Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

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.

Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

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.

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.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

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 subunit (also known as SEPTIN9). 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, for the first time, the 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 and microtubule cytoskeleton.

EB1-binding-myomegalin protein complex promotes centrosomal microtubules functions.

Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (γ-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression.

Eribulin targets a ch-TOG-dependent directed migration of cancer cells.

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 ß-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.

ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways.

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.

Characterization of a human ßV-tubulin antibody and expression of this isotype in normal and malignant human tissue.

There are seven distinct ß-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. ßV-tubulin, like ßIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human ßV-tubulin. The antibody did not cross-react with mouse ßV-tubulin or other human ß-tubulin isotypes and specifically labeled ßV-tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that ßV-tubulin was expressed in endothelial cells, myocytes and cells with muscle differentiation, structures with transport and/or secretory function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretory function such as prostate. ßV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. Initial studies in breast, lung and ovarian cancers indicated aberrant expression of ßV-tubulin, suggesting that this isoform may be associated with tumorigenesis. Thus, ßV-tubulin expression is a potentially promising prognostic marker of malignancy.

Tyrosine-dependent capture of CAP-Gly domain-containing proteins in complex mixture by EB1 C-terminal peptidic probes.

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.

Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast cancer progression.

INTRODUCTION: Altered expression of Septin 9 (SEPT9), a septin coding for multiple isoform variants, has been observed in several carcinomas, including colorectal, head and neck, ovarian and breast, compared to normal tissues. The mechanisms regulating its expression during tumor initiation and progression in vivo and the oncogenic function of its different isoforms remain elusive. METHODS: Using an integrative approach, we investigated SEPT9 at the genetic, epigenetic, mRNA and protein levels in breast cancer. We analyzed a panel of breast cancer cell lines, human primary tumors and corresponding tumor-free areas, normal breast tissues from reduction mammoplasty patients, as well as primary mammary gland adenocarcinomas derived from the polyoma virus middle T antigen, or PyMT, mouse model. MCF7 clones expressing individual GFP-tagged SEPT9 isoforms were used to determine their respective intracellular distributions and effects on cell migration. RESULTS: An overall increase in gene amplification and altered expression of SEPT9 were observed during breast tumorigenesis. We identified an intragenic alternative promoter at which methylation regulates SEPT9_v3 expression. Transfection of specific GFP-SEPT9 isoforms in MCF7 cells indicates that these isoforms exhibit differential localization and affect migration rates. Additionally, the loss of an uncharacterized SEPT9 nucleolar localization is observed during tumorigenesis. CONCLUSIONS: In this study, we found conserved in vivo changes of SEPT9 gene amplification and overexpression during human and mouse breast tumorigenesis. We show that DNA methylation is a prominent mechanism responsible for regulating differential SEPT9 isoform expression and that breast tumor samples exhibit distinctive SEPT9 intracellular localization. Together, these findings support the significance of SEPT9 as a promising tool in breast cancer detection and further emphasize the importance of analyzing and targeting SEPT9 isoform-specific expression and function.

Septin roles in tumorigenesis.

Septins are a family of cytoskeleton related proteins consisting of 14 members that associate and interact with actin and tubulin. From yeast to humans, septins maintain a conserved role in cytokinesis and they are also involved in a variety of other cellular functions including chromosome segregation, DNA repair, migration and apoptosis. Tumorigenesis entails major alterations in these processes. A substantial body of literature reveals that septins are overexpressed, downregulated or generate chimeric proteins with MLL in a plethora of solid tumors and in hematological malignancies. Thus, members of this gene family are emerging as key players in tumorigenesis. The analysis of septins during cancer initiation and progression is challenged by the presence of many family members and by their potential to produce numerous isoforms. However, the development and application of advanced technologies is allowing for a more detailed analysis of septins during tumorigenesis. Specifically, such applications have led to the establishment and validation of SEPT9 as a biomarker for the early detection of colorectal cancer. This review summarizes the current knowledge on the role of septins in tumorigenesis, emphasizing their significance and supporting their use as potential biomarkers in various cancer types.

MALDI in-source decay of high mass protein isoforms: application to alpha- and beta-tubulin variants.

Tubulin is one of the major targets in cancer chemotherapy and the target of more than twenty percent of the cancer chemotherapic agents. The modulation of isoform content has been hypothesized as being a cause of resistance to treatment. Isoform differences lie mostly in the C-terminus part of the protein. Extensive characterization of this polypeptide region is therefore of critical importance. MALDI-TOF fragmentation of tubulin C-terminal domains was tested using synthetic peptides. Then, isotypes from HeLa cells were successfully characterized for the first time by in-source decay (ISD) fragmentation of their C-terminus coupled to a pseudo MS(3) technique named T(3)-sequencing. The fragmentation occurred in-source, preferentially generating y(n)-series ions. This approach required guanidination for the characterization of the beta(III)-tubulin C-terminus peptide. This study is, to our knowledge, the first example of reflectron in-source decay (reISD) of the C-terminus of a 50 kDa protein. This potentially occurs via a CID-like mechanism occurring in the MALDI plume. There are now new avenues for top-down characterization of important clinical biomarkers such as beta(III)-tubulin isotypes, a potential marker of drug resistance and tumor progression. This paper raises the challenge of protein isotypes characterization for early cancer detection and treatment monitoring.

Methods in tubulin proteomics.

New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects of drugs on microtubules. The identification of tubulin isotypes and of regions of the microtubule involved in drug interactions has been advanced by proteomic methodologies. The diversity of tubulin sequences and posttranslational modifications (PTMs) can generate a complex mixture of heterodimers with unique molecular dynamics driving specific functions. Mass spectrometry (MS)-based approaches have been developed, and in combination with chromatographic and/or electrophoretic separation of tubulin polypeptides or peptides, they have contributed to our understanding of tubulin proteomics. We present protocols that we have used for the analysis of tubulin isotypes and PTMs present in tubulin isolated from cells in culture or tissues and for the identification of tubulin regions altered by microtubule-stabilizing agents. Tubulin proteomics complements structural and computer modeling information for a high-resolution view of microtubule dynamics and its alteration by drugs. These methodologies will help in providing insights into tubulin isotype-specific functions and in the design of drugs targeting either all tubulin heterodimers indiscriminately or only those containing specific isotypes.

Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation.

Toxoplasma gondii is an apicomplexan of both medical and veterinary importance which is classified as an NIH Category B priority pathogen. It is best known for its ability to cause congenital infection in immune competent hosts and encephalitis in immune compromised hosts. The highly stable and specialized microtubule-based cytoskeleton participates in the invasion process. The genome encodes three isoforms of both alpha- and beta-tubulin and we show that the tubulin is extensively altered by specific post-translational modifications (PTMs) in this paper. T. gondii tubulin PTMs were analyzed by mass spectrometry and immunolabeling using specific antibodies. The PTMs identified on alpha-tubulin included acetylation of Lys40, removal of the last C-terminal amino acid residue Tyr453 (detyrosinated tubulin) and truncation of the last five amino acid residues. Polyglutamylation was detected on both alpha- and beta-tubulins. An antibody directed against mammalian alpha-tubulin lacking the last two C-terminal residues (Delta2-tubulin) labeled the apical region of this parasite. Detyrosinated tubulin was diffusely present in subpellicular microtubules and displayed an apparent accumulation at the basal end. Methylation, a PTM not previously described on tubulin, was also detected. Methylated tubulins were not detected in the host cells, human foreskin fibroblasts, suggesting that this may be a modification specific to the Apicomplexa.

Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization.

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

Antiangiogenic vinflunine affects EB1 localization and microtubule targeting to adhesion sites.

The motile behavior of endothelial cells is a crucial event for neoangiogenesis. We previously showed that noncytotoxic concentrations of vinflunine inhibit capillary-like tube formation on Matrigel and endothelial cell migration with a concomitant increase in interphase microtubule dynamic instability. In this article, we further investigated the effects of vinflunine on migration and cytoskeleton interaction dynamics in HMEC-1 endothelial cells. We confirmed that vinflunine, at low and noncytotoxic concentrations (0.01-1 nmol/L), inhibited endothelial cell random motility by 54%. This effect was associated with a decrease in the percentage of stable microtubules and in the mean duration of pauses for dynamic ones. Moreover, we found that vinflunine altered adhesion site targeting by microtubules and suppressed the microtubule (+) end pause that occurs at adhesion sites during cell migration (from 151 +/- 20 seconds in control cells to 38 +/- 7 seconds in vinflunine-treated cells, P < 0.001). This effect was associated with the inhibition of adhesion site dynamics and the formation of long-lived stress fibers. Importantly, we found that vinflunine altered EB1 localization at microtubule (+) ends. These results highlight a new mechanism of action of vinflunine, which act by disrupting the mutual control between microtubule and adhesion site dynamics and strengthen the role of +TIPs proteins such as EB1 as key regulators of endothelial cell motility.

Increased levels of a unique post-translationally modified betaIVb-tubulin isotype in liver cancer.

Identifying changes at the molecular level during the development of hepatocellular carcinoma is important for the detection and treatment of the disease. The characteristic structural reorganization of preneoplastic cells may involve changes in the microtubule cytoskeleton. Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, vesicle transport, and motility. They are comprised of multiple isotypes of alpha- and beta-tubulin. Changes in the levels of these isotypes may affect not only microtubule stability and sensitivity to drugs but also interactions with endogenous proteins. We employed a rat liver cancer model that progresses through stages similar to those of human liver cancer, including metastasis to the lung, to identify changes in the tubulin cytoskeleton during carcinogenesis. Tubulin isotypes in both liver and lung tissue were purified and subsequently separated by isoelectric focusing electrophoresis. The C-terminal isotype-defining region from each tubulin was obtained by cyanogen bromide cleavage and identified by mass spectrometry. A novel post-translational modification of betaIVb-tubulin in which two hydrophobic residues are proteolyzed from the C-terminus, thus exposing a charged glutamic acid residue, was identified. The unique form of betaIVb-tubulin was quantified in the liver tissue of all carcinoma stages and found to be approximately 3-fold more abundant in nodular and tumor tissue than in control tissue. The level of this form was also found to be increased in lung tissue with liver metastasis. This modification alters the C-terminal domain of one of the most abundant beta-tubulin isotypes in the liver and therefore may affect the interactions of microtubules with endogenous proteins.

Aneuploidy acts both oncogenically and as a tumor suppressor.

An abnormal chromosome number, aneuploidy, is a common characteristic of tumor cells. Boveri proposed nearly 100 years ago that aneuploidy causes tumorigenesis, but this has remained untested due to the difficulty of selectively generating aneuploidy. Cells and mice with reduced levels of the mitosis-specific, centromere-linked motor protein CENP-E are now shown to develop aneuploidy and chromosomal instability in vitro and in vivo. An increased rate of aneuploidy does drive an elevated level of spontaneous lymphomas and lung tumors in aged animals. Remarkably, however, in examples of chemically or genetically induced tumor formation, an increased rate of aneuploidy is a more effective inhibitor than initiator of tumorigenesis. These findings reveal a role of aneuploidy and chromosomal instability in preventing tumorigenesis.