A structural view of microtubule dynamics

The essential microtubule property of dynamic instability is based on the binding, hydrolysis and exchange of GTP in each tubulin dimer. The recent high-resolution structures of tubulin and the microtubule have given us the first view at atomic level of properties such as nucleotide exchangeability, the linkage between polymerization and nucleotide hydrolysis. and the origin of microtubule destabilization, as well as the mode of action of antimitotic agents such as taxol.     

Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO^? present in TBCB, which is similar to the EEY/F-COO^? element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.     

Regulatory mechanisms of kinetochore–microtubule interaction in mitosis

Interaction of microtubules with kinetochores is fundamental to chromosome segregation. Kinetochores initially associate with lateral surfaces of microtubules and subsequently become attached to microtubule ends. During these interactions, kinetochores can move by sliding along microtubules or by moving together with depolymerizing microtubule ends. The interplay between kinetochores and microtubules leads to the establishment of bi-orientation, which is the attachment of sister kinetochores to microtubules from opposite spindle poles, and subsequent chromosome segregation. Molecular mechanisms underlying these processes have been intensively studied over the past 10 years. Emerging evidence suggests that the KNL1–Mis12–Ndc80 (KMN) network plays a central role in connecting kinetochores to microtubules, which is under fine regulation by a mitotic kinase, Aurora B. However, a growing number of additional molecules are being shown to be involved in the kinetochore–microtubule interaction. Here I overview the current range of regulatory mechanisms of the kinetochore–microtubule interaction, and discuss how these multiple molecules contribute cooperatively to allow faithful chromosome segregation.     

Microtubule dynamics

Microtubules are fibrous elements in the cytoplasm of eukaryotic cells, where they perform a wide variety of functions. Microtubules are major organizers of the cell interior and are vitally involved in motility events such as chromosome migration during cell division. To fulfill their physiological function, microtubule arrays have to undergo dramatic changes in their spatial arrangement, and this depends to a large extent on the complex and special dynamic properties of the individual polymers. In this review we first describe the intrinsic dynamic properties of microtubules assembled in vitro from purified tubulin and examine the relationships between these properties and microtubule functions. Subsequent sections concern microtubule dynamics in vivo, their similarity and differences with microtubule dynamics in vitro, and the nature of the cellular regulators which act on microtubule assemblies in physiological conditions. Received 2 May 2001; received after revision 10 July 2001; accepted 10 July 2001     

Calmodulin mediates melatonin cytoskeletal effects

In this article, we review the data concerning melatonin interactions with calmodulin. The kinetics of melatonin-calmodulin binding suggest that the hormone modulates cell activity through intracellular binding to the protein at physiological concentration ranges. Melatonin interaction with calmodulin may allow the hormone to modulate rhythmically many cellular functions. Melatonin's effect on tubulin polymerization, and cytoskeletal changes in MDCK and N1E-115 cells cultured with melatonin, suggest that at low concentrations (10^–9 M) cytoskeletal effects are mediated by its antagonism to Ca²⁺-calmodulin. At higher concentrations (10^–5 M), non-specific binding of melatonin to tubulin occurs thus overcoming the specific melatonin antagonism to Ca²⁺-calmodulin. Since the structures of melatonin and calmodulin are phylogenetically well preserved, calmodulin-melatonin interaction probably represents a major mechanism for regulation and synchronization of cell physiology.     

Back to the tubule: microtubule dynamics in Parkinson’s disease

Cytoskeletal homeostasis is essential for the development, survival and maintenance of an efficient nervous system. Microtubules are highly dynamic polymers important for neuronal growth, morphology, migration and polarity. In cooperation with several classes of binding proteins, microtubules regulate long-distance intracellular cargo trafficking along axons and dendrites. The importance of a delicate interplay between cytoskeletal components is reflected in several human neurodegenerative disorders linked to abnormal microtubule dynamics, including Parkinson’s disease (PD). Mounting evidence now suggests PD pathogenesis might be underlined by early cytoskeletal dysfunction. Advances in genetics have identified PD-associated mutations and variants in genes encoding various proteins affecting microtubule function including the microtubule-associated protein tau. In this review, we highlight the role of microtubules, their major posttranslational modifications and microtubule associated proteins in neuronal function. We then present key evidence on the contribution of microtubule dysfunction to PD. Finally, we discuss how regulation of microtubule dynamics with microtubule-targeting agents and deacetylase inhibitors represents a promising strategy for innovative therapeutic development.     

CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS–vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS–vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS–vinculin interaction in mechanosensing and focal adhesion dynamics.     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Antiviral strategies against influenza virus: towards new therapeutic approaches

Influenza viruses are major human pathogens responsible for respiratory diseases affecting millions of people worldwide and characterized by high morbidity and significant mortality. Influenza infections can be controlled by vaccination and antiviral drugs. However, vaccines need annual updating and give limited protection. Only two classes of drugs are currently approved for the treatment of influenza: M2 ion channel blockers and neuraminidase inhibitors. However, they are often associated with limited efficacy and adverse side effects. In addition, the currently available drugs suffer from rapid and extensive emergence of drug resistance. All this highlights the urgent need for developing new antiviral strategies with novel mechanisms of action and with reduced drug resistance potential. Several new classes of antiviral agents targeting viral replication mechanisms or cellular proteins/processes are under development. This review gives an overview of novel strategies targeting the virus and/or the host cell for counteracting influenza virus infection.     

Regulation of end-binding protein EB1 in the control of microtubule dynamics

The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of “endogenous EB1 antagonists” and emphasize the importance of finely regulating EB1 function at growing microtubule ends.     

Regulation of receptor function by cholesterol

Cholesterol influences many of the biophysical properties of membranes and is nonrandomly distributed between cellular organelles, subdomains of membranes, and leaflets of the membrane bilayer. In combination with the high dynamics of cholesterol distribution, this offers many possibilities for regulation of membrane-embedded receptors. Depending on the receptor, cholesterol can have a strong influence on the affinity state, on the binding capacity, and on signal transduction. Most important, cholesterol may stabilize receptors in defined conformations related to their biological functions. This may occur by direct molecular interaction between cholesterol and receptors. In this review, we discuss the functional dependence of the nicotinic acetylcholine receptor as well as different G protein-coupled receptors on the presence of cholesterol.     

Contrasting models for kinetochore microtubule attachment in mammalian cells

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or “kinetochore fibrils” have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules.     

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds α-tubulin and promotes α/β dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds α-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Δ mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Δ mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.     

Developing anti-neoplastic biotherapeutics against eIF4F

Biotherapeutics have revolutionized modern medicine by providing medicines that would not have been possible with small molecules. With respect to cancer therapies, this represents the current sector of the pharmaceutical industry having the largest therapeutic impact, as exemplified by the development of recombinant antibodies and cell-based therapies. In cancer, one of the most common regulatory alterations is the perturbation of translational control. Among these, changes in eukaryotic initiation factor 4F (eIF4F) are associated with tumor initiation, progression, and drug resistance in a number of settings. This, coupled with the fact that systemic suppression of eIF4F appears well tolerated, indicates that therapeutic agents targeting eIF4F hold much therapeutic potential. Here, we discuss opportunities offered by biologicals for this purpose.     

Sulfatide-tenascin interaction mediates binding to the extracellular matrix and endocytic uptake of liposomes in glioma cells

Tenascin-C is an extracellular matrix glycoprotein, whose expression is highly restricted in normal adult tissues, but markedly up-regulated in a range of tumors, and therefore serves as a potential receptor for targeted anticancer drug or gene delivery. We describe here a liposomal carrier system in which the targeting ligand is sulfatide. Experiments with tenascin-C-expressing glioma cells demonstrated that binding of liposomes to the extracellular matrix relied essentially on the sulfatide-tenascin-C interaction. Following binding to the extracellular matrix, the sulfatide-containing liposomes were internalized via both caveolae/lipid raft- and clathrin-dependent pathways, which would ensure direct cytoplasmic release of the cargoes carried in the liposomes. Such natural lipid-guided intracellular delivery targeting at the extracellular matrix glycoproteins of tumor cells thus opens a new direction for development of more effective anticancer chemotherapeutics in future. K. Shao & Q. Hou: These authors contributed equally to this work. Received 22 September 2006; received after revision 5 December 2006; accepted 9 January 2007     

Integrin and microtubule crosstalk in the regulation of cellular processes

Integrins engage components of the extracellular matrix, and in collaboration with other receptors, regulate signaling cascades that impact cell behavior in part by modulating the cell’s cytoskeleton. Integrins have long been known to function together with the actin cytoskeleton to promote cell adhesion, migration, and invasion, and with the intermediate filament cytoskeleton to mediate the strong adhesion needed for the maintenance and integrity of epithelial tissues. Recent studies have shed light on the crosstalk between integrin and the microtubule cytoskeleton. Integrins promote microtubule nucleation, growth, and stabilization at the cell cortex, whereas microtubules regulate integrin activity and remodeling of adhesion sites. Integrin-dependent stabilization of microtubules at the cell cortex is critical to the establishment of apical–basal polarity required for the formation of epithelial tissues. During cell migration, integrin-dependent microtubule stabilization contributes to front–rear polarity, whereas microtubules promote the turnover of integrin-mediated adhesions. This review focuses on this interdependent relationship and its impact on cell behavior and function.     

In vitro amiodarone protein binding and its interaction with warfarin

Summary The binding of amiodarone to human plasma protein and to bovine serum, albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.This work was partially funded by the CNR (National Research Council, Rome, Italy), Program on Clinical Pharmacology and Rare Diseases. The authors would like to thanks Drs E. Marzi and E. riva for their help.     

In vitro amiodarone protein binding and its interaction with warfarin

The binding of amiodarone to human plasma protein and to bovine serum albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.     

Proton motive force mediates a reorientation of the cytosolic domains of the multidrug transporter LmrP

LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.