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).
The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. Short stop (Shot), the sole Drosophila spectraplakin, os a model actin-microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, little is known about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. This study describes the intracellular dynamics of Shot and a structure-function analysis of its role as a cytoskeletal cross-linker. Shot was found to interact with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH(2)-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. The results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk (Applewhite, 2010).
This analysis revealed that Shot is able to interact with microtubules via two distinct mechanisms, association with the microtubule lattice mediated by the GAS2 domain of Shot, and an interaction with growing plus ends that is mediated by EB1, through multiple EB1-interacting motifs at the COOH terminus of Shot. The interaction of Shot with the microtubule lattice, but not with the plus end, requires an intact network of F-actin and a functional actin-binding domain at its NH2 terminus. The data also demonstrate that the actin- and microtubule-binding domains cooperate to regulate microtubule dynamics by cross-linking the two cytoskeletal networks. Shot cross-linking couples microtubules to actin in order to resist motor-mediated lateral sliding movements. The data suggest that Shot can exist in at least two different states. In the first state, observed after actin depolymerization or with the C isoform that exhibits low affinity for actin, Shot preferentially interacts with microtubule plus ends. In the second state, observed with the A, B, or Shot-ΔRod isoforms in the actin-rich region of the peripheral lamella, Shot is bound to the microtubule lattice via the GAS2 domain. The results suggest that the pool of Shot that is actively serving as a cross-linker is the lattice-bound population. It is speculated that the interconversion between these two states represents a regulated conformational change, so that the timing and placement of Shot-mediated cross-linking is precisely controlled within the cell. Understanding how Shot is regulated will be an important next step, but several potential mechanisms immediately present themselves. First, it is interesting to note that the Shot-GAS2-CTD construct decorated the microtubule lattice despite the presence of the EB1-interaction motif. This result suggests that GAS2 exerts a dominant effect when present in the same molecule and further suggests that it must be actively repressed when Shot is at microtubule plus ends. The hypothesis is favored that Shot is regulated by an intramolecular interaction that simultaneously represses the GAS2 and actin-binding CH domains during tip tracking. On activation, the molecule opens and is able to bind the lattice and actin. Second, the EF-hand motifs are closely positioned to the GAS2 domain, suggesting that calcium may participate in regulation of microtubule lattice binding. Third, there is evidence that Shot may act downstream of the small GTPase Rho; perhaps activation of this pathway leads to activation of the cross-linking activity. Fourth, binding of the CH domains to actin may itself be sufficient to trigger the transition from tip-tracker to lattice-binder. Understanding when and where Shot is activated to cross-link will be key to understanding its cellular functions (Applewhite, 2010).
Why does Shot possess these dual mechanisms (tip vs. lattice) for microtubule interaction? Microtubule dynamic instability is used as a mechanism to search intracellular space for docking and stabilization during kinetochore microtubule capture and polarization of migrating cells. Perhaps association with the microtubule plus end allows spectraplakins to search for activating inputs at the cell cortex or in actin-rich subregions of the cell. Different modes of microtubule association may also allow spectraplakins to regulate microtubule dynamics in different ways. A common theme emerging from studies of other +TIPs is that many of them function is as regulators of microtubule dynamic instability. Because the majority of subunit addition and loss occurs at the plus end, +TIPs are perfectly positioned to influence the conformation of tubulin at this site. This study presents evidence that Shot also can influence microtubule dynamics through a different mechanism, by cross-linking microtubules to the actin network to prevent lateral microtubule displacement by motor proteins (Applewhite, 2010).
Previous studies of the role of mouse ACF7 have clearly demonstrated a role for this protein as a 'guide' to direct microtubule growth along actin stress fibers to assist them in targeting to focal adhesions (Wu, 2008). The current data show that, in S2 cells, Shot-mediated cross-linking performs an additional function by cross-linking microtubules to actin in order to resist whip-like lateral displacements by motor proteins. Many Drosophila cell types exist without a dominant interphase microtubule organizing center, instead organizing acentriolar networks to organize their cytoplasm. It is speculated that, in the absence of a structural anchor at the minus end, acentriolar cells rely upon actin-microtubule cross-linking to maintain their organization and to resist forces produced by motor proteins. These forces may be generated during organelle transport, especially if the motors associated with the surface of one organelle engage multiple microtubules. Shot is, therefore, likely to play an important role in maintaining microtubule organization for the efficient motor-mediated delivery of organelles and other cargo to specific destinations within the cell. Furthermore, this activity is consistent with the observation that the microtubule network in mushroom body neurons in Shot mutants exhibits gross disorganization and an overall loss of microtubule polarity. Based on the current data a speculative model has been developed in which 'inactive' Shot associates with a growing microtubule plus end by its interaction with EB1. Dynamic instability allows microtubules to probe the cytoplasm for an activating signal in a 'search-and-capture' mechanism. On receiving the activating stimulus, Shot changes its conformation, releases from EB1, and engages actin with is dual CH domains and the microtubule lattice with its GAS2 domain. In this conformation, it is able to cross-link microtubules to actin to influence their growth trajectory or to stabilize the network against forces produced by motors. Testing these ideas will require careful observation of Shot dynamics in the developing embryo where cells are responding to extracellular cues, as well as genetic analyses to determine how the EB1-Shot interaction contributes to development (Applewhite, 2010).
Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors. The localization pattern of Spectraplakin, Drosophila Short stop, in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin's gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test. In summary, a unique localization of Spectraplakin was found in photoreceptors, and the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions was identified through genetic mutational analysis. These data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis (Mui, 2012).
This study investigated where Shot localizes compared to apical membrane domain, adherens junction, stable microtubule, and rhabdomere, in mid-stage pupal photoreceptors. The localization results of Shot in pupal eyes strongly indicate that Shot localizes in between the adherens junctions, at the basal side of the apical domain, and at the apical side of the stable microtubules. The rhabdomere terminal web (RTW), where the Shot localizes, may be the interface where the stable microtubules and F-actins of rhabdomere meet together. Since Shot has an actin-microtubule cross-linking activity, Shot might cross-link the two cytoskeletons of actin and microtubules at the RTW (Mui, 2012).
This genetic interaction data of shot and crb strongly suggests that Shot may provide an additional cue for Crb in photoreceptor development because the rough-eye phenotype caused by Crb overexpression was further enhanced by reduced shot gene dosage (shot/+). The relationship between crb and shot might be one of the following possibilities; (1) shot acts at the upstream of crb, (2) shot acts at the downstream of crb, or (3) shot and crb control the parallel pathway in photoreceptor development. From comparative genetic analysis, Crb and Shot require each other reciprocally to localize at their target sites of rhabdomere stalk (apical domain) and RTW (Mui, 2012).
Genetic analysis of the shot mutation strongly indicates that Shot modulates the apical Crb membrane domain during rhabdomere elongation. The apical membrane modulation activity of spectraplakin was further confirmed by spectraplakin overexpression which caused a dramatic misplacement of the apical membrane domain. It is postulated that the spectraplakin might affect the actin (rhabdomere) and/or microtubules based on its dual binding capacity for actin/microtubule, and its activity as an actin/microtubule cross-linker. Therefore, its role in photoreceptor morphogenesis might require its dual actin/microtubule binding, which will be supported by the absence of Crb-mislocalization activity of ShotC, which lacks the actin-binding domain. The Crb-mislocalization activity of ShotA might be based on its dual actin/microtubule binding ability (Mui, 2012).
One important point of the potential cross-talk between the crb and shot is their different spatial localizations in photoreceptors. How does one protein affect the other that is at a different localization in the cell? There might be at least several following possibilities; (1) a potential interaction when they co-localize during the trafficking before their final targeting, (2) a potential interaction in previous developmental time, or (3) a potential interaction at the interface where the two subcellular compartments meet. The 'RTW' is the place where the microtubules and F-actins (rhabdomere) meet each other. Therefore, the Shot might have a potential role in the regulation of stable microtubules and rhabdomere, and thereby the localizations of Crb and adherens junctions might be affected in photoreceptor cells (Mui, 2012).
Shot has a microtubule organizing activity. Therefore, the expected result in the loss of shot is the defects of stable microtubules which were observed in the loss-of-function study of shot mutation. Furthermore, the stable microtubules were mostly defected in the gain-of-function of ShotA-GFP overexpression. Therefore, the primary target of Shot seems to be the stable microtubules. Then, the defected microtubules may further affect the Crb and E-cad through the microtubule-based trafficking and/or other microtubule-based cell polarity. But the other possibilities of direct targeting of Shot toward Crb/E-cad or actin-based cell polarity cannot be excluded. ShotA-GFP overexpression results in ectopic localization of acetylated-tubulin (Acetub; stable microtubulin) around the cells which could be caused by the direct binding of ShotA-GFP to the Acetub. However, another possibility of the indirect mislocalization of Acetub caused by the mislocalized 'RTW' by the ShotA-GFP cannot be excluded (Mui, 2012).
Another potential possibility of Spectraplakin's function in the regulation of apical membrane domain might be through microtubule plus-end-tracking proteins (+TIPS). The +TIPS belong to the class of microtubule-associated proteins, and link microtubule ends with apical actin cytoskeleton. In Drosophila muscle-tendon junctions, Shot regulates microtubule cytoskeleton by forming a complex with the EB1 and APC of +TIPS. Therefore, there is a potential possibility of Shot and +TIPS interaction in apical domain control during photoreceptor morphogenesis (Mui, 2012).
This study has shown that Spectraplakin/Shot is required for correct localization of Crb, adherens junctions, and stable microtubules in the photoreceptors and disruption of Shot function affects photoreceptor morphogenesis. The data strongly suggests that Spectraplakin/Shot plays important functions in the modulation of cell membrane domains including the apical Crb domains of photoreceptors during pupal eye development. Evolutionary conservation in the structure and function of eye morphogenesis genes makes the Drosophila eye an excellent model for studying the genetic and molecular basis of retinal cell organization. Future work will help to uncover other genes that might affect the Crb positioning during the extensive morphological growth phase of the Drosophila pupal eye. Given the high degree of evolutionary conservation of Crb and Spectraplakin genes from Drosophila to higher mammals including humans, similar cooperative mechanism between Crb and Spectraplkain could have a role in the development and degeneration of human photoreceptor (Mui, 2012).
The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. This study shows that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. Binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, it is reported that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From these data a model is deduced that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth (Alves-Silva, 2012).
The regulation of MT networks is essential for many neuronal functions and processes, ranging from axonal growth to neurodegeneration. However, understanding how MTs are regulated in neurons remains a major challenge. Many MT-binding proteins have been identified and their potential roles in regulating the key processes of MT stabilization, MT polymerization, MT-based transport and MT-actin cross talk have been highlighted. But how these different functions merge into coordinated MT network regulation is poorly understood (Alves-Silva, 2012).
This work indicates spectraplakins as key integrators of different MT regulatory processes (see Model of Shot function in neuronal MT network organization). First, Shot stabilizes MTs, a role generally assigned to structural MAPs, such as Tau, MAP2 or MAP1b. Second, Shot regulates MT polymerization dynamics and guides them in the direction of axonal growth. In this function, Shot interacts with EB1, firmly establishing Shot-EB1 interaction as an important determinant of microtubule guidance as a crucial mechanism underpinning axon growth. In addition, Shot acts as an actin-MT linker during axonal growth, adding a third crucial MT regulatory role to the list. Therefore, spectraplakins work at the cross-roads of structural MAPs, +TIPs and actin-binding proteins, strongly suggesting that work on spectraplakins will provide exciting new opportunities to unravel the complexity of MT regulation in neurons (Alves-Silva, 2012).
At the molecular level, this work reveals the importance of the Ctail for Spectraplakin function. While GRD had previously been established as the domain of Shot which has the widest functional requirements, the current work suggested Ctail to be similarly important, as deduced from its crucial roles both in MT stabilization and guidance and its requirements in both neurons and tendon cells (Alves-Silva, 2012).
Shot localization along the shafts of axonal MTs is important for MT-stabilization, suggesting that spectraplakins might functionally overlap with structural MAPs. This finding might have important implications. For example, loss of structural MAP functions has relatively mild phenotypes even in double knock-out mice, and this could be caused by functional compensation through spectraplakins. It remains to be seen whether such potential functional overlap is general or context-specific. For example, different MAPs and spectraplakins might display individual traits, such as dependence on different MT modifications or selective antagonism to different destabilizing factors (Alves-Silva, 2012).
In support of these findings with Shot, MT-stabilizing roles of spectraplakins appear conserved in mammals. Thus, MT-stabilizing roles in neurons have similarly been reported for the Shot homolog BPAG1. Furthermore, axon shortening caused by knock-down of ACF7 in primary cortical neurons or N2A cells was rescued by taxol application. In agreement with these findings, also the domain requirements underlying MT stabilization are conserved. Thus, studies in fibroblasts have shown that related C termini of the mammalian spectraplakin ACF7/MACF1 and of human Gas2-like1/hGAR22 and Gas2-like2/hGAR17 all display MT-stabilizing properties that are dependent on their GRDs, and in each case the Ctails enhance their MT association. Therefore, this mechanistic principle appears conserved across a range of homologous proteins, and this work has provided first experimental proof that it is functionally relevant in vivo, such as in growing axons and tendon cells (Alves-Silva, 2012).
Despite their obvious functional conservation, Ctails are not conserved at the protein sequence level but display other commonalities instead. They are all unlikely to form an ordered secondary or tertiary structure, they all display a high content of arginines, serines and glycines, and most contain MtLS motifs. This study has identified a role for the arginines of Ctail and proposes that this positive net charge attracts Ctail to negatively charged MTs. Such a mechanisms is consistent with other models for MT association and can explain why Ctails are not conserved at the amino acid sequence level. In support of this model, the C terminus of mouse ACF7 has recently been demonstrated to detach from MTs, when negative surface charges of MTs were enzymatically removed (Alves-Silva, 2012).
The second subcellular role of Shot in MT guidance establishes EB1 and the EB1-Shot complex as important determinants of axonal growth. In the absence of Shot-EB1 complex function, MTs are disorganized and axons extend shorter. Similar phenotypes of curled, criss-crossed MTs correlating with axon shortening have been described in mammalian neurons lacking ACF7 function but also defective for Dynein/Lis1, GSK3 (as a regulator of APC downstream of Wnt3a/Dvl1 and of CLASP), or Spinophilin/Doublecortin (see Drosophila Spinophilin). The favored explanation for why MT disorganization attenuates axon growth is that MTs are less efficient in pushing along the axonal axis in the direction of axon growth. Notably, it has been shown previously that roles of Shot in MT guidance and axon growth also essentially require its linkage to actin. It is therefore proposed that Shot performs its MT guiding roles by linking MT plus ends to actin structures, such as the actin cortex in the axons or actin networks or bundles in growth cones. Such a function of spectraplakins is likely to coexist with parallel mechanisms of actin-MT linkage. For example, the F-actin-binding factor drebrin was shown to interact with EB3, and this interaction is likewise believed to be required to steer MT polymerization events in elongating axons (Alves-Silva, 2012).
Current models propose that +TIPs compete among each other for interaction with EB1 at MT plus ends. Other +TIPs, such as CLASP, APC, Dynein/Lis and CLIPs, are present in neurons and contribute to axonal growth regulation. Therefore, the +TIP functions of spectraplakins identified in this study can now be analyzed in the context of other +TIPs, providing new opportunities to gain an understanding of regulatory +TIP networks in the context of axonal growth (Alves-Silva, 2012).
Shot works at the cross-roads of different mechanisms of MT regulation, and this study has unraveled the underpinning mechanisms using a genetically and experimentally amenable and functionally well conserved Drosophila model of axon growth. These findings do not only advance the principal understanding of cytoskeletal regulation during axonal growth, but can be extrapolated to other functions of spectraplakins. Thus, spectraplakins play roles in clinically relevant cellular processes including neurodegeneration, skin blistering and cell migration in wound healing and brain development. The two modes of Shot function this study has proposed may very well be applicable. For example, the model for MT guidance displays interesting commonalities with models for ACF7 function in migrating keratinocytes during wound healing, where ACF7 is suggested to guide MTs along actin stress fibers to focal adhesions. Furthermore, MT-stabilizing roles are likely to explain Shot function in Drosophila tendon cells. Just like MT-stabilizing roles of Shot in axons and fibroblasts, GRD and Ctail are essential in tendon cells, where they enhance each others localization, whereas MtLS motifs and actin-binding calponin-homology domains of Shot are dispensable. Notably, tendon cells have been proposed as a cellular model for support cells of the vertebrate inner ear that are known to express the Shot homolog BPAG1. Therefore, the subcellular mechanisms of spectraplakins described in this study do not only have implications for the understanding of axonal growth but also for their other functions in neurons and non-neuronal cells (Alves-Silva, 2012).
Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. This study uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, a novel mechanism is proposed for maintaining myonuclear morphology, and its critical link to correct levels of nuclear factors in differentiated muscle fibers is revealed. These findings may shed light on the underlying mechanism of various muscular dystrophies (Wang, 2015).
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