Gene name - Lachesin
Cytological map position - 49A6--7
Function - cell adhesion
Symbol - Lac
FlyBase ID: FBgn0010238
Genetic map position - 2R
Classification - immunoglobulin domain cell adhesion molecule (cam) subfamily
Cellular location - surface
Organ morphogenesis requires the coordinated activity of many mechanisms involved in cell rearrangements, size control, cell proliferation and organ integrity. Lachesin (Lac), a cell surface protein, is required for the proper morphogenesis of the Drosophila tracheal system. Homozygous embryos for Lac mutations, which fail to complement the previous identified bulbous (bulb) mutation, display convoluted tracheal tubes and tube breaks. At the cellular level, enlarged cells are detected, suggesting that Lac regulates organ size by influencing cell length rather than cell number, and cell detachments, indicating a role for Lac in cell adhesion. Results from an in vitro assay further support that Lac behaves as a homophilic cell adhesion molecule. Lac co-localizes with Septate Junction (SJ) proteins, and ultrastructural analysis confirms that it accumulates specifically at this type of cellular junction. In Lac mutant embryos, previously characterized components of the SJs are mislocalized, indicating that the proper organization of SJs requires Lac function. In addition, mutations in genes encoding other components of the SJs produce a similar tracheal phenotype. These results point out a new role of the SJs in morphogenesis regulating cell adhesion and cell size (Llamargas, 2004).
There is a general role for SJs in tracheal morphogenesis and Lac contributes to this function. Previous experiments have suggested that the length of the tracheal tubes is controlled at the apical membrane of the tracheal cells, by a mechanism that is dependent on the activity of the Grainy head (Grh) transcription factor in response to Bnl/FGF signalling (Hemphala, 2003). This study indicated that an additional Grh-independent mechanism must also function in the lateral cell compartment to control tube length and integrity. The observation that Lac and other SJs proteins are also required for tube length and integrity led to the proposition that this independent lateral mechanism is based on the function of SJs. In this regard, the localization of none of the SJs proteins analyzed is dependent on grh (Hemphala, 2003). Very recently, an independent paper has been published on the role of the SJ protein Na+/K+ ATPse in tracheal development (Paul, 2003). Consistent with the current results, this paper also showed that SJs play a role in tracheal epithelial tube-size control (Llimargas, 2004).
The tracheal tubes seem particularly sensitive to defects in SJs; in particular, there is a loss of epithelial integrity not reported for other ectodermal tissues. In this regard, the specific and strong expression of Lac in tracheal cells from stage 13 might hint at a strong requirement for Lac, and possibly SJs, during tracheal morphogenesis. It is important to note that SJs start to form late in embryogenesis, only after most of the morphogenetic events have taken place; however, at these stages, the tracheal tree has not yet completed its morphogenesis and could require fully functional SJs. The integrity of tracheal tubes may be dependent on the ability of their cells to adhere to one another, since they are subjected to stronger pulling forces than most other ectodermal tissues. Interestingly, differences at the level of the cellular junctions have been reported between epidermal and tracheal cells (Llimargas, 2004).
Co-localization studies with confocal microscopy, immunoelectron microscopy analysis, functional studies on the permeability of the trans-epithelial barrier, and the mutual requirement of Lac and other SJ proteins for their correct subcellular localization indicate that Lac is a new component of the SJs. Localization or stabilization of some (but not all) SJ proteins has been found to be dependent upon that of other components. For example, Cora has a very faint staining and a diffuse localization in Nrx mutants, whereas localization of Dlg or Fas3 shows no obvious defect in those same mutants. SJ components whose proper localization is interdependent are thought to physically interact, and to belong to the same supramolecular complexes in the SJs. These results led to the proposal that SJs are formed by the recruitment of distinct components into different protein complexes. However, it still remains unknown which protein/s would initially be required to target these protein complexes to the SJs. The current results indicate that several SJ proteins are mislocalized in Lac mutants to a certain degree. One possibility is that Lac interacts with one of the components of the SJs, and that in the absence of this interaction SJs are not properly assembled. Lac is a cell surface protein, associated with the outer leaflet of the membrane bilayer via its GPI-tail, and could potentially interact, in cis and/or in trans, with transmembrane proteins belonging to SJ complexes. It has been shown that Contactin and TAG1, vertebrate GPI-linked proteins of the Ig superfamily, bind to vertebrate Nrx-like proteins in the context of axo-glial interactions in the region of the node of Ranvier. A similar hypothesis for Lac and Nrx was tested with the S2 cell aggregation assay. Although Lac-expressing transfected S2 cells form aggregates, consistent with the results of the bead-aggregation assay, they do not aggregate with untransfected S2 cells that endogenously express Nrx. This negative result suggests that Lac does not interact, at least in trans, with Nrx. Ig superfamily members often show heterophilic interactions with other family members, raising the possibility that Lac interacts with components of SJs carrying Ig domains (Llimargas, 2004).
However, the mislocalization of the analyzed SJ proteins in Lac mutants is not as severe as the one described for proteins that are thought to physically interact, as the levels of the proteins analyzed seemed normal. Therefore, the results do not necessarily point to a direct interaction of Lac with those proteins, and could suggest, instead, that SJs are not perfectly assembled in Lac mutants and, as a consequence, their components are less well localized or stabilized. In that scenario, Lac could recognize a preformed structure of the SJs in order to be properly localized -- Lac localization appears to require functional SJs because it is indeed abnormal in mutants that affect SJs structure, such as cora. It has been suggested that SJ multiprotein complexes present in adjacent cells have to interact with each other to ensure proper assembly of the junction. The nature of Lac as a homophilic cell adhesion protein suggests that it could mediate such intercellular interactions, and could have a role as a component of SJs specifically ensuring or reinforcing the SJs-mediated adhesion between neighboring cells (Llimargas, 2004).
The results suggest a new role for SJs in morphogenesis, by the control of cell length and adhesion. Organ and body size often depend on control of cell number, and SJs have been suggested to control cell proliferation. The current results suggest that, in the case of the tracheal system, SJs regulate organ size by influencing cell length rather than cell number. These cells do not need to be necessarily bigger to generate an increase in tracheal branch length. A major contribution to the increase in organ size could lie in the fact that cells become more elongated. Indeed, Lac mutant tracheal cells appear more elongated, identifying the control of cell shape as one of the regulatory mechanisms of tracheal tube size. It remains an open question whether, in this case, the control of cell length is a direct consequence of the role in cell adhesion. Thus, for example, in a pure mechanical model, the lessening of the tight contact between cells could abrogate a constraint for their elongation. Alternative explanations are also possible; for instance, tight contact among the tracheal epithelial cells would be required for coordinated signalling, allowing polarized conduction of information. Moreover, some of the SJ components themselves could control cell and tissue size via intercellular and intracellular cell signalling, as has been hypothesized, for example, for Cora and Nrg. An unexpected nuclear function for the vertebrate tight junction component ZO2, structurally and functionally related to Drosophila Dlg, has been recently suggested, possibly linking the permeability barrier and signalling functions of these structures. In this respect, other features of the Lac protein not necessarily related to its adhesion properties could be relevant to the control of tube size. For instance, Lac could also have a role in signalling, as is known for other GPI-linked Ig proteins (Llimargas, 2004).
Although the available data indicate that the mechanisms of tubulogenesis share many basic strategies across diverse animal groups, the generality of the current results could be hampered by the fact that SJs have not been reported in vertebrates. However, the localization of the Lac protein at the SJs, and the function of SJs in morphogenesis, could be of functional significance to other systems for several reasons. First, some of the roles of SJs are assumed to be performed by tight junctions in vertebrates. Second, some tight junction components are structurally related to, and others are homologs of, SJs proteins. Third, many homologs of the Drosophila SJ proteins, as well as structures that are similar to the SJs, have been identified in mammals. In this context, SJs-like proteins could be similarly involved in defining proper organ size and morphogenesis in these other systems, by influencing cell adhesion properties and cell shape (Llimargas, 2004).
Initial comparisons to the DNA sequence data base indicated grasshopper Lachesin was similar to the Drosophila Amalgam protein. In order to determine whether Lachesin was the grasshopper homolog of Amalgam, PCR primers were used to amplify sequences from Drosophila cDNA. Products of the expected size were amplified and were verified by DNA sequencing. The PCR fragment was then used to isolate a Drosophila Lachesin cDNA clone. Sequence comparisons showed that the isolated Drosophila cDNA is not Amalgam and is remarkably similar to grasshopper Lachesin. Based on the high degree of sequence similarity it is concluded that the Drosophila homolog of grasshopper Lachesin had been isolated. When the three Ig domains which make up 90% of the proteins are compared, the predicted Lachesin amino acid sequences are 75% identical. This is much higher than the identities seen between other genes that have been cloned in both grasshopper and Drosophila such as Fasciclin 1 (50% identical), Fasciclin 2 (41%), and neuroglian (65%). Considering the large evolutionary distance separating grasshopper and Drosophila (on the order of 300 million years), the high degree of sequence conservation indicates that a large portion of the Lachesin sequence may be important for its function. A C-terminal hydrophobic domain characteristic of GPI-linked proteins suggests that the protein is linked to the membrane by a GPI anchor (Karlstrom, 1993).
Lac was first identified in the grasshopper embryo as a membrane protein specifically expressed in neurogenic cells and subsets of neurons and axons; subsequently, a fly homolog was identified (Karlstrom, 1993), but its role during development has not been examined. The Lac protein contains three immunoglobulin (Ig) domains, and a C-terminal hydrophobic domain characteristic of GPI-linked proteins. Vertebrate proteins displaying the same domain arrangement are known as IgLONs (Llimargas, 2004).
date revised: 20 April 2004
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