short stop/kakapo
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 (Rogers, 2002) 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).
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).
Axon extension and guidance require a coordinated assembly of F-actin and microtubules as well as regulated translation. The molecular basis of how the translation of mRNAs encoding guidance proteins could be closely tied to the pace of cytoskeletal assembly is poorly understood. Previous studies have shown that the F-actin-microtubule crosslinker Short stop (Shot) is required for motor and sensory axon extension in the Drosophila embryo. This study provides biochemical and genetic evidence that Shot functions with a novel translation inhibitor, Krasavietz (Kra, Extra bases, Exba), to steer longitudinally directed CNS axons away from the midline. Kra binds directly to the C-terminus of Shot, and this interaction is required for the activity of Shot to support midline axon repulsion. shot and kra mutations lead to weak robo-like phenotypes, and synergistically affect midline avoidance of CNS axons. shot and kra dominantly enhance the frequency of midline crossovers in embryos heterozygous for slit or robo, and in kra mutant embryos, some Robo-positive axons ectopically cross the midline that normally expresses the repellent Slit. Finally, Kra also interacts with the translation initiation factor eIF2β and inhibits translation in vitro. Together, these data suggest that Kra-mediated translational regulation plays important roles in midline axon repulsion and that Shot functions as a direct physical link between translational regulation and cytoskeleton reorganization (Lee, 2007).
Kra and its human homolog BZAP45 contain an N-terminal leucine-zipper
domain of unknown function and a C-terminal W2 domain. This study shows that Kra
can bind to eIF2ß through its W2 domain and inhibit translation in vitro.
It is very likely that Kra competes with eIF5 and eIF2Bepsilon for the common
binding partner eIF2ß, thus inhibiting the assembly of functional
preinitiation complexes. A similar mode of translation inhibition has been proposed for DAP-5/p97, which may compete with its homolog eIF4G for eIF3 and eIF4A, thus reducing both cap-dependent and -independent translation. However, the step in translation initiation that is regulated by Kra remains to be addressed experimentally (Lee, 2007).
Kra-mediated translational repression appears to be an important mechanism
underlying midline axon guidance. In the kra mutant embryos, Fas
II-positive CNS axons that normally remain ipsilateral cross the midline
ectopically. This phenotype is observed with the pCC axons from early stages
(stages 12 and 13) of axogenesis when they pioneer one of the Fas II pathways.
The introduction of multiple alanine substitutions (12A and 7A) into Kra
significantly reduces its ability to bind eIF2ß and abolishes its
activity to rescue the kra mutant phenotype, suggesting that the
function of Kra in axon guidance depends on its interaction with eIF2ß.
Consistent with this conclusion, mutations in the eIF2ß gene
also lead to the ectopic midline crossing of Fas II-positive axons (Lee, 2007).
There is a growing body of evidence that F-actin and microtubules are
coordinately assembled to each other during axon extension and guidance.
Interactions of filopodial actin bundles and microtubules are key features of
filopodial maturation into an axon and of growth cone turning. Shot, a
conserved molecule that scaffolds F-actin, microtubules and the microtubule
plus end-binding protein EB1, is a strong candidate to bring microtubule plus
ends into contact with F-actin bundles. Indeed, Shot is required for the
extension of sensory and motor axons, and a mammalian homolog of Shot, ACF7, is
required for microtubules to track along F-actin cables towards the leading
edge of spreading endodermal cells. Thus, previous studies have suggested that
Shot/ACF7 coordinately organizes F-actin and microtubules to support the
motility of neuronal growth cones and nonneuronal cells (Lee, 2007).
These findings suggest that Shot also functions together with the translation
inhibitor Kra to control midline axon repulsion. Shot physically associates
with Kra in vivo. The shot loss-of-function phenotype at the CNS
midline is reminiscent of the kra loss-of-function phenotype. The
major Kra-binding domain in Shot is required for its role in midline axon
repulsion. Moreover, shot and kra genetically interact in a
dosage-sensitive manner for the midline phenotype. These data also support the
idea that cytoskeletal assembly and translational regulation can occur in a
coordinated way. Midline axon repulsion requires both the
activity of Kra to recruit eIF2ß and the activity of Shot to bind to
F-actin. Thus, it is likely that local levels of eIF2ß available for
protein synthesis can be spatially regulated with regard to actin cytoskeleton
remodeling during axon guidance (Lee, 2007).
In Drosophila, Slit is the key ligand driving midline axon repulsion, and therefore midline crossing of CNS growth cones is primarily controlled by the Robo receptor of Slit. How then do neurons regulate levels of Robo on the surface of their axons and growth cones? The transmembrane protein Commissureless (Comm) has been shown to dynamically regulate Robo expression. Comm functions as an intracellular sorting receptor to target newly made Robo for lysosomal degradation, thereby blocking its transport to the growth cone that is crossing the midline (Lee, 2007).
Translational regulation has also been shown to alter the responsiveness of
growth cones to the midline repellents. In vitro
studies of cultured embryonic retinal ganglion cells (RGCs) provided an
insight into how translation is regulated in axons and growth cones in
response to midline guidance cues. Treatment of these neurons with netrin-1
leads to the rapid activation of signaling pathways that phosphorylate the
translation initiation factor eIF4E and its binding protein eIF4E-BP1 and thus
induces axonal protein synthesis. The data presented in this study indicate that the role of Kra in midline axon repulsion depends on its ability to recruit the translation
initiation factor eIF2ß to Shot. Thus, protein complexes containing Shot,
Kra and eIF2ß may function as additional targets for signaling systems
that critically control axon guidance at the CNS midline. Regulation of
Shot-Kra-eIF2ß complexes may occur in neuronal cell bodies, where Kra is
concentrated, or in axons and growth cones, which may require local protein
synthesis to meet developmental requirements. In the latter case, since Kra is
not detectable in the CNS axons of the Drosophila embryo, even a low
amount of Kra may be sufficient for guiding axons (Lee, 2007).
Intriguingly, Robo was aberrantly detected on commissural axons in
kra1/kra2 mutant embryos. Given the increased
frequency of ectopic crossovers in these embryos, as well as the documented
role of Robo in preventing axons from crossing the midline, this
finding is somewhat paradoxical. One possibility is that Kra, induced by
interaction with Shot and eIF2ß, could repress the synthesis of as yet
unidentified proteins that transduce or modulate Robo signaling. In
shot or kra mutant embryos, perhaps this translational
regulatory circuit is not activated, and thus Robo-expressing growth cones
abnormally cross the midline because of a decrease in the strength of Robo
signaling output. Alternatively, Kra may function to finely tune the
expression levels of their multiple targets that mediate attractive or
repulsive responses. In this scenario, impairment of the Shot-Kra-eIF2ß
circuit could disturb the precise balance between repulsion and attraction
signaling at the midline, thereby decreasing the overall sensitivity of
Robo-expressing growth cones to Slit. Therefore, efforts to reveal the direct
targets of Kra-mediated repression in the future may provide better insights
into the immediate mechanisms by which translational regulation plays an
essential role for midline axon repulsion (Lee, 2007).
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