pollux

Protein Interactions

Based on Plx's expression on cell surfaces and the presence of putative CAM and ECM adhesion sites within the primary structure of Plx, a series of experiments was carried out to determine if Plx could promote cell adhesion. To determine if Plx's integrin recognition motif was functionally active and could initiate cell-attachment, an assay was carried out of the ability of cultured human MG63 cells to adhere to microtiter wells coated with affinity purified full-length recombinant Plx. MG63 cells, which express the alpha5 and beta3 integrin subunits, require a substratum containing functionally active RGD recognition sites for attachment. Like the positive control (fibronectin-coated wells), the MG63 cells attach to Plx-coated wells. When MG63 cells are pretreated with the pentapeptide Gly-Agr-Gly-Asp-Pro-Ser, attachment is blocked. However, pretreatment with a similar peptide (Gly-Agr-Gly-Glu-Pro-Ser) that lacks an RGD recognition motif fails to block cell-attachment to the Plx substratum, demonstrating MG63's attachment requirement, namely, the Plx RGD integrin recognition site. Although Plx appears late in CNS development, indicating that it may not play an active role in fasciculation, its restricted expression in subsets of axon fascicles suggests that it may function to maintain fascicle structure via homophilic interactions. To determine if Plx can promote cell aggregation via homophilic interactions, cell aggregation experiments were performed with transformed cultured Drosophila Schnieder S2 cells. Cell lines that express a full-length Plx, under the control of the metallothionein promoter or in transiently transfected S2 cells using the same copper activated plx transgene, fail to produce significant cell aggregation. Immunolocalization of Plx on the surface of the transformed S2 cells, in the absence of membrane permeablizing detergents, demonstrates that Plx accumulates asymmetrically on plasma membranes and displays an extracellular domain(s). The asymmetry of Plx expression on the surface of the S2 cells is consistent with its in vivo regionalization and may indicate a selective interaction with other membrane proteins and/or with cytoskeletal components. Moreover, immunostaining experiments fail to reveal any significant membrane-membrane associations mediated by Plx, suggesting that under the culture conditions used, Plx does not function as a homophilic adhesion molecule. However, Plx may require additional cofactors or modifications to promote cell adhesion, conditions not provided by the cultured S2 cells (Zhang, 1996).

DEVELOPMENTAL BIOLOGY

Maternal and Embryonic expression

During embryogenesis, Plx uniformly covers the apical surface of cellular blastoderm cells. It is later found regionally concentrated along subsets of central nervous system axon pathways and on the apical surface of the trachea's tubular epithelium. During oocyte development, PLX mRNA is maternally expressed in the egg chamber's nurse cells. PLX mRNA expression is first detected in the oocyte support cells shortly after they are formed; as development progresses, the PLX in situ signal intensifies, suggesting a cumulative increase in steady-state mRNA levels. Starting at oocyte maturation stage 10, when the cytoplasmic contents of the nurse cells are transported into the oocyte, PLX transcripts appear evenly distributed throughout the oocyte. In situ hybridizations carried out on embryos undergoing cellularization indicate that PLX mRNA is taken up into the newly formed blastoderm cells. Shortly thereafter, Plx protein is first detected in the embryo. Serial sections through immunostained cellular blastoderm embryos reveal that Plx protein is uniformly distributed on the apical surface of the blastoderm cells. No PLX message or protein is observed in the embryo's pole cells. During gastrulation, levels of the evenly distributed mRNA and protein diminish, such that by stage 9 little or no staining above background is observed (Zhang, 1996).

plx transcription is next detected in the CNS of late stage 11 embryos. Identified by their position within the developing ganglia, as observed in serial transverse sections, newly formed neurons throughout the CNS express PLX message. PLX transcripts are not detected in neuroblasts or ganglion mother cells at any stage of CNS development. No message or protein is observed in the peripheral nervous system. As the number of post-mitotic cells increases during CNS development, so does the number of plx expressing cells: by stage 14, most if not all neurons contain PLX transcripts. Plx protein is first detected in the CNS on the surface of neuronal cell bodies, during late stage 12. Starting at stage 13, Plx begins to accumulate uniformly along axon tracts that extend the entire length of the ventral cord's longitudinal connectives. As ventral cord contraction continues, serial sections through whole-mount immunostained stage 14 or older embryos, reveal immunostaining on cell surfaces outlining subsets of cell bodies and show that most axon fascicles traversing the longitudinal connectives accumulate Plx. At the light-microscopic level of resolution, Plx immunostaining within the fascicles appears to be associated with axonal plasma membranes and the peri-axonal space between the bundled axons. However, ultrastructural localization will be required to identify its exact position within fascicles (Zhang, 1996).

No appreciable Plx immunostaining was observed in the ventral cord's commissures or in the lateral projecting axon tracts that exit via its segmental or intersegmental axon pathways. Many of the axon fascicles that make up the commissures and segmental tracks are also part of the longitudinal connectives. The restricted or regional deposition of Plx within subsets of axon pathways that traverse both commissures and connectives is a characteristic shared by many axonal CAMs and ECM glycoproteins in both vertebrates and invertebrates. The regionalization of Plx may be due to directed deposition via a specific transport/translocation mechanism and/or its target receptor molecule is regionalized within axon fascicles. In contrast to the axon fascicles residing in the ventral cord commissures, fascicles within the brain's supraoesphageal commissure, which interconnect the cephalic lobes, accumulate high levels of Plx. Serial transverse sections show that Plx is again regionally distributed within the fascicles. Within the developing brain's supraoesphageal ganglion, little or no Plx is found on axon surfaces before they enter or after they exit this commissure. In addition to the heavy labeling on the supraoesophageal commissure, subsets of symmetrically positioned cell bodies, presumably neurons, within the cephalic lobes also have detectable levels of Plx on their surfaces. During the initial phase of axonogenesis, when the first longitudinal and commissural pathways are established, Plx protein is not found on the pioneering axons or associated with newly formed axon fascicles. This late appearance of Plx within the developing CNS, relative to membrane proteins involved in axonogenesis, suggests that its role in the CNS is one of maintenance of function rather than in establishing its structure. Plx's motor neuron-selective adhesive site (Hunter, 1989) may function to maintain proper motor neuron axonal-ECM contact within these fascicles (Zhang, 1996).

Like other Drosophila axonal adhesion glycoproteins (such as Neuroglian), Plx is also expressed in the tubular tracheal epithelium. Similar to plx's late activation during CNS development, PLX message is first detected in cells lining the major, large diameter tracheal tubes shortly after fusion of the dorsal trunk metamers. No expression is detected during the primary phase of tracheal development, particularly in the tracheal pits or during initial branch formation and early outgrowth of the dorsal and lateral tubes. In addition to the dorsal and lateral trunks, PLX message is detected in other major branches. However, expression is not detected throughout the tracheal tree. Little or no PLX message is found in cells making up the smaller tubes nor is it detected in the smallest branches of the trachea, the tracheole. Plx protein is first observed in the trachea shortly after its message is detected. Examination of stage 15 embryos, in whole-mount and in serial transverse sections, reveals that Plx localizes to the apical surface of the epithelium. The level of Plx immunostaining corresponds with tracheal tube diameters. While the dorsal and lateral trunks and their immediate branches show consistently high levels of Plx immunostaining, little or no staining is observed on, or in, the smaller branches or associated with tracheoles. At embryonic stage 15, when the tracheal tubes are filled with fluid, Plx is also detected in the lumen of the dorsal and lateral trunks, suggesting that all or a cleaved portion of Plx may dissociate from the epithelial cell's plasma membrane. The late appearance of Plx in the trachea and its presence in the lumen is consistent with it being part of, or associated with, the trachea's cuticular ECM, which is formed during stages 15 and 16 of development. The regionalized distribution, confined to predominantly the major, larger diameter tubes, further suggests that Plx may associate with or make up a particular part of the cuticular ECM substructure known as the endocuticle (or procuticle). The cuticular ECM intima of large diameter tubes is comprised of an outer (luminal) epicuticle and an inner procuticle layer. The procuticle consists of a filamentous matrix that fills the taenidial folds between the epicuticle and the epithelium. The procuticle is not part of the cuticular intima of smaller diameter tubes or in tracheoles (Zhang, 1998).

Effects of Mutation or Deletion

Essential for viability, plx mutant analysis indicates that larval death is attributable to asphyxiation brought on by fluid-congested tracheal tubes. Ultrastructural examination of mutant tracheae reveals defects in cell-extracellular matrix contacts (Zhang, 1996).

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date revised: 25 May 2021

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