End-tracking Proteins Research Articles

Models for actin polymerization motors

Actin polymerization drives cell membrane protrusions and the propulsion of intracellular pathogens. The molecular mechanisms driving actin polymerization are not yet fully understood. Various mathematical models have been proposed to explain how cells convert chemical energy released upon actin polymerization into a pushing force on a surface. These models have attempted to explain puzzling properties of actin-based motility, including persistent attachment of the network to the membrane during propulsion and the interesting trajectories of propelled particles. These models fall generally into two classes: those requiring filament (+)-ends to fluctuate freely from the membrane to add subunits, and those where filaments elongate with their (+)-ends persistently associated with surface through filament end-tracking proteins ("actoclampin" models). This review compares and contrasts the key predictions of these two classes of models with regard to force-velocity profiles, and evaluates them with respect to experiments with biomimetic particles, and the experimental evidence on the role of end-tracking proteins such as formins and nucleation-promoting factors in actin-based motility.

Coordination of Actin Filament and Microtubule Dynamics during Neurite Outgrowth

Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskeletal dynamics, direct characterization of the corresponding molecular events has remained a challenge. Here, we address this outstanding problem by examining neurite outgrowth stimulated by local application of cell adhesion substrates. During acute outgrowth, the advance of organelles and underlying microtubules was correlated with regions of attenuated retrograde actin network flow in the periphery. Interestingly, as adhesion sites matured, contractile actin arc structures, known to be regulated by the Rho/Rho Kinase/myosin II signaling cascade, became more robust and coordinated microtubule movements in the growth cone neck. When Rho Kinase was inhibited, although growth responses occurred with less of a delay, microtubules failed to consolidate into a single axis of growth. These results reveal a role for Rho Kinase and myosin II contractility in regulation of microtubule behavior during neuronal growth.

Dynamic behavior of GFP–CLIP-170 reveals fast protein turnover on microtubule plus ends

Microtubule (MT) plus end–tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood–testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150 Glued, EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.

CLIP170 autoinhibition mimics intermolecular interactions with p150Glued or EB1

CLIP170 and p150(Glued) localize to the plus ends of growing microtubules. Using crystallography and NMR, we show that autoinhibitory interactions within CLIP170 use the same binding determinants as CLIP170's intermolecular interactions with p150(Glued). These interactions have both similar and distinct features when compared with the p150(Glued)-EB1 complex. Our data thus demonstrate that regulation of microtubule dynamics by plus end-tracking proteins (+TIPs) occurs through direct competition between homologous binding interfaces.

Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition

Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.

IQGAP1 Stimulates Actin Assembly through the N-Wasp-Arp2/3 Pathway

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.

Nematode Sperm Motility: Nonpolar Filament Polymerization Mediated by End-Tracking Motors

In nematode sperm cell motility, major sperm protein (MSP) filament assembly results in dynamic membrane protrusions in a manner that closely resembles actin-based motility in other eukaryotic cells. Paradoxically, whereas actin-based motility is driven by addition of ATP-bound actin subunits onto actin filament plus-ends located at the cell membrane, MSP dimers assemble from solution into nonpolar filaments that lack a nucleotide binding site. Thus, filament polarity and on-filament ATP hydrolysis, although essential for actin-based motility, appear to be unnecessary for membrane protrusions by MSP. As a potential resolution to this paradox, we propose a model for MSP filament assembly and force generation by MSP filament end-tracking proteins. In this model, ATP hydrolysis drives affinity-modulated, processive interactions between membrane-associated proteins and elongating filament ends. However, in contrast to the “actoclampin” model for actin filament end-tracking motors, ATP activates the tracking protein (or a soluble cofactor) rather than the MSP subunits themselves (in contrast to activation of actin subunits by ATP binding). The MSP end-tracking model predicts properties that are consistent with several key observations of MSP-based motility, including persistent membrane attachment, polymerization of filament ends at the membrane with depolymerization of free-filament ends away from the membrane, as well as a saturating dependence of polymerization rate on the concentration of non-MSP soluble cytoplasmic components.

The Schizosaccharomyces pombe EB1 Homolog Mal3p Binds and Stabilizes the Microtubule Lattice Seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

Diffusion Rate Limitations in Actin-Based Propulsion of Hard and Deformable Particles

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.

CLASPing the Cell Cortex

CLASPs are spatially regulated microtubule plus end tracking proteins involved in forming polarized microtubule arrays. Work in this issue of Developmental Cell identifies the protein LL5beta as a key CLASP binding platform that mediates communication between the cell cortex and the microtubule cytoskeleton.

A +TIP for a smooth trip

Is there a cellular mechanism for preventing a depolymerizing microtubule track from “slipping out from under” its cargo? A recent study in budding yeast indicates that when a chromosome is transported to the minus end of a spindle microtubule, its kinetochore-bound microtubule plus end–tracking protein (+TIP) Stu2 may move to the plus end to promote rescue; i.e., to switch the depolymerizing end to a polymerizing end. The possibility that other +TIPs may play a similar role in sustaining a microtubule track during vesicular transport deserves investigation.

Signaling Mechanisms Underlying Slit2-Induced Collapse of Xenopus Retinal Growth Cones

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

Mechanisms for focusing mitotic spindle poles by minus end–directed motor proteins

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end–directed motor proteins. Here, we have characterized the roles of two minus end–directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end–tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end–directed motors cooperate to ensure spindle pole coalescence during mitosis.

Melanophilin and myosin Va track the microtubule plus end on EB1

In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end–tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp–microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end directly by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p–Kar9p–Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

EB1 Is Essential duringDrosophilaDevelopment and Plays a Crucial Role in the Integrity of Chordotonal Mechanosensory Organs

EB1 is a conserved microtubule plus end tracking protein considered to play crucial roles in microtubule organization and the interaction of microtubules with the cell cortex. Despite intense studies carried out in yeast and cultured cells, the role of EB1 in multicellular systems remains to be elucidated. Here, we describe the first genetic study of EB1 in developing animals. We show that one of the multiple Drosophila EB1 homologues, DmEB1, is ubiquitously expressed and has essential functions during development. Hypomorphic DmEB1 mutants show neuromuscular defects, including flightlessness and uncoordinated movement, without any general cell division defects. These defects can be partly explained by the malfunction of the chordotonal mechanosensory organs. In fact, electrophysiological measurements indicated that the auditory chordotonal organs show a reduced response to sound stimuli. The internal organization of the chordotonal organs also is affected in the mutant. Consistently, DmEB1 is enriched in those regions important for the structure and function of the organs. Therefore, DmEB1 plays a crucial role in the functional and structural integrity of the chordotonal mechanosensory organs in Drosophila.

Force Generation by Cytoskeletal Filament End-Tracking Proteins

Force generation in several types of cell motility is driven by rapidly elongating cytoskeletal filaments that are persistently tethered at their polymerizing ends to propelled objects. These properties are not easily explained by force-generation models that require free (i.e., untethered) filament ends to fluctuate away from the surface for addition of new monomers. In contrast, filament end-tracking proteins that processively advance on filament ends can facilitate rapid elongation and substantial force generation by persistently tethered filaments. Such processive end-tracking proteins, termed here filament end-tracking motors, maintain possession of filament ends and, like other biomolecular motors, advance by means of 5′-nucleoside triphosphate (NTP) hydrolysis-driven affinity-modulated interactions. On-filament NTP hydrolysis/phosphate release yields substantially more energy than that required for driving steady-state assembly/disassembly of free filament ends (i.e., filament treadmilling), as revealed by an energy inventory on the treadmilling cycle. The kinetic and thermodynamic properties of two simple end-tracking mechanisms (an end-tracking stepping motor and a direct-transfer end-tracking motor) are analyzed to illustrate the advantages of an end-tracking motor over free filament-end elongation, and over passive end-trackers that operate without the benefit of NTP hydrolysis, in terms of generating force, facilitating rapid monomer addition, and maintaining tight possession of the filament ends. We describe an additional cofactor-assisted end-tracking motor to account for suggested roles of cofactors in the affinity-modulated interactions, such as profilin in actin-filament end-tracking motors and EB1 in microtubule end-tracking motors.

Microtubules Direct Axon Growth

Two groups investigated mechanisms by which microtubules may be regulated in response to signals that promote axonal movement (see Kalil and Dent). Lee et al. identified the microtubule-associated protein orbit (also known as mast and the ortholog of vertebrate CLASPs) in a genetic screen in Drosophila as interacting with the kinase Abl in the retina. Orbit was implicated in both central and peripheral axon migration in Drosophila and interacted genetically with the Slit-Robo signaling pathway. Analysis of the vertebrate ortholog in Xenopus by visualization of green fluorescent protein (GFP)-tagged CLASP showed that CLASP associated with a subset of microtubules in the growth cone, and these microtubules penetrated into the leading edge of the actin-rich filopodia. Overexpression of GFP-CLASP resulted in microtubule looping and a reduction in the advance of the leading edge of the growth cone. Thus, orbit (or CLASP in vertebrates) appears to be poised between the repellent receptor (robo) and the cytoskeleton to coordinate the movement of microtubules in the growth cone in response to axon guidance cues. Zhou et al. investigated the mechanism by which nerve growth factor (NGF) promoted axon growth in embryonic mouse dorsal root ganglia (DRG) neurons. Various pharmacological treatments and analysis of cells expressing mutants of the signaling components verified that signaling stimulated by NGF proceeded from phosphoinositide 3-kinase (PI3K) to integrin-linked kinase (ILK) to glycogen synthase kinase 3β (GSK-3β) and that this pathway was necessary for NGF-mediated axon elongation. APC (adenomatous polyposis coli), a microtubule-binding protein and target of GSK-3β, was found in axons associated with the cytoskeleton (based on insolubility to detergent extraction) and was visually localized to the tips of growing microtubules. APC localization to the distal ends of axons was lost under conditions that blocked NGF signaling and caused growth cone collapse. Expression of mutants of APC that lacked critical domains for mediating direct or indirect microtubule interactions inhibited axon elongation. Thus, NGF-mediated axon elongation appears to follow a path to APC to control microtubule assembly in the growth cone. K. Kalil, E. W. Dent, Hot +TIPS: Guidance cues signal directly to microtubules. Neuron 42 , 877-883 (2004). [Online Journal] H. Lee, U. Engel, J. Rusch, S. Scherrer, K. Sheard, D. Van Vactor, The microtubule plus end tracking proteins Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance. Neuron 42 , 913-926 (2004). [Online Journal] F. Q. Zhou, J. Zhou, S. Dedhar, Y.-H. Wu, W. D. Snider, NGF-induced axon growth is mediated by localized inactivation of GSK-3β and functions of the microtubule plus end binding protein APC. Neuron 42 , 897-912 (2004). [Online Journal]

The Microtubule Plus End Tracking Protein Orbit/MAST/CLASP Acts Downstream of the Tyrosine Kinase Abl in Mediating Axon Guidance

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.

A Novel Localization Pattern for an EB1-like Protein Links Microtubule Dynamics to Endomembrane Organization

A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins [1], label growing microtubule ends specifically in diverse organisms [2, 3] and are implicated in spindle dynamics [4], chromosome segregation [5], and directing microtubules toward cortical sites [6]. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems [7, 8] but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules [3, 9], a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.