The microtubule cytoskeleton is the backbone of neuronal morphogenesis, driving the development of the dendrites and axon, and supporting trafficking to distant compartments. How neuronal microtubules are maintained and renewed in the face of this dynamic endeavor and long-term commitment remains unclear. Recently, in-lattice repair has emerged as an alternative mechanism of microtubule renewal that could allow for continuity of the existing network and for the emergence of cell polarity. Here, we directly assessed microtubule turnover using microinjection of labeled tubulin in cultured hippocampal neurons that exhibit well defined stages of development and polarization during the first 24 hours in culture. Sizeable tubulin integration was visible minutes after microinjection, indicating fast turnover of microtubules in developing neurons. Once it appeared, a longer neurite that would become the nascent axon showed decreased turnover, both for its acetylated and non-acetylated populations of microtubules. Combining microinjection with expansion microscopy allowed us to visualize tubulin integration sites along single microtubules, unambiguously demonstrating the existence of in-lattice integration along neurites. In thick neurites, we observed preferential integration within non-acetylated cortical microtubules, but in-lattice integration sites were also visible in the deeper core bundles of acetylated microtubules. Overall, our results link previous observations of microtubule stabilization patterns in developing neurons to their actual turnover. Mapping these patterns of turnover strengthens the notion that microtubules establish an organized network that participates in axon emergence and the establishment of neuronal polarity. ### Competing Interest Statement The authors have declared no competing interest. French National Research Agency, ANR-24-CE13-7996 NeuroSchool, ANR-16-CONV000X, ANR-17-EURE-0029 Excellence Initiative of Aix Marseille University AMIDEX, AMX-22-RE-AB-137, AMX-19-IET-004, AMX-19-IET-002 CPER-FEDER, PA0014842

We identified AB8939, a novel small synthetic molecule that exhibits strong and broad antiproliferative activity against a panel of various cancer cell types with IC₅₀ values in the nanomolar range. In vitro investigations showed that AB8939 is a novel microtubule-targeting agent that interacts with the colchicine-binding site of tubulin. AB8939 disrupts the microtubule network, leading to mitotic arrest in G2/M phase and subsequent apoptosis. Importantly, AB8939 overcomes drug resistance mechanisms, including overexpression of efflux transporters such as P-glycoprotein (P-gp) and aberrant expression of β3-tubulin. AB8939 displays high cytotoxicity against blasts from AML patients, including blasts resistant to cytarabine (Ara-C). In vivo, AB8939 shows strong antitumor activity in MOLM-14, an Ara-C-resistant AML model, as evidenced by tumor growth inhibition and substantial increase in mouse survival. Further experiments performed on an AML PDX TG-AML-36 model demonstrated that AB8939 efficiently kills leukemic stem cells (CD34+/CD38−). Reverse proteomic experiments revealed that AB8939 inhibits ALDH1 and ALDH2, enzymes often overexpressed in tumors and tumor stem cells, thereby favoring tumor progression and relapse. AB8939 is a novel dual-targeting drug that acts on both tubulin and ALDH enzymes, with potential activity against various cancer types, especially refractory AML with complex karyotypes such as those displaying MECOM rearrangement and AML with mutations associated with poor prognosis, such as ASXL1 and TP53. ### Competing Interest Statement M.H., S.L., A.G., B.H., M.N, S.S., E.R., D.P., J.M., A.B., A.M., C.M., C.A., P.D and .L.G., are employees or shareholders of AB Science. AB8939 is a drug candidate under Phase 1 clinical development by AB Science. This does not alter our adherence to BioRxiv policies on sharing data and materials.

Among epigenetic modifiers, telomeres represent attractive modulators of the genome in part through position effects. Telomere Position Effect-Over Long Distances (TPE-OLD) modulates gene expression by changes in telomere-dependent long-distance loops. To gain insights into the molecular mechanisms of TPE-OLD, we performed a genome-wide transcriptome and methylome analysis in proliferative fibroblasts and myoblasts or differentiated myotubes with controlled telomere lengths. By integrating omics data, we identified a common TPE-OLD dependent cis-acting motif that behaves as an insulator or enhancer. Next, we uncovered trans partners that regulate these activities and observed the consistent depletion of one candidate factor, RBPJ, at TPE-OLD associated loci upon telomere shortening. Importantly, we confirmed our findings by unbiased comparisons to recent Human transcriptomic studies, including those from the Genotype-Tissue Expression (GTEx) project. We concluded that TPE-OLD acts at the genome-wide level and can be relayed by RBPJ bridging Alu-like elements to telomeres. In response to physiological (i.e., aging) or pathological cues, TPE-OLD might coordinate the genome-wide impact of telomeres through recently evolved Alu elements acting as enhancers in association with RBPJ.

Supplementary Fig. S1 from Antiangiogenic vinflunine affects EB1 localization and microtubule targeting to adhesion sites

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin–membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

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.

Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (Sept9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to Sept9 isoform 1 (Sept9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring Sept9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from Sept9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin associated pathologies.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton. Summary Human septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.

Measuring 3D orientation properties of single fluorescent emitters including their angle wobbling, as well as their position, is a challenge that would enrich super-resolution techniques with structural molecular information. We present a polarized microscopy technique that provides all 3D orientation parameters unambiguously, using four-polarization splitting of the image plane and intensity filtering in the back focal plane. Using an inverse problem approach we can retrieve 3D orientation, wobbling and 2D position of the fluorophores with high precision. We validated the technique using fluorescent nano-beads and applied it to the structural study of fluorescently labelled F-actin filaments.

The microtubule plus-end binding protein EB1 is the core of a complex protein network which regulates microtubule dynamics during important biological processes such as cell motility and mitosis. We found that iASPP, an inhibitor of p53 and predicted regulatory subunit of the PP1 phosphatase, associates with EB1 at microtubule plus-ends via a SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the leading edge of migrating cells, and at the cortex of mitotic cells leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of Myosin-Ic (Myo1c), identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up during mitosis because of defective cortical rigidity. We propose that iASPP, together with EB1 and Myo1c, contributes to mitotic cell cortex rigidity, allowing astral microtubule capture and appropriate positioning of the mitotic spindle.

We present a polarized microscopy technique that allows the measurement of orientation properties of single fluorescent molecules, as well as their localization with tens of nm precision. Orientation properties encompass both mean orientation and orientational fluctuations extent (wobbling) averaged over the time scale of the image recording (30-100ms). These characteristics are not perceptible in single molecule super-resolution imaging techniques such as PALM or STORM, which rely on pure position localization. The measurement of orientational behaviour of single fluorescent probes in addition to their localization enriches these techniques, since providing that the fluorophores are rigidly linked to the proteins of interest, they can report protein organization at the nanoscale. In particular, the local conformational properties of proteins can be addressed with the orientation information, while wobbling can probe local constraints due to charges, viscosity and steric effects. Our approach is based on polarization splitting of the imaging plane, which provides high precision determination of 2D in-plane orientation, wobbling, and 2D positions of single molecules. We have developed an inverse problem approach to retrieve the full orientation and wobbling information in a fast and robust way, from the intensity of the single molecules in four split polarized images. The intrinsic off-plane orientation tilt of molecules can introduce a bias in the estimation of their wobbling. We have estimated this bias from simulations and propose a solution to reduce it, using a lower detection NA. We have used this technique to report and classify the orientational behaviour of different fluorescent probes in in vitro reconstructed single F-actin filaments labelled with phalloidin conjugates. This study shows the importance of the probe structure in its orientational behaviour and permits now to evaluate the structural properties of F-actin in complex actin structures and networks in cells.

Functions of septin cytoskeletal polymers in tumorigenesis are still poorly defined. Their role in the regulation of cytokinesis and cell migration were proposed to contribute to cancer associated aneuploidy and metastasis. Overexpression of Septin 9 (Sept9) promotes migration of cancer cell lines. SEPT9 mRNA and protein expression is increased in breast tumors compared to normal and peritumoral tissues and amplification of SEPT9 gene was positively correlated with breast tumor progression. However, the existence of multiple isoforms of Sept9 is a confounding factor in the analysis of Sept9 functions. In the present study, we analyze the protein expression of Sept9_i2, an uncharacterized isoform, in breast cancer cell lines and tumors and describe its specific impact on cancer cell migration and Sept9 cytoskeletal distribution. Collectively, our results showed that, contrary to Sept9_i1, Sept9_i2 did not support cancer cell migration, and induced a loss of subnuclear actin filaments. These effects were dependent on Sept9_i2 specific N-terminal sequence. Sept9_i2 was strongly down-regulated in breast tumors compared to normal mammary tissues. Thus our data indicate that Sept9_i2 is a negative regulator of breast tumorigenesis. We propose that Sept9 tumorigenic properties depend on the balance between Sept9_i1 and Sept9_i2 expression levels.

Significance Microtubule dynamics is tightly regulated during fundamental biological processes such as mitosis, thereby representing a major target for anticancer therapies. To better understand the molecular mechanisms underlying the organization of the microtubule network, we systematically investigated proteins interacting with EB1, a major regulator of microtubules dynamics. We identified a specific isoform of myomegalin, which we termed “SMYLE,” that assembles a macromolecular complex associated with the centrosome, the major microtubule-organizing center in cells, and also connected to the microtubule nucleating complex. SMYLE promoted microtubule assembly from the centrosome and subsequent stabilization of microtubules at the cell periphery. This had consequences on cell motility, mitosis, and cell-cycle progression, suggesting that SMYLE might be an important player in tumor progression.

Non-cytotoxic concentrations of microtubule targeting agents (MTAs) interfere with the dynamics of interphase microtubules and affect cell migration, which could impair tumor angiogenesis and metastasis. The underlying mechanisms however are still ill-defined. We previously established that directed cell migration is dependent on stabilization of microtubules at the cell leading edge, which is controlled by microtubule +end interacting proteins (+TIPs). In the present study, we found that eribulin, a recently approved MTA interacting with a new class of binding site on β-tubulin, decreased microtubule growth speed, impaired their cortical stabilization and prevented directed migration of cancer cells. These effects were reminiscent of those observed when +TIP expression or cortical localization was altered. Actually, eribulin induced a dose-dependent depletion of EB1, CLIP-170 and the tubulin polymerase ch-TOG from microtubule +ends. Interestingly, eribulin doses that disturbed ch-TOG localization without significant effect on EB1 and CLIP-170 comets, had an impact on microtubule dynamics and directed migration. Moreover, knockdown of ch-TOG led to a similar inhibition of microtubule growth speed, microtubule capture and chemotaxis. Our data suggest that eribulin binding to the tip of microtubules and subsequent loss of ch-TOG is a priming event leading to alterations in microtubule dynamics and cancer cell migration.

Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks at growing microtubule ends in an EB1-dependent manner. The interaction between the C-terminal domain of EB1 and the CAP-Gly domains of the +TIP CLIP-170 depends on the last tyrosine residue of EB1. In the present study, we generated peptidic probes corresponding to the C-terminal tail of EB1 to affinity-capture binding partners from cell lysates. Using an MS-based approach, we showed that the last 15 amino-acid residues of EB1, either free or immobilized on beads, bound recombinant CAP-Gly domains of CLIP-170. We further demonstrate that this binding was prevented when the C-terminal tyrosine of EB1 was absent in the peptidic probes. Western blotting in combination with a label-free quantitative proteomic analysis revealed that the peptidic probe harboring the C-terminal tyrosine of EB1 effectively pulled-down proteins with CAP-Gly domains from endothelial cell extracts. Additional proteins known to interact directly or indirectly with EB1 and the microtubule cytoskeleton were also identified. Our peptidic probes represent valuable tools to detect changes induced in EB1-dependent +TIP networks by external cues such as growth factors and small molecules.