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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.

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 themechanisms underlying septin-associated pathologies. [ABSTRACT FROM AUTHOR]

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 themechanisms underlying septin-associated pathologies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.sup.3] technique named [T.sup.3]-sequencing. The fragmentation occurred in-source, preferentially generating [y.sub.n]-series ions. This approach required guanidination for the characterization of the [[beta].sub.III]-tubulin C-terminus peptide. This study is, to our knowledge, the first example of reflectron in-source decay (re-ISD) 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].sub.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. 10.1021/ac100996v

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the [alpha]/[beta]-tubulin heterodimer, GTP bound to [beta]-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to [beta]-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both [beta]- and [alpha]-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography--electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both [beta]-and [alpha]-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimmer--dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography--electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers. hydrogen/deuterium exchange

Tubulin from Hey cells by reverse-phase HPLC coupled to ESI ion-trap MS is analyzed to confirm that the sequence of human betaV-tubulin entered in the NCBI protein database is correct beyond the C- terminal sequence. The results demonstrate that the human betaV-tubulin sequence present in the NCBI protein database is effectively expressed at the protein level in epithelial cells.

A study was conducted analyze the inhibition effects of allothiocochicinoids on tubulin polymerization in vivo. Conformer ratios in different solvents and coupling constants for allocolchicinoids in a number of configurations were considered in the study. Results showed that the 7S enantiomer configuration was less active than the 7R isomer. Findings also indicated that molecular chirality in esters supporting a 7-O-aroyl substituent was reversed.

Hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques were used to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another microtubule stabilizing agent (MSA), discodermolide, whose synthetic analogues might have potential use in the clinic. The complementary stabilizing effects of taxol and discodermolide on the microtubules might provide better insight into the synergy observed between the two drugs in vivo.

A rat liver cancer model is employed to study the various changes taking place in the tubulin cytoskeleton during carcinogenesis. The model is shown to exhibit a novel post-translational modification of a unique form of [beta]IVb-tubulin isotype in the patient.

The liquid chromotography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines are described. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein.

Research delineates expression of tubulin mutations at the protein level and their role in drug resistance. Results indicate that in the cell lines exhibiting resistance to microtubule-stabilizing drugs such as taxol, betaIII-tubulin expression is observed, which represents tubulin isotype-related resistance marker.