Jaguar physically intereacts with Miranda. Anti-Miranda antibodies were used to extract protein complexes from Drosophila embryos and more than 90% of Miranda was immunoprecipitated. In addition to a quadruplet of Miranda isoforms identified by mass spectrometry, two proteins of approximately 250 and 140 kDa were reproducibly immunoprecipitated with anti-Miranda antiserum. Mass spectrometry determination of tryptic peptide sequences and a database search identified these proteins as the nonmuscle myosin II Zip and the unconventional myosin VI Jar. Immunoprecipitation with anti-Jar antibodies further verified that Jar is associated with Miranda. In addition to Jar, Staufen, Prospero, and prospero mRNA were also coimmunoprecipitated with Miranda using anti-Miranda antibodies, demonstrating in vivo association of Miranda with myosin VI Jar as well as several components that require Miranda for basal localization (Petritsch, 2002).
To further examine the role of Jar in asymmetric division, its distribution was compared with the dynamic Miranda localization in neuroblasts. Miranda appears around the apical cortex and in the cytoplasm in early prophase, in the cytoplasm and on the cortex in late prophase, and is translocated at metaphase to a tight basal crescent, as well as to puncta around the aster microtubules and a faint lining of the microtubules of the mitotic spindle. In telophase, Miranda is mainly inherited by the ganglion mother cell (Petritsch, 2002).
Jar is localized in small particles mainly in the cytoplasm and less frequently at the cortex in a dynamic pattern. The density of Jar particles in neuroblasts is highest in prophase and metaphase, coinciding with Miranda translocalization and spindle rotation. Jar particles accumulate preferentially to the basal half in 45% of metaphase neuroblasts, whereas they are more homogeneously distributed in the rest. In telophase, Jar particles are inherited by the ganglion mother cell preferentially but are also seen in the neuroblast. This dynamic pattern is reminiscent of the linear movement of Jar-containing particles in syncytial blastoderm embryos (Mermall, 1994). The partial overlap between Jar and Miranda puncta in the cytoplasm in prophase and in the cytoplasm and at the cortex in metaphase and telophase is consistent with a role for Jar in the basal translocation of Miranda. Given that Jar interacts with a number of proteins besides Miranda (Buss, 2001; Geisbrecht, 2002), it is perhaps to be expected that Jar does not colocalize with Miranda to a greater extent. Jar has never been seen concentrated to a tight basal crescent, suggesting that Jar itself is most likely not an anchor for the cell fate determinants at the basal cortex. The transient accumulation of Jar particles on the basal side in metaphase neuroblasts is consistent with its involvement in the transport of Miranda to the basal pole (Petritsch, 2002).
A 195-kD protein coimmunoprecipitates with Jaguar. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homolog identified of cytoplasmic linker protein (CLIP)-170, a protein that links endocytic vesicles to microtubules. This protein has been named CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between Drosophila CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. Jaguar and CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They also colocalize at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homolog of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, suggests that these two proteins may functionally link the actin and microtubule cytoskeletons (Lantz, 1998).
Because 95F myosin has been implicated in transport and the vertebrate homolog of Drosophila CLIP-190, CLIP-170, is suspected of being involved in transport, the colocalization in axons, where vesicle/organelle transport along both actin and microtubules has been observed, is particularly intriguing. The particulate distribution of both Jaguar and CLIP-190 in nerve processes in the embryo and also in cultured cells from Drosophila embryos is consistent with these structures being vesicles. CLIP-170 has been suggested to participate in loading endocytic vesicles on to the plus ends of microtubules. No previous data exist that suggest an interaction of CLIP-170 with the actin cytoskeleton. However, the plus ends of peripheral microtubules to which the CLIP-170-associated vesicles appear to bind are adjacent to the actin filament-rich cortex. One function for CLIP-190 and Jaguar in the same structures may be to coordinate the transport of specific cargoes along both microtubules and actin filaments to facilitate their proper localization in the cell (Lantz, 1998).
Myosin VI localizes to the leading edges of growth-factor-stimulated fibroblast cells and has been suggested to be involved in cell motility. There has been no direct test of this hypothesis, however. Drosophila melanogaster MyoVI is expressed in a small group of migratory follicle cells, known as border cells. Depletion of MyoVI specifically from border cells severely inhibits border cell migration. Similar to MyoVI, E-cadherin is required for border cell migration. E-cadherin and Armadillo (Arm, Drosophila beta-catenin) protein levels are specifically reduced in cells lacking MyoVI. In addition, MyoVI protein levels are reduced in cells lacking DE-cadherin or Arm. MyoVI and Arm co-immunoprecipitated from ovarian protein extracts. These data suggest that MyoVI is required for border cell migration where it stabilizes E-cadherin and Arm. Mutations in MyoVIIA, another unconventional myosin protein, also lead to deafness, and MyoVIIA interacts with E-cadherin through a membrane protein called vezatin. Multiple biochemical mechanisms may exist, therefore, for cadherins to associate with diverse unconventional myosins that are required for normal stereocilium formation or maintenance (Geisbrecht, 2002).
Echinoid (Ed) is a homophilic immunoglobulin domain-containing cell adhesion molecule (CAM) that localizes to adherens junctions (AJs) and cooperates with Drosophila epithelial (DE)-cadherin to mediate cell adhesion. This study shows that Ed takes part in many processes of dorsal closure, a morphogenetic movement driven by coordinated cell shape changes and migration of epidermal cells to cover the underlying amnioserosa. Ed is differentially expressed, appearing in epidermis but not in amnioserosa cells. Ed functions independently from the JNK signaling pathway and is required to regulate cell morphology, and for assembly of actomyosin cable, filopodial protrusion and coordinated cell migration in dorsal-most epidermal cells. The effect of Ed on cell morphology requires the presence of the intracellular domain (Edintra). Interestingly, Ed forms homodimers in vivo and Edintra monomer directly associates with unconventional myosin VI/Jaguar (Jar) motor protein. ed genetically interacts with jar to control cell morphology. It has previously been shown that myosin VI is monomeric in vitro and that its dimeric form can associate with and travel processively along actin filaments. Thus, it is proposed that Ed mediates the dimerization of myosin VI/Jar in vivo which in turn regulates the reorganization and/or contraction of actin filaments to control changes in cell shape. Consistent with this, it was found that ectopic ed expression in the amnioserosa induces myosin VI/Jar-dependent apical constriction of this tissue (Lin, 2007).
Dorsal closure involves cell shape changes and migration of dorsal-most epithelial (DME) cells over the apically constricted amnioserosa. At later stages of dorsal closure, myosin II/Zipper is the major motor protein generating force to drive the contraction of both DME and amnioserosa cells. However, its role in establishing/maintaining early DME cell morphology (prior to the assembly of visible actomyosin cable) has not yet been documented. This study showns that Ed is required to regulate cell morphology at an early stage. Ed forms homodimers and monomeric Ed can directly associate with myosin VI/Jar. Moreover, the data suggest that the effect of ed on cell shape changes is mediated through jar. How does Ed cooperate with Jar to cause cell shape changes? Since myosin VI is monomeric in vitro and cannot itself efficiently initiate dimerization unless two molecules are held in close proximity (Park, 2006), it is proposed that Ed, via homodimerization, might promote the assembly of functional myosin VI/Jar dimer in vivo that either tethers Ed to actin filaments (as an anchor) or moves processively along actin filaments (as a transporter). Interestingly, Jar also associates with Arm to stabilize DE-cadherin/Arm during border cell migration. Thus, it might be feasible that Ed recruits dimeric Jar to AJs where Jar associates with the actomyosin network and stabilizes the DE-cadherin/Arm complex that in turn also interacts with the actomyosin network. In this scenario, myosin VI/Jar acts as an anchor molecule to link homophilic CAMs like Ed and DE-cadherin of AJs to actin filaments. Due to the large step size of myosin VI, the resulting dimeric Ed/Jar complex might simultaneously associate with and cross-link neighboring actin filaments. In this aspect, the function of Jar in DME cells would be similar to Canoe, which links Ed to actin filaments in wing disc cells. Interestingly, Ed associates with Canoe and Jar via different domains (C-terminal PDZ domain-binding motif and N-terminal 80 amino acids, respectively). However, unlike Jar, the distribution of Canoe at AJs of DME cells is unaffected in edlF20 M/Z embryos, indicating the importance of tissue-specific interactions (Lin, 2007).
Following the engagement of myosin VI/Jar to actin filaments at AJs, myosin II/Zipper, myosin VI/Jar or other myosin motors might be responsible for the force generation to establishing/maintaining early DME cell morphology. For example, the plus end-directed myosin II/Zipper generates pulling force to drive contraction of DME cells during the zippering phase of dorsal closure. Moreover, at stage 12, the apical constriction caused by ectopic ed expression in amnioserosa cells is also associated with the accumulation of myosin II. However, its role in early DME cell morphology remains unknown. In contrast, the minus end-directed myosin VI/Jar might theoretically generate a pushing force to cause cell expansion. This is, however, in contrast with the observation that Ed cooperates with Jar to cause cell contraction. It is likely that myosin VI/Jar functions only as an anchor to link Ed to actin filaments but not as a force-generating motor. However, since the organization and orientation of actin filaments in early DME cells are currently unknown, the possibility cannot be completely excluded that myosin VI plays an additional role in force generation (Lin, 2007).
It has been shown that loss of Ed in the ed mutant clones induces apical constriction in the wing imaginal disc and this study demonstrates that ectopic ed expression in the amnioserosa also induces apical constriction of this tissue. While both loss of ed and ectopic ed expression can induce apical constriction, the mechanisms, however, differ in these two systems. In the former case, apical constriction of ed−/− cells is caused by the accumulation of a higher density of DE-cadherin, Arm and actin (and by their interaction with myosin II). According to the differential adhesion hypothesis, these ed−/− cells thus achieve stronger adhesiveness (affinity) and self-sort out from the surrounding wild-type cells. In contrast, overexpression of ed in amnioserosa might promote, via myosin VI/Jar dimer, the assembly of actin filaments that in turn interact with myosin II/Zipper, myosin VI/Jar or other myosin motors to produce apical constriction.
The strong genetic interaction between ed and jar is detectable not only during dorsal closure but also during germband retraction, a process that is associated with dramatic cell shape change of germband cells. Thus, Ed might also cooperate with Jar to regulate germband cell elongation along the D/V axis (Lin, 2007)
There are two types of AJs present in DME cells. The AJs facing LE contain only DE-cadherin, while the AJs connecting adjacent DME cells possess both DE-cadherin and Ed which both associate with actin filaments. The difference in AJ composition might regulate their stability and the strength of cell-cell adhesion. For example, AJs possessing both DE-cadherin and Ed might be more stable and rigid. This scenario, together with the tension exerted by Ed in the DME cells, can prevent each connecting DME cell from moving prematurely even at stage 12. In contrast, the presence of only DE-cadherin at AJs of the LE front might allow faster turnover of AJs that in turn results in more efficient cell migration. Upon removal of Ed, DME cells lose their tension and contain only DE-cadherin at their AJs, which together permit uncoordinated and premature migration of these cells at an earlier stage (Lin, 2007)
This study demonstrated that ed is also required for the assembly of actomyosin cable from stage 13 of dorsal closure onwards. Because the presence of the actomyosin cable can maintain a taut LE front, the irregular migration defect of ed mutant DME cells during early dorsal closure will become even more obvious when actomyosin cable assembly also fails in these cells. Thereafter, all DME cells migrate toward the dorsal midline with different speeds. The faster-moving DME cells of a given segmental stripe may extend filopodia to sense and preferentially zip up with the other faster-moving DME cells derived from either an adjacent segmental stripe or another stripe not directly opposite it (instead of in the opposing stripe) to cause misalignment. Although Ed is critical for actomyosin cable formation, Ed accumulates only at ANC but not at LE. It is possible that Ed might recruit additional factors to promote actomyosin cable assembly. Alternatively, Ed might function as a scaffold protein to regulate ANC formation, as all ANC-associated molecules tested mislocalize in the absence of Ed (Lin, 2007)
During the course of this study, Laplante (2006) proposed that differential Ed expression in lateral epidermis and amnioserosa (some vs. no Ed expression) promotes the generation of actomyosin cable and therefore, dorsal closure. In contrast, this study demonstrated that the elimination of Ed expression border by ectopic expression of Ed in amnioserosa (i.e., Ed expression at both sides of the border) still induces assembly of actomyosin cable in DME cells. It is possible that different levels of Ed expression across the border, but not necessary 'some vs. no Ed expression', are sufficient to trigger the generation of actomyosin cable (Lin, 2007).
Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. The Androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The Androcam N lobe noncanonically binds a single Ca(2+) and is locked in a 'closed' conformation, causing Androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered Androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, Androcam replacing calmodulin at IQ will abolish a Ca(2+)-regulated, calmodulin-mediated myosin VI structural change. It is proposed that the N lobe prevents androcam from interfering with other calmodulin-mediated Ca(2+) signaling events. Androcam exemplifies how duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins (Joshi, 2012).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.