Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs), have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Gα subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation. Using Drosophila S2 cells as a model system, this study shows that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, it was found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. It is proposed that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Gα activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development (Rogers, 2004).
The cellular functions of the microtubule plus-end binding protein EB1 has been characterized in Drosophila S2 cells; this protein plays an important role in regulating microtubule dynamics and in the assembly and dynamics of the mitotic spindle (Elliott, 2005). In order to learn more about EB1's functions, attempts were made to identify EB1 binding partners with affinity purification. Recombinant Drosophila GST-EB1 bound to glutathione Sepharose beads was used as an affinity chromatography matrix to bind interacting partners from S2 cell extracts. Bound proteins were eluted from the beads and separated by SDS-PAGE, and excised bands were subjected to tryptic digestion and mass spectrometry fingerprinting (Rogers, 2004).
Twenty 'EB1-specific' proteins were identified over the course of five independent pull-down experiments. However, of these, only six candidates were identified in all five trials: CLIP190, the Drosophila ortholog of vertebrate CLIP-170, which localizes to the plus ends of microtubules, Orbit/MAST, a microtubule plus-end-associated protein that interacts with CLIP-170, nonmuscle myosin II heavy chain, the minus-end-directed kinesin, Ncd, Shortstop, a member of the spectraplakin family of actin/microtubule cross-linking proteins, and DRhoGEF2. This paper focuses on DRhoGEF2 for further study (Rogers, 2004).
The association of DRhoGEF2 with EB1 in vitro raised the possibility that this protein may localize to the tips of microtubules. To test this idea, polyclonal antibodies were generated against the C-terminal 720 amino acid residues of DRhoGEF2. These antibodies recognized a ~280 kDa polypeptide on immunoblots of S2 cell extracts; this polypeptide was eliminated after DRhoGEF2 RNAi treatment, indicating that the antibodies were reacting with the correct polypeptide (Rogers, 2004).
By immunofluorescence, anti-DRhoGEF2 antibodies recognized punctate structures distributed throughout the cell. Superimposed upon this punctate pattern, however, were short (~1 μm) linear tracks that colocalized with the tips of microtubules. Moreover, immunofluorescent staining of DRhoGEF2 in S2 cells expressing low amounts of EB1-EGFP indicated that these two proteins colocalize exactly at microtubule tips. In the perinuclear region of many cells, DRhoGEF2 antibodies also stained larger spots that costained with γ-tubulin, a centrosome marker. Depletion of DRhoGEF2 with RNAi eliminated antibody staining of all of these structures in S2 cells. Thus, these immunofluorescence experiments reveal that DRhoGEF2 exists in three pools within S2 cells: punctate throughout the cell, at microtubule tips, and on centrosomes (Rogers, 2004).
DRhoGEF2 tagged with green fluorescent protein (GFP) was tested to examine its dynamic behavior through time-lapse imaging with a spinning-disk microscope. As predicted from immunofluorescence data, 'comet-like' structures of DRhoGEF2-GFP moved from the cell center toward the periphery in a manner that was very similar to that observed for EB1-GFP. In many cells, an intense spot of DRhoGEF2-GFP was observed near the perinuclear region. This spot likely corresponded to the centrosome staining because the tips of nucleated microtubules emanated from this point in a radial pattern. Microtubule dynamics are essential for this movement because it could be eliminated with either 10 μM colchicine or 10 μM taxol. Thus, it is concluded that DRhoGEF2 associates with the tips of growing microtubules and exhibits plus-end tracking that is qualitatively similar to that described for EB1 (Rogers, 2004).
Because DRhoGEF2 was isolated based upon its association, direct or indirect, with EB1, EB1 was deleted from cells with RNAi and whether the association of DRhoGEF2 with microtubule tips was examined. In cultures treated with control dsRNA, scoring of fixed cells stained for DRhoGEF2 and microtubules revealed that 94% of the cells (n = 300) had DRhoGEF2 associated with the microtubule tip. In contrast, in S2 cells treated for 7 days with EB1 dsRNA, only 5% of the cells retained DRhoGEF2 at the plus ends. These results demonstrate that targeting of DRhoGEF2 to growing microtubule plus ends is an EB1-dependent process (Rogers, 2004).
To further understand DRhoGEF2 functions, how overexpression and depletion of the protein affects the morphology of S2 cells was examined. When S2 cells are plated on concanavalin A, they adopt a 'fried-egg' appearance with a dome-like central domain defined by the nucleus and perinuclear organelle-rich region and an extended, symmetrical lamella. In contrast, overexpressing DRhoGEF2 caused many cells to adopt a smaller, contracted footprint on the substrate and to become significantly taller than control cells. These overexpressing cells formed a skirt of abnormally large membrane ruffles that tapered to the base of a raised, organelle-rich compartment, and the overall morphology resembled a 'bonnet' shape. This result suggests that DRhoGEF2 can induce contractility, in agreement with the genetic phenotype of DRhoGEF2 mutations (Rogers, 2004).
Several genetic studies implicate DRhoGEF2 as a positive regulator of Rho1. To test whether Rho activation is involved in generating the unusual phenotype associated with DRhoGEF2 overexpression, cells were transfected with constitutively active Rho1V14 and transfected cells were identified with an antibody raised against Drosophila Rho1. As predicted, most of the Rho1V14-expressing cells duplicated the morphology produced by DRhoGEF2 overexpression. In order to next test if inhibition of Rho1 prevented DRhoGEF2-induced shape change, DRhoGEF2-EGFP was transfected into cells that had been treated with Rho RNAi. Depletion of Rho1 by RNAi produced large multinucleate cells that did not contract in response to DRhoGEF2 overexpression. In contrast, RNAi inhibition of the other six Rho family members did not block DRhoGEF2-induced contraction (Rogers, 2004).
Active Rho is known to stimulate nonmuscle myosin II, and a genetic interaction has been demonsrated between DRhoGEF2 and myosin II during Drosophila morphogenesis. One well-characterized mechanism by which Rho1 activates myosin II is Rho kinase (DROK in Drosophila) stimulation, which activates the motor by phosphorylating the myosin light chain and by inactivating myosin light chain phosphatase. In order to determine if DROK is indeed downstream of DRhoGEF2, DROK was depleted with RNAi or kinase activity was inhibited with Y-27632, a pharmacological inhibitor, and then cell morphology was examined after DRhoGEF2 overexpression. Both treatments significantly reduced the numbers of cells exhibiting the contracted morphology. From these data, it is concluded that DRhoGEF2 changes S2 cell morphology through Rho1 and its downstream effector, DROK (Rogers, 2004).
To confirm that myosin II is a downstream effector in the DRhoGEF2 pathway in this system, the behavior of GFP-tagged myosin II in control S2 cells on concanavalin A was compared with that of S2 cells overexpressing DRhoGEF2. To perform this analysis, a stable cell line was generated expressing the myosin II regulatory light chain (RLC), known by Drosophila nomenclature as Spaghetti squash, under the control of the gene's endogenous promoter. Ectopic expression of RLC-GFP did not produce observable defects in actin organization or behavior; its distribution exactly coincided with the myosin II distribution determined by immunofluorescence staining of the same cells. RLC-GFP typically incorporated into punctae in the cell periphery and into higher-order structures in the central region of the cells. Time-lapse spinning-disk confocal microscopy revealed that punctae of RLC-GFP formed in the distal cell periphery and then translocated centripetally at a constant rate of 4.0 ± 0.3 μm/min toward the cell center. Such behavior of RLC-GFP is qualitatively very similar to the behavior of fluorescently labeled myosin II in cultured mammalian cells (Rogers, 2004).
Upon overexpression of DRhoGEF2, punctae of RLC-GFP were rarely observed. Instead, the majority of RLC-GFP signal was present in circular 'purse string' structures surrounding the organelle-dense region at the center of the cell. Time-lapse observation revealed that peripheral formation of RLC-GFP punctae and retrograde flow were infrequent in DRhoGEF2-overexpressing cells and that these RLC-GFP-containing purse strings were stable over a span of hours. The location and concentration of the myosin II suggests that actomyosin contraction is responsible for producing the bonnet-shaped appearance of these cells. From these observations, it is propose that DRhoGEF2 regulates myosin II dynamics and contractility in S2 cells (Rogers, 2004).
Genetic analyses of epithelial-sheet invagination in the early Drosophila embryo suggest that DRhoGEF2 may act downstream of the heterotrimeric Gα protein Concertina (Cta). To examine directly whether Concertina can activate DRhoGEF2, cells were transfected either with Myc-tagged wild-type Cta or Myc-tagged Cta bearing a constitutively activating point mutation (R277H) that inactivates GTPase activity, and the morphology of the transfected cells was examined. Cells expressing Myc-Cta were morphologically indistinguishable from untransfected cells, and only 3% of cells displayed a mildly contracted phenotype. In contrast, the majority of cells expressing Myc-CtaR277H exhibited the contracted morphology and myosin II purse string reminiscent of DRhoGEF2 overexpression. Similar results were obtained with three other constitutively activated Concertina constructs. However, the shape change was prevented in 88% of these cells (if they were pretreated for 7 days with dsRNA so that DRhoGEF2 was depleted. These results suggest that Concertina can act upstream of DRhoGEF2 to regulate S2 cell morphology (Rogers, 2004).
Next it was determined whether activation of DRhoGEF2 through Concertina affected its association with the microtubule cytoskeleton. Cells expressing Myc-Cta or Myc-CtaR277H were fixed and double stained for the Myc epitope tag and for DRhoGEF2. Overexpression of wild-type Concertina did not affect DRhoGEF2 association with microtubule plus ends or with the centrosome. However, constitutively activated Concertina resulted in DRhoGEF2 dissociation from microtubule tips; only 10% of the cells showed any colocalization of DRhoGEF2 with microtubule plus ends. Instead, DRhoGEF2 exhibited a diffuse staining pattern throughout the cell; this pattern likely represents association with the plasma membrane. Targeting of EB1 to the plus ends was not perturbed by CtaR277H, suggesting that Concertina signaling regulates the interactions between DRhoGEF2 and factors at microtubule tips (Rogers, 2004).
In an attempt to identify novel cellular factors that interact with EB1, this study unexpectedly discovered that DRhoGEF2, a key regulator of morphogenesis in Drosophila, associates with the tips of growing microtubules. This interesting type of intracellular motility required EB1 in a manner analogous to the EB1-dependent microtubule plus-end tracking of the vertebrate adenomatous polyposis coli (APC) tumor suppressor protein. This finding represents the first example of a regulator of the actin cytoskeleton that tracks along microtubule plus ends. Moreover, the dissociation of DRhoGEF2 from microtubule tips upon activation of Concertina also represents the first example of a regulated association of a protein with the microtubule plus end (Rogers, 2004).
The dissection of the DRhoGEF2 pathway at a cellular level is also consistent with genetic studies of Drosophila morphogenesis. These studies implicate Concertina in myosin II contractility through the Rho/Rho kinase pathway. The Rho1/Rho kinase/myosin II system is a widely employed module for bundling and contraction of actin filaments; it is involved in the formation of adhesion structures and stress fibers, retraction of the trailing edge in migrating cells, muscular contraction, morphogenetic cell shape changes, and construction of the cleavage furrow at the end of mitosis. Context- and location-specific activation of the Rho1/Rho kinase/myosin II module is likely to reside in the activation of specific RhoGEFs, over 20 of which reside within the Drosophila genome. This hypothesis is consistent with observations that inhibition of Rho1 or its downstream effectors causes a dramatic cytokinesis failure in S2 cells and embryos, but inhibition of DRhoGEF2 does not. Instead, DRhoGEF2 has been implicated in morphogenetic cell shape changes only in epithelial cells. Thus, it is believed that the signaling pathway that was engineered in S2 cells recapitulates events involved in the cellular shape changes preceding gastrulation in Drosophila blastula epithelia cells (Rogers, 2004).
However, in Drosophila development, this signaling pathway must be activated in a polarized manner by an unidentified receptor and its ligand so that myosin contraction occurs locally at the apical surface. In such a setting of asymmetric signaling, it is proposed that the intracellular transport of DRhoGEF2 on microtubule plus ends may play an important role in localized activation of the pathway. It is speculated that inactive DRhoGEF2 interacts with the tips of microtubules, whereupon these growing microtubules deliver 'packets' of DRhoGEF2 in the vicinity of the actin cortex. If DRhoGEF2 does not receive an activating input, it diffuses back into the cytoplasm to begin the transport cycle anew. However, if DRhoGEF2 is delivered to a subcortical region containing a high concentration of receptor-activated Concertina, DRhoGEF2 can locally activate the Rho1/Rho kinase/myosin II module. Moreover, because DRhoGEF2 possesses potential lipid (pleckstrin homology) and protein-protein (PDZ, RGS, and DH [Dbl homology]) interaction domains, microtubule-delivered DRhoGEF2 may be retained at the cortex if activated by Concertina. Although a microtubule-assisted activation of the Rho pathway during cellular shape changes during morphogenesis (such as in epithelial cells) is proposed, similar models that account for small GTPase activation during cellular motility have been suggested as well (Rogers, 2004).
In principle, interactions between DRhoGEF2 and its cortical activators could occur through diffusion within the cytoplasm. The evolution of this elaborate microtubule polymerization-based transport mechanism undoubtedly reflects some important property of the signaling pathway that is not yet understood. Perhaps the amount of DRhoGEF2 carried on the tip of a microtubule represents some quanta -- a critical concentration of the protein required either to respond to upstream inputs or to locally activate Rho1 in a cortical subdomain. This idea is supported by the observation that, at very low expression levels and without Concertina signaling, DRhoGEF2-GFP efficiently tracks microtubule ends without activating cellular contraction. Alternatively, it is possible that interaction with EB1 or some other protein at the microtubule plus end primes DRhoGEF2 for activation at the cortex. A third possibility is that microtubule dynamic instability is not uniform within a polarized cell but is locally modulated in order to deliver DRhoGEF2 to the cortex in a nonrandom manner. Testing between these hypotheses will require identification of the signaling components (i.e., the ligand-receptor pair) that act upstream of Concertina, reconstitution of the complete pathway in S2 cells, and the selective disruption of the association of DRhoGEF2 with microtubule tips in Drosophila embryos (Rogers, 2004).
Shortstop (Shot) is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. To address how Shot exerts its activity at the molecular level, the molecular interactions of Shot with candidate proteins was investigated in mature larval tendon cells. Shot colocalizes with the complex between EB1 and APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. It is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. These results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex (Subramanian, 2003).
Tendon cells undergo maturation during larval stages. In third instar larvae, different tendon cells acquire distinct shapes according to their orientation and the type of muscles to which they are connected. Initially, the localization of Shot was characterized relative to MT and F-actin organization in mature tendon cells of flat, opened third instar larvae. Shot and Tubulin staining overlapped within the entire cell. A unique domain at the focal plane of the muscle-tendon junction exhibits a compact MT array, which overlapped Shot staining. This domain was not detected when the optical section was taken 0.5 microm more internal to the junction focal plane. An optical cross-section perpendicular to the muscle-tendon junction site shows that the MT-Shot array extends from the muscle-tendon junction to the cuticle. Moreover, the MT array is oriented in the same direction as the microfilaments of the muscle cells, as shown by EM analysis. Thus, Shot and MTs are colocalized within a unique subcellular domain in the tendon cell that connects the muscle-tendon junction and the cuticle (Subramanian, 2003).
These immunofluorescent localization studies suggest a distinct abundance of MTs and MFs at both sides of the muscle-tendon junction. While MFs are highly enriched at the muscle side, MTs are detected mainly at the tendon side. To address whether this organization reflects differences in the distribution of additional junction-associated proteins, the larval flat preps were stained for PSß-integrin, Paxillin, and P-tyrosine, and their relative distribution at the focal plane of the muscle-tendon junction was analyzed. In all preparations, Shot marks the outlines of the tendon cell. About half of the PSß-integrin staining overlaps Shot staining, whereas the other half is located at the muscle membrane. Paxillin is more abundant on the muscle side. Interestingly, staining for P-tyrosine, which marks the extent of tyrosine-phosphorylated proteins (including Paxillin, Src, and others) is restricted to the tendon side of the junction and is tightly associated with the plasma membrane. Thus, although PSß-integrin distribution appears to be equal at both sides of the muscle-tendon hemiadherens junction, the molecular composition on both sides of these junctions appears to be distinct, as demonstrated for EB1, APC1, Paxillin, and the extent of P-tyrosine reactivity. It is tempting to speculate that these unequal protein distributions might relate to the enrichment of MTs and Shot on the tendon cell side (Subramanian, 2003).
EB1 is an evolutionarily conserved protein that binds the plus ends of growing MTs. It was first identified as a binding partner for the adenomatous polyposis coli tumor suppressor, APC. The EB1/APC complex is involved in regulation of MT polymerization and MT association with distinct subcellular domains. For example, in yeast, the EB1 homolog (BIM1) has been shown to modulate MT dynamics and link MTs to the cortex. Drosophila EB1 (Elliott, 2005) is important for the proper assembly, dynamics, and positioning of the mitotic spindle. Its association with APC2 in apical cell-cell adherens junctions is suggested to be essential for parallel spindle orientation (Lu, 2002) and for neuroblast asymmetric cell division (Subramanian, 2003).
Biochemical data suggest that the association of the C-terminal EF-GAS2 domain with EB1 is MT independent. A direct physical interaction between the C-terminal EF-GAS2 domain and alpha-tubulin had been suggested by a yeast two-hybrid screen and by the ability to precipitate purified Tubulin by the GAR and GSR domains (both included within the C terminus of the mammalian ACF7). These domains lack the EF-hands motif. EB1 is detected in association with the entire Shot C-terminal domain containing EF-hands. At this stage, no additional information is available regarding the site responsible for EB1 association (Subramanian, 2003).
The data suggest that Shot association with the basal muscle-tendon junction is EB1 independent (since it is detected even in the absence of EB1/APC1); hence, it is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. The assembly of the MT-rich domain may be induced by either their direct association with Shot or with EB1/APC1, or with both. Interestingly, EB1, APC1, and Shot are not observed at the cell-cell adherens junctions formed between the tendon cell and its neighboring ectodermal cells (Subramanian, 2003).
There is no direct evidence for the polarity of MTs within the compact Shot/MT-rich domain, since no differential EB1 localization was detected in this domain. Other studies suggest that MTs in the entire epidermis are arranged at a polar orientation in which their plus ends face the basal pole and their minus ends face the cuticle. Similarly, in the pupal wing, MTs have been shown to be arranged with their plus ends facing the basal hemiadherens junctions. Thus, it is likely that in the tendon cells (which are part of the epidermal layer), MTs are similarly arranged, i.e., with their plus ends facing the basal hemiadherens junction (Subramanian, 2003).
The experiments show that reduced Shot activity leads to a significant tendon cell elongation, occurring presumably following muscle contractions. What is the mechanism allowing the MTs to elongate in the mutant tendon cell? An interesting possibility is that the MTs are connected to the muscle-tendon junction through their plus ends via their association with EB1 and Shot, and that this arrangement arrests further MT polymerization and maintains the MTs in a polarized arrangement. Following dissociation of the Shot/EB1/APC1 complex and the reduction of Shot activity, MTs undergo further polymerization and extension, leading to the significant elongation of the tendon cell. The newly formed MTs are not well connected to the cell cortex, thus leading to cell breakdown upon further muscle contraction. Support for the involvement of Shot in mediating MT-polarized organization emerges from recent analysis of Shot function in mushroom body neurons of the Drosophila adult brain. Distinct Nod-ßgal reactivity suggests that MT polarity within the axons is distinct from that of dendrites in wild-type mushroom body neurons. In neurons mutant for shot, the polarity of MTs in the axons is reversed and resembles that of dendrites (Subramanian, 2003).
Shot may perform a similar function in the organization of a compact and polarized array of MTs in the adult wing epithelium, as well as within the ligament cells of embryonic sensory chordotonal organs (Subramanian, 2003).
Studies with the different Shot domains show that the Actin binding domain, but not the Plakin domain, is capable of driving specific localization of both domains to the F-actin layer at the muscle-tendon junction. The thin Actin layer at the muscle-tendon junction may therefore be essential for the recruitment of Shot into the cytoplasmic faces of the hemiadherens domains. The C terminus containing the EF-hands and GAS2 domains is also capable of localizing at the muscle-tendon junction domain. This localization may be attributed to its association with endogenous EB1, as well as with MTs that are already arranged in the larval tendon cells, or with endogenous Shot. The Plakin domain on its own did not show specific subcellular localization, suggesting that it does not bind to proteins that are highly localized in the tendon cell. Alternatively, proteins that may form a complex with this domain may be engaged in existing protein complexes and therefore are not accessible to the exogenous Plakin protein. None of the Shot structural domains show a dominant-negative effect when overexpressed in tendon cells or in wing imaginal discs; this finding suggests that the proteins to which they bind are not present in limited amounts (Subramanian, 2003).
Interestingly, in larvae tendon cells, both the Actin-Plakin domain of Shot and the Shot C-terminal EF-GAS2 domain exhibit similar distribution. When transfected into Schneider cells, each domain shows a distinct subcellular localization. The Actin-Plakin-GFP was detected at the leading edge and in most cases did not overlap EB1, while the EF-GAS2 domain overlapped EB1 and decorated MTs. Similar studies with Drosophila Shot and ACF-7, the mammalian Shot ortholog, show that the Actin binding domain and the EF-GAS2 domain are associated with Actin MFs and with MTs, respectively, in transfected cells. However, the M1 domain in ACF-7 (similar to the Plakin domain) has been shown to be associated with MTs in the transfected cells, while the Shot-Plakin domain does not exhibit significant association with MTs. These differences may reflect differential distribution of yet uncharacterized ACF-7 binding proteins within the mammalian cells (Subramanian, 2003).
What could be the connection between Shot activity and reduced F-actin content? Recent studies suggest that MT disassembly activates Rho by the release of GEFs that are specifically associated with and inhibited by MTs. In tendon cells, no unique association was detected of GEF (Pebble) or Rho with the MT-rich domain. Therefore, the relevance of these factors is not clear. Recently, it was shown that in Drosophila embryonic tracheal cells, activated RhoA mimicks the Shot loss-of-function phenotype; this finding suggests a similar inverse correlation between Actin polymerization by RhoA and the loss of shot function. Thus, the activity of Shot in organizing MTs to special subcellular sites via its association with EB1/APC1, and the inhibition of F-actin in these sites, may be relevant to other tissues in which Shot plays an essential role (Subramanian, 2003 and references therein).
The MT network is essential for a wide array of cellular functions. Shot, a multidomain Plakin family member, is essential for arranging a compact network of MTs in tendon cells. This is achieved by the association of Shot with the cytoplasmic faces of the muscle-tendon junction and presumably by the subsequent recruitment of the EB1/APC1 complex to these sites. In tendon cells, this unique MT organization is essential to resist muscle contraction (Subramanian, 2003).
CLIP-170 was the first microtubule plus-end-tracking protein to be described, and is implicated in the regulation of microtubule plus-ends and their interaction with other cellular structures. The cell-cycle-dependent mechanisms which localise the sole Drosophila melanogaster homologue CLIP-190 have been studied. During mitosis, CLIP-190 localises to unattached kinetochores independently of spindle-checkpoint activation. This localisation depends on the dynein-dynactin complex and Lis1 which also localise to unattached kinetochores. Further analysis revealed a hierarchical dependency between the proteins with respect to their kinetochore localisation. An inhibitor study also suggested that the motor activity of dynein is required for the removal of CLIP-190 from attached kinetochores. In addition, CLIP-190 association to microtubule plus-ends is regulated during the cell cycle. Microtubule plus-end association is strong in interphase and greatly attenuated during mitosis. Another microtubule plus-end tracking protein, EB1, directly interacts with the CAP-Gly domain of CLIP-190 and is required to localise CLIP-190 at microtubule plus-ends. These results indicate distinct molecular requirements for CLIP-190 localisation to unattached kinetochores in mitosis and microtubule ends in interphase (Dzhindzhev, 2005; full text of article).
The CLIP family of proteins is implicated in regulating microtubule dynamics and linking microtubule plus-ends with other cellular structures. To understand these functions, it is crucial to elucidate where and how these proteins localise within cells. This study investigated the molecular mechanisms of CLIP-190 localisation using RNAi in Drosophila cells, rather than using the expression of dominant proteins, to gain a clearer and more comprehensive view. The study revealed that distinct, cell-cycle-dependent mechanisms localise CLIP-190 to microtubule plus-ends and unattached kinetochores (Dzhindzhev, 2005).
CLIP and EB1 proteins are two major families of microtubule plus-end-tracking proteins. This study is the first demonstration of both a physical interaction and localisation-dependency between CLIP and EB1 proteins in higher eukaryotes. CLIP-190 requires EB1 to localise to microtubule plus-ends, and CLIP-190 directly interacts with EB1 through its CAP-Gly domain, which also binds to microtubules. Considering that this has previously been reported for fission yeast homologues, these findings demonstrate that this interaction and dependency are conserved among eukaryotes. The most obvious interpretation would be that EB1 simply bridges microtubule plus-ends and CLIP-190. However, this is thought not be the case. First, CLIP-170 has been shown to bind directly to microtubule plus-ends in vitro. Secondly, localisation studies show incomplete overlapping of the two proteins on microtubule plus-ends. Also, other EB1 interacting proteins, such as RhoGEF2 and the spectraplakin Short stop, which both require EB1 for their localisation to microtubule plus-ends (Rogers, 2004; Slep, 2005), show distinct localisation from that of EB1. Therefore, it is more likely that EB1 acts as a loading factor for these proteins, rather than a simple bridge. In addition, it seems that multiple microtubule plus-end-binding proteins, such as CLIPs, CLASPs, p150Glued, EB1, Lis1, Dynein, Short stop, APC and RhoGEF2, directly interact with each other and with microtubules. It is an exciting challenge to understand the regulatory network acting on microtubule plus-ends (Dzhindzhev, 2005).
In addition, it was found that the association of CLIP-190 to microtubule plus-ends is greatly reduced during mitosis. This is in contrast to EB1, which is associated with plus-ends throughout the cell cycle. This cell-cycle-regulation has not been described in other systems, possibly because of a lack of co-examination with EB1, and leads to the conclusion that it is not the consequence of a change in microtubule dynamics. It might be possible that CLIP-190 is modified during the cell cycle. Since EB1 is essential for CLIP-190 localisation to microtubule ends, EB1 activity or interaction between EB1 and CLIP-190 might also be regulated. Alternatively, other inhibitory proteins might be activated during mitosis to attenuate CLIP-190 association with microtubule ends. Phosphorylation of the EB1 homologue mal3p and its inhibitory effects on the interaction with the CLIP-190 homologue tip1p have been shown in fission yeast (Busch, 2004). It remains to be examined whether this phosphorylation is cell-cycle-regulated. Cell-cycle regulation might be important for releasing CLIP-190 for kinetochore function or preventing the plus-end-binding activity from interfering with CLIP-190 function at kinetochores. This report is the first to describe the cell-cycle regulation of the plus-end-binding of CLIP proteins. Elucidation of the precise mechanism and significance of this regulation may lead to further understanding of the temporal and spatial regulation of microtubules in cells (Dzhindzhev, 2005).
CLIP-190 localises to unattached kinetochores in mitosis. The localisation of CLIP proteins to kinetochores has been shown in mammalian, Drosophila and budding-yeast cells. Studies of CLIP-170 localisation to kinetochores in mammalian cells suggest an intricate physical and functional relationship with the dynein-dynactin complex. CLIP-170 binds directly to and requires Lis1 for kinetochore localisation. In turn, Lis1 interacts with multiple subunits of dynein-dynactin and is displaced from kinetochores when the motor complex is disrupted. Most of these studies relied on the overexpression of dominant-negative proteins (Dzhindzhev, 2005).
A previously unreported dependency was found, namely the requirement of Lis1 for dynein localisation to kinetochores. In mammalian cells, dynein is required for Lis1 localisation, whereas the overexpression of full-length or truncated Lis1 does not prevent dynein localisation to kinetochores. These results lead to the idea that Lis1 might be an auxiliary protein that bridges the dynein complex to cargo proteins. The RNAi results clearly indicate that Lis1 is required for dynein localisation to kinetochores. Combined with previous results, Lis1 and dynein seem to depend on each other for their localisation. This is the first report of such dependency in any eukaryote, and it gives the Lis1 protein a more integral part in dynein function (Dzhindzhev, 2005).
The results also suggest that microtubule attachment directly removes CLIP-190 from kinetochores rather than through spindle-checkpoint signalling. Dynein seems to be responsible for the removal of CLIP-190 from kinetochores in addition to its role in localising CLIP-190 to kinetochores. It has been shown that dynein removes several kinetochore proteins along microtubules upon the attachment of microtubules. This study provides the first evidence that a member of the CLIP family also utilises dynein-motor-activity to leave attached kinetochores. Interestingly, it was found that unlike in interphase, EB1 is not required for the mitotic localisation of CLIP-190 to unattached kinetochores. This is intriguing in the light of recent evidence (Tirnauer, 2002b) that EB1 associates with attached kinetochores when the kinetochore microtubules are polymerizing (Dzhindzhev, 2005).
In conclusion, these results indicate that CLIP-190 localisation is regulated during the cell cycle and requires distinct mechanisms in mitosis and interphase. Spatial and temporal regulation of CLIP-190 localisation probably play crucial roles in the regulation of microtubule dynamics and their interaction with other cellular structures (Dzhindzhev, 2005).
Regulation of microtubule polymerization and depolymerization is required for proper cell development. Two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. The data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. These findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization (Mennella, 2005).
EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. This study defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, the crystal structure of the EB1 COOH-terminal domain was solved. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity (Slep, 2005; full text of article).
This study has elucidated a motif shared by mammalian APC and several mammalian spectraplakin MACFs. Although these two families bear no apparent sequence homology outside the identified EB1 binding site, they do share a similar composite domain architecture and cellular functions. Both proteins are extremely large (1,000-5,000 amino acids) due to repeated domains in their central regions (ß-catenin and axin binding sites in APC and dystrophin-like spectrin repeats in MACFs. Both proteins also are characterized by microtubule binding motifs followed immediately after by the COOH-terminal EB1 recognition motif. In recent studies, both APC and the spectraplakins ACF7 and Shot have been shown to play roles in axon growth, cell polarity, and migration. In these studies, overexpression of the EB1 COOH-terminal domain resulted in a dominant-negative disruption of polarity signaling, indicative that an interaction with full-length EB1 is required for proper signal transduction/cytoskeletal stabilization by mammalian APC and spectraplakins. GSK-3ß, a kinase implicated in cell polarity signal transduction pathways, also has been implicated in the regulation of APC and MACFs. It is possible that this kinase might, at least in part, control polarity by regulating EB1 interaction because in vitro studies have shown that phosphorylation of APC abrogates binding to EB1 (Slep, 2005).
Interestingly, the two APC genes in Drosophila have the ß-catenin/axin binding domains, but lack the COOH-terminal region in mammalian APC that contains the EB1 binding motif. To date, Drosophila APCs have not been observed tracking along microtubule tips, although DmAPC2 has been found to tether mitotic spindles to cortical actin. Furthermore, the spectraplakin interaction with EB1 and microtubule tips appears to be widely conserved in all metazoans. Previous work has implicated the MACF Shot as an EB1-associated factor based on colocalization and coimmunoprecipitation, but a direct interaction has not been shown. This study definitively demonstrates that the in vivo association of Shot with the tips of microtubules is mediated by EB1. Thus, it is speculated that the spectraplakins may have represented the original metazoan invention of a microtubule tracking, cell polarity factor and that vertebrates subsequently incorporated this feature into APC by duplication and gene integration. Consistent with this idea, the EB1 binding motifs are more similar between vertebrate MACF and APC than they are between MACFs from vertebrates and invertebrates (Slep, 2005).
In addition to the EB1 interaction, localization studies suggest other features about the mechanism by which spectraplakins and APC interact with microtubules. Shot colocalizes only with the third of EB1 furthest from the microtubule plus end. This pattern, which is not observed for other proteins such as RhoGEFs, suggests that Shot does not coassemble with EB1 on the growing end of the microtubule but recognizes EB1 after a temporal delay, perhaps also suggesting some additional mechanism of regulation (Slep, 2005).
X-ray crystallographic studies show that the conserved EB1 COOH-terminal domain constitutes a unique coiled coil-four-helix bundle motif that confers both dimerization and cargo binding. Although EB1 and Bim1p were suggested to have a coiled coil domain based on sequence analysis, this is the first physical evidence that EB1 proteins are indeed dimers. Dimerization appears to rely not only on the coiled coil but also on the four-helix bundle motif, which provides nearly half of the buried surface area formed in dimerization. Consistent with this idea, destabilization of the four-helix bundle with the I224A mutation abolishes dimerization (Slep, 2005).
Alanine scanning mutagenesis, based on the crystal structure, was used to uncover a bivalent cargo binding site in the EB1 COOH-terminal domain. Both subunits of the homodimer appear to contribute residues to the cargo binding site, which is also supported by the finding that abrogating dimerization prevents cargo binding. Although the EB1 COOH-terminal domain is highly negatively charged, many of the key determinants in MACF2 binding are hydrophobic and form a discrete exposed hydrophobic pocket in the center of the domain. In comparison to the high conservation of EB1's COOH-terminal domain (from yeast to man), the repeat binding motifs found in APC and MACF2 are less well conserved, especially if one compares Drosophila to human. However, general features are the presence of several hydrophobic residues (mainly prolines) and a net positive charge. It is speculated that these prolines and other hydrophobics dock into EB1's hydrophobic pocket, whereas the charged residues may augment the binding interaction by providing a peripheral gasket (Slep, 2005).
The conservation of solvent-exposed residues in EB1's COOH-terminal domain extends from vertebrates down to the yeast homologues Bim1 and Mal3. Kar9 is functionally homologous to APC and Shot in its general role of microtubule search and capture; however, Kar9 does not have a repeat motif architecture similar to APC/spectraplakins. A weak segment of homology with hydrophobic character between regions of Kar9 and the EB1 binding motif has been reported, but this site has not been experimentally confirmed. It will be interesting to compare and contrast the molecular basis for the EB1-APC interaction and the Bim1-Kar9 interaction to determine if a similar or unique set of determinants govern these binding interactions (Slep, 2005).
The results also help to explain previous mutagenesis results of EB1 that were performed before the availability of a crystal structure. Wen (2004) mutated conserved hydrophilic residues within aa 211-229 in EB1 and found that the mutations E211A/E213A, E211A/D215A, K220A/R222A, and E211A/E213A/K220A/R222A inhibited EB1-APC interaction in vitro and yielded supporting phenotypic behavior in vivo in an mDia-mediated cell polarity pathway. The current results predict the strongest effect for constructs containing the K220A mutation and milder effects for the one with the E213A mutation, which agrees with the observations by Wen (2004). This correlation between EB1 mutants binding to MACF21595-1637 and APC also substantiates the likelihood of a similar binding architecture between these two classes of proteins and the EB1 COOH-terminal domain (Slep, 2005).
Several EB1 family members exist in metazoan genomes (four in Drosophila and three in humans. Although the crystal structures reported in this paper describe a homodimeric structure, the possibility that EB1 heterodimers exist and are used for diverse cargo recognition cannot be ruled out. The length of the coiled coil domains and the residues in the COOH-terminal domain that constitute the hydrophobic core and mediate dimer contacts are highly conserved across family members, especially within species. Because each bivalent cargo binding site is formed from residues contributed by each of the EB1 molecules, heterodimers could confer complex, pair-wise cargo recognition motifs. Thus, the possibility of whether or not heterodimers of EB1 can form in vitro and within cells merits investigation (Slep, 2005).
The EB1 COOH-terminal domain also may contain cargo binding sites other than the one that defined in this study for APC and MACF at the coiled coil-four-helix bundle junction. Several proteins, including the dynein-dynactin complex, Ncd, the microtubule-destabilizing kinesin Klp10A, and RhoGEF2, have been reported to track along microtubule tips and associate with EB1. However, only in the cases of the p150Glued dynactin subunit and Klp10A has a direct interaction with EB1 been shown. In the case of p150Glued, deletion studies have suggested that p150Glued and APC use overlapping yet unique binding sites on EB1's COOH-terminal domain. The p150Glued binding site maps to EB1's coiled-coil-four-helix bundle but also requires EB1's COOH terminus (13 residues not included in the crystallization construct). Thus, it is possible that EB1 contains multiple cargo binding sites, some of which might be independent and others which might be mutually exclusive. The crystal structure reported in this study should provide a valuable tool for probing these future questions concerning EB1-cargo interactions (Slep, 2005).
Microtubule plus end binding proteins (+TIPS) localize to the dynamic plus ends of microtubules where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1 and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. This study reports crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically-induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. It is proposed that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end (Slep, 2007).
This work analyzes the biochemical mechanisms of three plus end tracking protein families (EB1, CLIP-170 and XMAP215) using x-ray crystallography, in vitro tubulin binding/assembly assays, and in vivo plus end tracking assays. Five crystal structures are presented of +TIP tubulin binding domains across multiple species and families illustrating structural conservation within families and functional convergence across families. In vitro and in vivo analysis reveal an underlying general theme to these unrelated domains. While tubulin binding can be conferred by a single +TIP domain, the promotion of microtubule nucleation and polymerization in vitro and plus end tracking in vivo require multiple domains acting in concert. In the case of EB1 and CLIP-170 members, two domains are required for robust plus end localization and these two structurally unrelated domains can even cooperate if artificially joined together to achieve this activity. XMAP215 members are more complicated, perhaps due to the relative weaker association between TOG domains and tubulin in higher eukaryotic members. However, multimerization of TOG domains clearly facilitates tubulin nucleation in vitro and overall microtubule association in living cells. From these data, a model is presented in which unrelated tubulin binding domains have convergently evolved a similar mechanism for enhancing tubulin assembly on growing microtubule ends by acting as multivalent tubulin polymerization chaperones (Slep, 2007).
The tubulin binding domains of these three +TIP proteins are quite diverse in their architecture and show no evidence of a common evolutionary origin. However, the structural conservation within a +TIP class is very high, as evidenced by the near superimposition of yeast Bim1p with human EB1 and yeast Stu2p with Drosophila Mini spindles. The different architectures also suggest varying interactions modes with tubulin and potential synergy between the +TIPs. XMAP215 has a very flat binding interface created by a series of rigid, highly conserved loops on one face of the TOG domain; the length of the TOG domain is fairly closely matched to a tubulin monomer (α or β) and likely makes extensive contacts through a combination of hydrophobic and electrostatic interactions. In contrast to the elongated TOG domain, the CH domain of EB1 is spherical and approximately half the size. Mutagenesis suggests that several residues around one hemisphere contribute to tubulin binding. This data is consistent with recent work suggesting that EB1 might nestle in the groove between tubulin protofilaments. CLIP-170 also has a globular structure and the data suggests that it might bind tubulin’s disordered C-terminal tail as part of its binding interface (Slep, 2007).
Comparison of the +TIPS and protein engineering studies suggest a considerable structural variation in how +TIP proteins can be interconnected to achieve their activities in plus end tracking and promoting tubulin assembly. In some cases, tubulin binding domains are arrayed in tandem along a polypeptide chain with presumably unstructured linkers in between (e.g., Mini spindles, Ch-TOG, CLIP-170). The cis linking domains within a polypeptide appears to be critical, since it is shown that TOG domains added in trans do not reconstitute function. In other cases, single α/β tubulin binding domains are found in a polypeptide and multivalent tubulin binding is achieved by polypeptide dimerization [e.g., EB1 family and Bik1 (the CLIP-170 homologue from yeast)]. Hybrid strategies are also employed (e.g. CLIP-170, which has tandem Cap-Gly domains and is dimerized via a coiled coil and Stu2p which has tandem TOG domains, a dimerization domain and an additional C-terminal microtubule binding domain). It was also found that native dimerization sequences are not essential, as a variety of artificial dimerization strategies (e.g. GCN4 leucine zippers, gluthione S-transferase, and FKBP-rapamycin-FRB) are capable of reconstituting +TIP protein function (Slep, 2007).
Collectively, these studies reveal structural variation in how multiple +TIP tubulin binding domains can be combined to promote tubulin oligomerization for microtubule assembly. However, cis versus trans arrangements of +TIP domains may produce certain unique outcomes. For example, the linear arrays of TOG domains in the extended XMAP215 structure may generate a pseudo protofilament-like arrangement that is particularly effective for microtubule nucleation. Indeed, XMAP215 has a potent microtubule nucleation ability compared with CLIP-170, potentially related to an in vivo nucleation activity given XMAP215’s TACC-dependent localization to the centrosome. Analogous mechanisms exist for the actin cytoskeleton where the Spire protein utilizes several arrayed actin binding domains to template the nucleation of an actin filament (Slep, 2007).
The data show that single +TIP tubulin binding domains do not promote microtubule nucleation or growth in vitro (and in some cases are inhibitory), nor do they localize to microtubule plus ends in vivo. In contrast, multimerized +TIP tubulin binding domains are potent microtubule nucleator in vitro, promote microtubule growth in vitro and are requisite in vivo for microtubule plus end localization. For EB1 and CLIP-170, it was dynamically shown that plus end tracking requires an ability to bind more than one tubulin subunit. These results suggest a model in which +TIPs bind multiple tubulin dimers in solution and then deliver these larger tubulin oligomers to the ends of microtubules. This general idea was first introduced two decades ago by (Gard, 1987) to explain the high rates of tubulin assembly induced by XMAP215. Multimerization overcomes the inherent polymerization barrier tubulin heterodimers face due to single longitudinal and lateral tubulin:tubulin affinities estimated to be on the order of mM and M respectively. +TIP-induced multimerization of tubulin would increase the effective affinity for the microtubule lattice through cooperative binding, thereby decreasing the critical concentration for polymerization. By stabilizing tubulin-tubulin interactions prior to their full incorporation into a mature, cylindrical lattice, the +TIPs would act as polymerization chaperones. Physiologically, such chaperones would become particularly important for enhancing the growth of microtubules when free tubulin dimers are below the critical concentration of microtubule assembly or when microtubule destabilizing proteins are active (e.g in mitosis). This mechanism also enhances the spatial and temporal regulation of microtubule assembly in the cell, in part by regulating the localization and/or activity of +TIPs without modifying tubulin, the basic unit of polymerization (Slep, 2007).
Support for this general model comes from a number of other laboratories. Tandem Cap-Gly domains in CLIP-170 have been shown to plus end track whereas single Cap-Gly domains of the homolog CLIP-115 show greatly reduced microtubule association. Optical trapping studies analyzing microtubule growth showed step-wise growth that increased in size in the presence of full length XMAP215, a result they interpreted as XMAP215-facilitated incorporation of tubulin oligomers onto the microtubule end (Slep, 2007).
An apparent exception to the multiple tubulin binding rule for +TIPs is the CLASP family, which contains only one TOG domain at its N-terminus. However, informed by TOG structures and secondary structure predictions several TOG-like (TOGL) domains can be identified in the CLASP family. Two additional dodeca-helical domains are predicted with alternating loops that exhibit high homology to TOG domain intra-HEAT loops. If folded into a HEAT-like structure, the conservation profile of alternating loops would be localized to one face of the domain, suggesting that TOGL domains may bind tubulin by a mechanism similar to what is described in this study for the TOG domain. Thus, although poorly conserved at the primary structure level, XMAP215 and CLASP appear to be ancient +TIP relatives that may employ a similar general strategy for plus end tracking. Support for CLASP’s possible polymerization chaperone role comes from studies that observed CLASP-dependent microtubule subunit incorporation into fluxing kinetochore fibres (Slep, 2007).
The model for +TIPs as multimeric tubulin chaperones undoubtedly oversimplifies the complexity of interactions that are occurring at the microtubule plus end in vivo. +TIP families show divergent effects on microtubule dynamics, some promoting growth while others act as anti-pause, destabilization, rescue and perhaps even nucleation factors. Some of these differences in activity may reside in variations in the affinity constants of +TIP domains with tubulin monomers, oligomers and microtubules. As an example, non-EB1 family microtubule-binding CH domain proteins, including CLAMP and HEC1 appear to have higher affinity for the microtubule lattice than EB1 and localize along the entire length of the microtubule rather than just at plus ends. The spatial arrangement of +TIP domains, arrayed versus dimerized, may lead to distinct effects on microtubule dynamics by preferentially stabilizing lateral versus longitudinal tubulin-tubulin interactions. Finally, the intricate web of +TIP-+TIP interactions may generate unique outcomes on tubulin assembly. Multi-component in vitro assays, high resolution EM analysis of microtubules, and x-ray structures of +TIP-tubulin complexes will be required to further understand how +TIPs regulate the microtubule lattice (Slep, 2007).
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