-seq data in conjunction with the connectivity map from taxane-sensitive and -resistant GC cell lines, predicted imatinib as the top candidate drug to overcome drug resistance. Imatinib treatment completely reversed taxane resistance, experimentally confirming the prediction, and did so unexpectedly by selective depletion of CLIP-170S. Other RTK inhibitors, such as Sunitinib, Ponatinib, Axitinib, and Cediranib also depleted CLIP-170S, suggesting a class effect, however, the mechanism is unknown. To investigate the mechanism of RTKi induced depletion of CLIP-170S, we sought to investigate the involvement of proteasomal and lysosomal cellular compartments. Preliminary results indicated that treatment of CLIP-170S expressing cells with Imatinib or Cediranib led to an increase in lysosome size, suggesting increased lysosomal activity. In parallel, we are testing in-vivo the synergistic activity of the RTKi, Cediranib, in combination with docetaxel in GC tumors expressing CLIP-170S. Together, these results will allow us to implement a novel clinical trial for patients with GC by using Cediranib as a treatment to increase taxane efficacy through CLIP-170S depletion. Citation Format: William G. Stone, Urko del Castillo, Prashant V. Thakkar, Manish A. Shah, Paraskevi Giannakakou. Cediranib reverses taxane resistance in CLIP-170S expressing gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1742.

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.

Abstract P6-14-07: Genetic ablation of ACF7, a member of the +TIP family of microtubule-binding proteins, impairs metastasis of HER2-positive breast cancer

Abstract Breast cancer is the leading cause of cancer death in women worldwide. Among the different subtypes of this disease, HER2-positive breast cancer is known to be one of the most aggressive and is linked to poor prognosis. This subtype is prone to develop metastases, reflecting a significant cellular plasticity. While the molecular machinery controlling actin dynamics is well implicated in cell invasion, the contribution of the microtubules is ill-defined. We recently conducted a functional screen to identify such regulators, and characterized ACF7, a microtubule plus-end binding protein (+TIP), as a new promoter of invasion. Here, we aim to determine the in vivo contribution of ACF7 to tumor progression and to reveal the cellular and molecular mechanisms allowing this +TIP to promote metastasis. We analyzed the expression of ACF7 at the protein level across a panel of tumor microarrays and found that it is detectable in all breast cancer subtypes with the highest levels seen in HER2-positive. To study the role of ACF7 during tumor progression, we generated an ACF7 genetically engineered mouse model of breast cancer. We exploited a HER2-positive breast cancer mouse model (MMTV-NIC) that we bred with ACF7Flox mice. This approach allows for conditional deletion of ACF7 in mammary glands and represents a powerful model to define the roles of ACF7 in HER2-driven breast cancer. Our results show that ACF7 is not involved in tumor initiation and tumor growth as no difference between number of nodules per mouse, tumor mass or number of mammary intraepithelial neoplastic lesions was shown. However, our results show a significant decrease in lung metastasis in the ACF7cKO mice. To gain mechanistic insights into the roles of ACF7 in this specific process, we derived primary cell lines from tumors isolated from MMTV-NIC-ACF7WT and MMTV-NIC-ACF7cKO mice. We explored if ACF7 contributes to migration and invasion in a HER2 context. By using live-cell imaging, we demonstrated that ACF7-null cells display defects in both random and directed cell migration, as they show a delay in wound closure. We determined that ACF7KO cells show an increase in focal adhesions size, and exhibit defects in reorienting their actin and microtubules cytoskeletons parallel to the direction of migration. ACF7 was also found to promote cell invasion since ACF7KO cells were less efficient to cross a Matrigel membrane. To investigate the metastatic potential of ACF7-null tumors, we performed RNA-sequencing using RNA isolated from MMTV-NIC-ACF7WT and MMTV-NIC-ACF7cKO tumors. We identified a striking change in expression of genes involved in epithelial to mesenchymal transition (EMT), such as for example E-cadherin, Vimentin or the transcription factor Twist. These results suggest that ACF7 promotes metastasis through maintaining an EMT state in HER2 breast cancer cells. Collectively, our work demonstrates ACF7 as an important player in HER2 positive breast cancer progression via its functions in both cell migration and cell invasion. This works also suggests that developing approaches to therapeutically target regulators of microtubule dynamics may reveal new opportunities to decrease tumor progression and metastatic expansion. Citation Format: Rebecca Cusseddu, Jean-François Côté. Genetic ablation of ACF7, a member of the +TIP family of microtubule-binding proteins, impairs metastasis of HER2-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-07.

MAPping tubulin mutations.

Microtubules are filamentous structures that play a critical role in a diverse array of cellular functions including, mitosis, nuclear translocation, trafficking of organelles and cell shape. They are composed of α/β-tubulin heterodimers which are encoded by a large multigene family that has been implicated in an umbrella of disease states collectively known as the tubulinopathies. De novo mutations in different tubulin genes are known to cause lissencephaly, microcephaly, polymicrogyria, motor neuron disease, and female infertility. The diverse clinical features associated with these maladies have been attributed to the expression pattern of individual tubulin genes, as well as their distinct Functional repertoire. Recent studies, however, have highlighted the impact of tubulin mutations on microtubule-associated proteins (MAPs). MAPs can be classified according to their effect on microtubules and include polymer stabilizers (e.g., tau, MAP2, doublecortin), destabilizers (e.g., spastin, katanin), plus-end binding proteins (e.g., EB1-3, XMAP215, CLASPs) and motor proteins (e.g., dyneins, kinesins). In this review we analyse mutation-specific disease mechanisms that influence MAP binding and their phenotypic consequences, and discuss methods by which we can exploit genetic variation to identify novel MAPs.

The Neuron Navigators: Structure, function, and evolutionary history.

Neuron navigators (Navigators) are cytoskeletal-associated proteins important for neuron migration, neurite growth, and axon guidance, but they also function more widely in other tissues. Recent studies have revealed novel cellular functions of Navigators such as macropinocytosis, and have implicated Navigators in human disorders of axon growth. Navigators are present in most or all bilaterian animals: vertebrates have three Navigators (NAV1-3), Drosophila has one (Sickie), and Caenorhabditis elegans has one (Unc-53). Structurally, Navigators have conserved N- and C-terminal regions each containing specific domains. The N-terminal region contains a calponin homology (CH) domain and one or more SxIP motifs, thought to interact with the actin cytoskeleton and mediate localization to microtubule plus-end binding proteins, respectively. The C-terminal region contains two coiled-coil domains, followed by a AAA+ family nucleoside triphosphatase domain of unknown activity. The Navigators appear to have evolved by fusion of N- and C-terminal region homologs present in simpler organisms. Overall, Navigators participate in the cytoskeletal response to extracellular cues via microtubules and actin filaments, in conjunction with membrane trafficking. We propose that uptake of fluid-phase cues and nutrients and/or downregulation of cell surface receptors could represent general mechanisms that explain Navigator functions. Future studies developing new models, such as conditional knockout mice or human cerebral organoids may reveal new insights into Navigator function. Importantly, further biochemical studies are needed to define the activities of the Navigator AAA+ domain, and to study potential interactions among different Navigators and their binding partners.

Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration

The microtubule (MT) plus-end binding protein Clip170 is associated closely with breast cancer invasion and migration. In this study, Clip170 tension observed by a newly designed cpstFRET tension probe was suggested to be positive related to breast cancer aggressiveness, which could be regulated by α-tubulin detyrosination-induced MT disassembly. Clip170 phosphorylation induced by Ribosomal protein S6 kinase (RSK) could also increase its tension and promote the conversion of a discrete comet-like Clip-170 distribution into a spotty pattern during cancer metastasis. Heightened Clip170 tension was correlated with the formation of cortactin-associated filopodia and lamellipodia, and then promoted invasion and metastasis both in vitro and in vivo. Meanwhile, Clip170 tension enhanced at the leading edge in directional migration, accompanying with IQGAP1 subcellular distribution variation. Our work indicates that the malignancy and directionality during breast cancer migration depend on the magnitude and polarization of Clip170 tension, and we suggest Clip170 tension as a new potential drug target for breast cancer therapy.

Abstract 2575: Avanbulin, the active moiety of the tumor checkpoint controller lisavanbulin (BAL101553), has anti-lymphoma activity

Abstract Background: Avanbulin (BAL27862) is a microtubule-targeting agent (MTA) that promotes tumor cell death by modulating the spindle assembly checkpoint. Preclinical studies in glioblastoma (GBM) have shown that avanbulin is also active in human cancer cells resistant to other MTAs used in the clinic. Lisavanbulin (BAL101553) is a water-soluble, oral, lysine prodrug of avanbulin, currently in phase 1/2 for patients with recurrent GBM and, in combination with radiotherapy, for newly diagnosed GBM patients. Preclinical studies and clinical signals have also suggested an association between lisavanbulin activity and expression of microtubule plus-end binding protein (EB1). MTAs are active agents for lymphoma patients, as exemplified by the inclusion of vincristine in the R-CHOP regimen, standard treatment for diffuse large B cell lymphoma (DLBCL). Thus, our aim was to evaluate avanbulin, the active moiety of lisavanbulin, for its potential anti-lymphoma activity. Methods: Lymphoma cell lines derived from activated B cell like (ABC) and germinal center B cell type (GCB) DLBCL were exposed to increasing doses of avanbulin; cell proliferation was measured with MTT. Apoptosis induction was performed with annexin V staining on cells incubated in a live cell imaging system (Incucyte) treated with 5, 10, 20 or 40nM avanbulin. Images were taken every 4h for 72h. Cell cycle analysis was performed after 24, 48 and 72h of drug exposure at 20nM. Results: 26 DLBCL cell lines, treated for 72h with increasing avanbulin doses, showed high anti-proliferative activity of the compound, with a median IC50 = 11nM. No differences in terms of IC50 were observed comparing ABC (n=8) to GCB (n=18) DLBCLs, nor based on MYD88, TP53 and BCL2 status. Cell lines with MYC translocation were less sensitive than the ones without translocation. All lymphoma cell lines exhibited high EB1 RNA expression, with no correlation with sensitivity to avanbulin.Live cell imaging demonstrated a strong apoptosis induction in 15 out of 17 cell lines tested at 20 and 40nM. Among the 15 cell lines undergoing apoptosis, 8 cell lines already showed an apoptosis induction within 24h of drug exposure, 6 lines between 24 and 48h and 1 line after 48h exposure. These data confirmed the high activity of avanbulin already seen with the MTT assay.Cell cycle experiments performed at 24, 48 and 72h confirmed the strong cytotoxic effect of avanbulin in 4 DLBCL cell lines, including 2 ABC (OCILY3 and TMD8) and 2 GCB (SUDHL5 and SUDHL16) lines. Sub-G0 accumulation was already present at 24h in all 4 cell lines tested, with an increase over time. Especially at 24h, where we also observed a G2-M arrest in 3 out of 4 cell lines. Conclusions: Our data demonstrate the high cytotoxic anti-lymphoma activity of avanbulin, suggesting a potential activity of its prodrug lisavanbulin in lymphoma patients. Citation Format: Filippo Spriano, Luca Aresu, Luciano Cascione, Alberto J. Arribas, Nicole Forster-Gross, Felix Bachmann, Heidi Lane, Francesco Bertoni. Avanbulin, the active moiety of the tumor checkpoint controller lisavanbulin (BAL101553), has anti-lymphoma activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2575.

Abstract 2623: Variants of CLIP-170 associated with taxane resistance in tumors

Abstract Despite its widespread use, the majority of patients with gastric cancer (GC) will not respond to taxane chemotherapy due to resistance mechanisms. Recently, we reported the discovery of a novel truncated variant of the microtubule plus-end binding protein CLIP-170, hereafter CLIP-170S, whose expression is enriched in taxane resistant cell lines and patients with GC. Mass-spec proteomics and 5’-RACE further showed that CLIP-170S lacked the first 155 amino acids, including the Cap-Gly motif required for microtubule plus-end localization. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Unlike canonical CLIP-170, we showed that CLIP-170S was mislocalized from the MT plus-end to the MT lattice. Computational analysis of RNA-seq data in conjunction with the connectivity map from taxane-sensitive and resistant GC cell lines, predicted imatinib as the top candidate drug to overcome drug resistance. Imatinib treatment completely reversed taxane resistance, as predicted, and did so unexpectedly by selective depletion of CLIP-170S. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. We are currently unraveling the molecular mechanisms by which a) CLIP-170S impairs taxane association to its microtubule binding site and b) Imatinib and other RTK inhibitors selectively deplete CLIP-170S. Our data show that CLIP-170S is a clinically prevalent variant that confers taxane resistance in tumors, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Urko del Castillo, William G. Stone, Giuseppe Galletti, Neel Madhukar, Elena Vila Navarro, Elena Barasoain, Holly V. Goodson, Dan Sackett, José Fernando Díaz, Yao Lu, Arindam RoyChoudhury, Henrik Molina, Olivier Elemento, Manish A. Shah, Paraskevi Giannakakou. Variants of CLIP-170 associated with taxane resistance in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2623.

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.

Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end-directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end-directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end-directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end-directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

PTEN Regulates Dendritic Arborization by Decreasing Microtubule Polymerization Rate.

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway. Loss-of-function mutations in PTEN have been found in a subset of patients with macrocephaly and autism spectrum disorder (ASD). PTEN loss in neurons leads to somal hypertrophy, aberrant migration, dendritic overgrowth, increased spine density, and hyperactivity of neuronal circuits. These neuronal overgrowth phenotypes are present on Pten knock-out (KO) and reconstitution with autism-associated point mutations. The mechanism underlying dendritic overgrowth in Pten deficient neurons is unclear. In this study, we examined how Pten loss impacts microtubule (MT) dynamics in both sexes using retroviral infection and transfection strategies to manipulate PTEN expression and tag the plus-end MT binding protein, end-binding protein 3 (EB3). We found Pten KO neurons sprout more new processes over time compared with wild-type (WT) neurons. We also found an increase in MT polymerization rate in Pten KO dendritic growth cones. Reducing MT polymerization rate to the WT level was sufficient to reduce dendritic overgrowth in Pten KO neurons in vitro and in vivo Finally, we found that rescue of dendritic overgrowth via inhibition of MT polymerization was sufficient to improve the performance of Pten KO mice in a spatial memory task. Taken together, our data suggests that one factor underlying PTEN loss dependent dendritic overgrowth is increased MT polymerization. This opens the possibility for an intersectional approach targeting MT polymerization and mTOR with low doses of inhibitors to achieve therapeutic gains with minimal side effects in pathologies associated with loss of neuronal PTEN function.SIGNIFICANCE STATEMENT Loss of Pten function because of genetic deletion or expression of mutations associated with autism spectrum disorder (ASD), results in overgrowth of neurons including increased total dendritic length and branching. We have discovered that this overgrowth is accompanied by increased rate of microtubule (MT) polymerization. The increased polymerization rate is insensitive to acute inhibition of mechanistic target of rapamycin (mTOR)C1 or protein synthesis. Direct pharmacological inhibition of MT polymerization can slow the polymerization rate in Pten knock-out (KO) neurons to rates seen in wild-type (WT) neurons. Correction of the MT polymerization rate rescues increased total dendritic arborization and spatial memory. Our studies suggest that phosphatase and tensin homolog (PTEN) inhibits dendritic growth through parallel regulation of protein synthesis and cytoskeletal polymerization.

IASPP contributes to cell cortex rigidity, mitotic cell rounding, and spindle positioning.

iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.

Drebrin regulates cytoskeleton dynamics in migrating neurons through interaction with CXCR4

Stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR4) are regulators of neuronal migration (e.g., GnRH neurons, cortical neurons, and hippocampal granule cells). However, how SDF-1/CXCR4 alters cytoskeletal components remains unclear. Developmentally regulated brain protein (drebrin) stabilizes actin polymerization, interacts with microtubule plus ends, and has been proposed to directly interact with CXCR4 in T cells. The current study examined, in mice, whether CXCR4 under SDF-1 stimulation interacts with drebrin to facilitate neuronal migration. Bioinformatic prediction of protein-protein interaction highlighted binding sites between drebrin and crystallized CXCR4. In migrating GnRH neurons, drebrin, CXCR4, and the microtubule plus-end binding protein EB1 were localized close to the cell membrane. Coimmunoprecipitation (co-IP) confirmed a direct interaction between drebrin and CXCR4 using wild-type E14.5 whole head and a GnRH cell line. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain. A decrease in hippocampal granule cells was also detected, and co-IP confirmed a direct interaction between drebrin and CXCR4 in PN4 hippocampi. Migration assays on primary neurons established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1 on neuronal movement. Bioinformatic prediction then identified binding sites between drebrin and the microtubule plus end protein, EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during central nervous system development.

SOCS3–microtubule interaction via CLIP-170 and CLASP2 is critical for modulation of endothelial inflammation and lung injury

Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3–MT association. We discovered that—in addition to its known partners gp130 and JAK2—SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3–CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3–JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.