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Keywords:

  • adhesion complex assembly;
  • cell differentiation;
  • cell sorting;
  • morphogenesis;
  • unconventional protein transport

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

The social amoeba Dictyostelium discoideum is a simple but powerful model organism for the study of cell–cell adhesion molecules and their role in morphogenesis during development. Three adhesive systems have been characterized and studied in detail. The spatiotemporal expression of these adhesion proteins is stringently regulated, often coinciding with major shifts in the morphological complexity of development. At the onset of development, amoeboid cells express the Ca2+-dependent cell–cell adhesion molecule DdCAD-1, which initiates weak homophilic interactions between cells and assists in the recruitment of individuals into cell streams. DdCAD-1 is unique because it is synthesized as a soluble protein in the cytoplasm. It is targeted for presentation on the cell surface by an unconventional protein transport mechanism via the contractile vacuole. Concomitant with the aggregation stage is the expression of the contact sites A glycoprotein csA/gp80 and TgrC1, both of which mediate Ca2+/Mg2+-independent cell–cell adhesion. Whereas csA/gp80 is a homophilic binding protein, TgrC1 binds to a heterophilic receptor on the cell. During cell aggregation, csA/gp80 associates preferentially with lipid rafts, which facilitate the rapid assembly of adhesion complexes. TgrC1 is synthesized at low levels during aggregation and rapid accumulation occurs initially in the peripheral cells of loose mounds. The extracellular portion of TgrC1 is shed and becomes part of the extracellular matrix. Additionally, analyses of knockout mutants have revealed important biological roles played by these adhesion proteins, including size regulation, cell sorting and cell-type proportioning.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

Multicellularity most likely evolved via clonal development from unicellular spore, zygote or aggregative development (Bonner 1998; Grosberg & Strathmann 2007). Examples of cell-aggregatory multicellular lineages are the myxobacteria and the Dictyostelid slime molds. The development of ordered structures in multicellular organisms proceeds through a complex series of cellular interactions, in which cells sort out and associate into specific multicellular groups. Cells frequently migrate from one site to another and reorganize at their final destination to establish the various tissue primordia. Individual cells or groups of cells must continuously change their relative positions and mutual adhesiveness during morphogenesis. The analysis of a number of independently evolved pairs of unicellular and multicellular relatedness has led to the proposal that proteins involved in cell adhesion, cell–cell signaling and cell differentiation are genetic tool kits for multicellularity (Vogel & Chothia 2006; King et al. 2008; Abedin & King 2010).

The search for the origin of multicellularity often begins with cell–cell adhesion molecules (CAMs) and substratum adhesion proteins (Bowers-Morrow et al. 2004; Harwood & Coates 2004). Recent studies of CAMs show that, in addition to cell–cell adhesion, CAMs play key roles in sensing environmental cues and in generating signals that regulate a diversity of cellular processes, including gene expression, cell proliferation, cell polarity, cell motility, and apoptosis. Differential temporal and spatial expression of cell adhesion molecules in specific groups of cells or tissues is stringently regulated during development. The formation or dissolution of specific adhesion complexes frequently lead to signals that serve as a major driving force behind cell migration, cell sorting, differentiation and tissue formation. Therefore, CAMs have been recognized as major morphoregulators and signaling molecules (Edelman & Crossin 1991; Gumbiner 1996) because of their crucial participation in many dynamic physiological and pathological processes, such as tissue architecture, organ regeneration and cancer metastasis.

The social amoeba Dictyostelium discoideum has been adopted as a model organism to address many important biological processes, ranging from social biology to medical sciences (Williams 2010). The use of D. discoideum to investigate the nature and functions of CAMs was popularized by the pioneering work of Gerisch and his colleagues about half a century ago (Müller-Taubenberger & Bozzaro 2006). There are many experimental advantages associated with this organism. Dictyostelium has a simple and well-defined life cycle and is amenable to various molecular and cellular manipulations. The amoeboid cells feed on bacteria and multicellular development is triggered upon the depletion of food. Development can be synchronized and cells at specific developmental stages are readily available in large quantities for biochemical studies. The developmental cascade can be divided into an aggregation phase and a post-aggregation phase, each lasting for ∼12 h. During the first 6–8 h, cells exit the solitary state to adopt a social behavior, which allows them to interact and undergo chemotaxis in response to extracellular cyclic adenosine monophosphate (cAMP) (Parent & Devreotes 1996). Cell–cell contacts are established via CAMs in cell streams, which eventually give rise to loose mound structures. Then cells secrete extracellular matrix material, which forms a slime sheath to encase the developing structure (Loomis 1972). The appearance of tight mounds demarcates the beginning of the post-aggregation phase. Cell-type differentiation within the mound is accompanied by cell sorting mediated by the differential spatiotemporal expression of CAMs. The sorting out of cells within the slug eventually leads to a spatial pattern with prestalk cells (∼20%) in the anterior region and prespore cells (∼80%) in the posterior. Cell movement continues to drive the morphogenesis of the tipped mound, which extends into a long finger-like structure and transforms into a migratory slug (Weijer 2009). Culmination starts with the slug rearing up on its posterior end and the prestalk cells migrating downward in a tubular structure to form a nascent stalk, giving rise to a fruiting body composed of a sorus of spores atop a filamentous stalk.

The participation of CAMs in diverse biological processes is evident throughout Dictyostelium development. The initial work of Gerisch led to the identification of two classes of cell–cell adhesion sites based on their sensitivity to ethylenediaminetetraacetic acid (EDTA) (Beug et al. 1970; Gerisch 1980). Subsequent work from several laboratories has identified four adhesion systems and three of them have been characterized in detail (Coates & Harwood 2001; Siu et al. 2004). The EDTA-sensitive adhesion sites consist of two subtypes, the Ca2+-dependent sites and the Mg2+-dependent sites (Fontana 1993; Wong et al. 2002). While the Ca2+-dependent adhesion sites are mediated by the CAM DdCAD-1 (Wong et al. 1996, 2002), the molecular identity of the Mg2+-dependent sites remains unknown. Both EDTA-sensitive adhesive systems promote initial contacts among cells in the early stages of chemotactic migration and cell stream formation (Wong et al. 2002). The other two adhesion systems are made up of csA/gp80 and TgrC1/LagC/gp150, respectively. Cell–cell contacts mediated by these CAMs are stable in up to 15 mmol/L of EDTA. While csA/gp80 contributes to the stability of cell–cell contacts in cell aggregates, TgrC1/LagC/gp150 is responsible for cell–cell cohesion in the post-aggregation stages. Thus, temporal and spatial expression of CAMs is stringently controlled, corresponding to major shifts in their developmental stage (Fig. 1). This review will focus on the regulation of the spatiotemporal expression of these proteins and their involvement in morphogenesis.

image

Figure 1.  Life cycle of Dictyostelium discoideum and the expression of cell–cell adhesion molecules (CAMs). The Ca2+-dependent CAM DdCAD-1 is expressed soon after the initiation of development and its cellular level remains more or less constant throughout development. Expression of the Ca2+/Mg2+-independent CAM csA/gp80 coincides with the onset of aggregation and exhibits a biphasic pattern of accumulation at the aggregation stage. Transcription of TgrC1/LagC/gp150 is initiated at mid-aggregation at a low level and rapid accumulation occurs at the mound stage.

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Regulation of temporal expression of CAMs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

Dictyostelium CAMs show a distinct temporal pattern of expression and they are regulated by factors that correlate with major shifts to higher orders of morphological complexity (Fig. 1). At the onset of development, cells express EDTA-sensitive contact sites mediated by the Ca2+-dependent homophilic binding CAM DdCAD-1, which is encoded by the gene cadA (Brar & Siu 1993; Wong et al. 1996). When cultured in association with bacteria, DdCAD-1 displays a unique temporal pattern of expression, which combines the characteristics of early genes and aggregation stage genes (Yang et al. 1997). DdCAD-1 is synthesized soon after the initiation of development together with a group of early developmentally regulated proteins (Knecht et al. 1987). At the cell streaming stage, DdCAD-1 expression is stimulated by nanomolar pulses of exogenous cAMP. An examination of the cadA 5′-flanking region has revealed the presence of several G/C-rich elements. Deletion analysis has led to the identification of an 80 bp sequence between −359 and −280, which harbors the major prestarvation factor (PSF)- and cAMP-response activity (Sriskanthadevan et al. 2007). This 80 bp region contains three G/C-rich elements, two of which contain the core sequence GTGTG of the cAMP response elements found in the promoter of the csaA gene (Desbarats et al. 1992). The same region also contains four TTG boxes with sequences corresponding closely to the consensus sequence (TTGXTTG) found in the PSF-response elements of discoidin-1γ and α-mannosidase genes (Vauti et al. 1990; Schatzle et al. 1993).

Shortly afterwards, cells begin to synthesize the contact sites A glycoprotein csA/gp80, which is encoded by the csaA gene (Noegel et al. 1986; Wong & Siu 1986). The cell binding activity of csA/gp80 is stable in low concentrations of EDTA and it mediates cell–cell adhesion via homophilic binding in a Ca2+/Mg2+-independent manner. Transcription of the csaA gene occurs 2–3 h after the initiation of development. The csaA gene shows a biphasic pattern of expression with basal transcription induced by a cAMP-independent mechanism, followed by a cAMP-induced rise in transcription rate (Siu et al. 1988; Mann & Firtel 1989; Ma & Siu 1990). Several G/C-rich cAMP response elements have been mapped in the csaA promoter (Desbarats et al. 1992). Also, perturbation of the EDTA-sensitive contact sites substantially reduces the level of csA/gp80, suggesting that csA/gp80 expression is also influenced by prior cell–cell contact (Desbarats et al. 1994).

The level of csA/gp80 has a major influence on the size of aggregates. Slug size has been found to positively correlate with the level of csA/gp80 expressed on cells (Kamboj et al. 1990). Recent studies by the Gomer laboratory have led to the identification of a large secreted protein complex named counting factor (CF), which is part of a negative feedback loop that regulates the expression of DdCAD-1 and csA/gp80 (Roisin-Bouffay et al. 2000). Although CF affects the cAMP-induced signaling cascade, changing the size of exogenous cAMP pulses does not phenocopy the effects of CF on cell–cell adhesion, suggesting that CF regulates CAMs via a pathway independent of cAMP pulses (Tang et al. 2001). Mutational analysis showed that cells overexpressing CF have a much lower level of DdCAD-1 and csA/gp80 expression, resulting in the formation of tiny aggregates (Roisin-Bouffay et al. 2000). Similarly, a recent report on DNG1, a Dictyostelium homologue of the tumor suppressor ING1, shows that dng1-null cells express a much reduced level of csA/gp80 and form very small aggregates (Mayanagi et al. 2005). These observations are consistent with the notion that CAMs are involved in size regulation.

A second EDTA-resistant cell adhesion system is mediated by the Ca2+/Mg2+-independent CAM, LagC/gp150 (Gao et al. 1992; Dynes et al. 1994), which is encoded by the gene tgrC1, a member of the tgr gene family (Benabentos et al. 2009). LagC/gp150 is therefore referred to as TgrC1 hereafter. TgrC1 mediates cell–cell adhesion by heterophilic binding and plays an important role in morphogenesis and cell differentiation in the post-aggregation stages of development (Wang et al. 2000). TgrC1 is expressed at low levels in the mid-aggregation stage, and accumulates quickly during mound formation (Wang et al. 2000; Iranfar et al. 2001). Sequences similar to known cAMP response elements can be detected in the promoter region of the tgrC1 gene. However, tgrC1 transcription is not affected by low concentrations of cAMP pulses, but is stimulated by high levels of cAMP that arise in the post-aggregation stage (Dynes et al. 1994). Analysis of the expression profiles of post-aggregation genes in wildtype and mutant cells show that accumulation of the tgrC1 mRNA is dependent on GBF, the G-box binding transcription factor and both GBF and TgrC1 are required for the transcription of many post-aggregation genes. It is proposed that TgrC1 and GBF form a feed-forward loop that can integrate temporal information with morphological signals to activate post-aggregation genes after cell–cell contacts have been made (Iranfar et al. 2006). Finally, it is of interest to note that the mRNA level of tgrC1 in prestalk cells is three times higher than that in prespore cells (Dynes et al. 1994). This may explain why prestalk cells are more resistant to dissociation by antibodies raised against TgrC1 (Lam et al. 1981).

Studies of CAM-knockout mutants have revealed an interesting relationship among CAMs. In cadA cells, csA/gp80 is expressed precociously at a higher level than in wildtype cells (Wong et al. 2002), resulting in an increase in adhesiveness among cadA cells. The synthesis of csA/gp80 is known to be highly augmented by cAMP pulses (Desbarats et al. 1992). Since the formation of cell–cell contacts affects cAMP metabolism and cAMP signaling (Fontana & Price 1988; Fontana et al. 1991a,b), it is possible that the loss of DdCAD-1 expression may somehow enhance cAMP signaling and stimulate a higher level of csA/gp80 expression. However, inhibition of cell–cell adhesion by EDTA leads to reduced levels of csA/gp80 expression (Desbarats et al. 1994). These results suggest that the loss of DdCAD-1 expression and the inhibition of EDTA-sensitive cell adhesion sites may elicit different intracellular signals that can lead to opposite outcomes. It is also possible that the inhibition of gp80 expression by EDTA is due to the inhibition of the Mg2+-dependent sites or other pleiotropic effects of EDTA.

In csaA cells, TgrC1, which is normally expressed at a basal level at the aggregation stage, is expressed at a much higher level at that stage (Wang et al. 2000). In tgrC1 cells, csA/gp80 is expressed for another 15 h after its normal termination time (Liansheng Hou and Chi-Hung Siu, unpubl. data, 2009). The expression of CAMs appears to be coupled to the transcriptional regulation of one another, suggesting that CAMs might participate in a network of feedback loops that regulate its expression as well as other developmental processes.

Unique structural features of DdCAD-1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

DdCAD-1 is unique among CAMs because it contains neither a hydrophobic signal peptide nor a transmembrane domain (Wong et al. 1996). The nuclear magnetic resonance (NMR) solution structures of DdCAD-1 show two β-sandwich domains linked by a short bridge. The N-terminal domain exhibits remarkable resemblance to βγ-crystallins and the C-terminal domain adopts an Ig-like fold (Fig. 2A) (Lin et al. 2004, 2006). The protein contains three Ca2+-binding pockets, two of which are located in the N-terminal domain and play a crucial role in the function of DdCAD-1 (Fig. 2B). DdCAD-1 is tethered to the cell membrane via the C-terminal domain while the N-terminal domain extends outward to engage in homophilic interactions (Fig. 2C) (Lin et al. 2006). Molecular modeling based on NMR structures and mutational analysis have led to a novel model of Ca2+-dependent cell–cell adhesion, which is distinct from mechanisms proposed for mammalian Ca2+-dependent CAMs (Gumbiner 2005). The forward binding reaction of DdCAD-1 involves charge interactions between two N-terminal domains. In the absence of Ca2+, the two protein surfaces repel each other due to the negative-charge clusters on them. Upon Ca2+ binding, the charged properties of apposing surfaces change, allowing interactions between them. The binding is further stabilized by hydrophobic interactions between the N-terminal domain of one protein and the C-terminal domain of the partner protein.

image

Figure 2.  Nuclear magnetic resonance (NMR) solution structures of DdCAD-1. (A, B) Ribbon drawing of the lowest-energy conformers of the Ca2+-free form (A) and Ca2+-bound form (B) DdCAD-1. Blue, N-terminal domain; green, C-terminal domain; red, helical structures in the N-terminal domain. In each domain, β-strands are sequentially labeled with letters and numbers. Strands A1, B1, D1 and G1 form motif 1; C1, E1, F1 and H1 form motif 2; A2, D2, I2 and H2 form sheet 1; B2, C2, E2, F2 and G2 form sheet 2. (C) Schematic drawing of cell–cell adhesion mediated by the trans-interactions between DdCAD-1 molecules. N, N-terminal domain; C, C-terminal domain; Gray barrels, anchoring proteins on the membrane. (Reprinted from Lin et al. 2006.)

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Additionally, DdCAD-1 has been found to contain phosphorylated threonine residues (Secko et al. 2004). The expression of constitutively active RasG(G12T) protein in cells leads to a reduction of threonine phosphorylation in DdCAD-1, which is accompanied by a concomitant increase in DdCAD-1-mediated cell cohesion, suggesting that the phosphorylation state of DdCAD-1 has a major influence on the membrane presentation and/or the adhesive activity of DdCAD-1 (Secko et al. 2006).

DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

DdCAD-1 is synthesized in the cytoplasm as a soluble protein of 213 amino acids (Brar & Siu 1993). This unexpected observation raises the question of how DdCAD-1 is presented on the cell surface to participate in cell–cell binding. In order for DdCAD-1 to function as an adhesion protein, it must be translocated from the cytoplasm to the cell membrane. How can this be achieved in the absence of a signal peptide and a transmembrane domain? Interestingly, an increasing number of soluble cytoplasmic proteins are now known to be secreted from a variety of cells. So far, cells have been found to utilize several different strategies to target soluble proteins for secretion, including ABC transporters, the endosomal pathway, the formation of exosomes, and a flipflop mechanism (Nickel & Seedorf 2008). In the case of DdCAD-1, it is transported to the plasma membrane by contractile vacuoles (Fig. 3A) (Sesaki & Siu 1996). Contractile vacuoles constitute the osmoregulatory organelle inside Dictyostelium cells. Water and other solutes inside the cell are collected by a tubular network that feeds into vacuolar structures for release from the cell upon fusion with the plasma membrane. Therefore, the appearance of DdCAD-1 on the plasma membrane is influenced by the osmotic environment of the cells. The suppression of contractile vacuole activity under hyperosmotic conditions leads to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the loss of cell cohesiveness (Sesaki et al. 1997). Shifting cells back to a hypotonic condition induces a rapid increase in DdCAD-1-positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell surface. Pharmacological studies suggest the involvement of the vacuolar-type H+-ATPase in this process (Sesaki et al. 1997).

image

Figure 3.  Membrane targeting of DdCAD-1 by an unconventional protein mechanism via the contractile vacuoles. (A) Confocal image showing the association of DdCAD-1 with contractile vacuoles inside the cell. Bar, 5 μm. (B) Different patterns of DdCAD-1 signal associated with the luminal surface of contractile vacuoles (a, b). Upon fusion of contractile vacuoles with the plasma membrane, a contiguous staining pattern of DdCAD-1 spanning between the contractile vacuole membrane and the plasma membrane (c, d). Bars, 3 μm. (Reprinted from Sesaki et al. 1997.) (C) Confocal micrographs showing the association of DdCAD-1-GFP with the contractile vacuole network. Cells expressing DdCAD-1-GFP were collected at 3 h of development, fixed and labeled with mouse anti-calmodulin (CaM) mAb (red). Arrows indicate contractile vacuoles filled with DdCAD-1-GFP but devoid of CaM. Bar, 5 μm. Asterisk indicates a large contractile vacuole beginning to fuse with the plasma membrane. (D) Budding of vesicles into the lumen of the contractile vacuole. Cells expressing DdCAD-1-GFP were developed in 17 mmol/L phosphate buffer and then deposited on slides for attachment. The styryl dye FM4-64 (red) was added at 1 mg/mL to visualize both the contractile vacuoles and the plasma membrane. Time-lapse sequences of confocal images were recorded between 5 and 20 min after dye addition. Confocal images of the boxed area in the light micrograph are shown. Arrows point to membrane protrusions in the contractile vacuole lumen, where DdCAD-1-GFP and FM4-64 co-localized. Schematic drawings of the contractile vacuole are shown below the confocal images. (Reprinted from Sriskanthadevan et al. 2009.)

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In vitro reconstitution studies using purified contractile vacuoles and recombinant proteins indicate that this unconventional transport process involves at least four distinct steps: (i) targeting of DdCAD-1 to contractile vacuoles; (ii) translocation of DdCAD-1 into the lumen of contractile vacuoles; (iii) association of DdCAD-1 with an “anchoring” protein on the lumenal side of the vacuole; and (iv) diffusion of the anchored DdCAD-1 from the vacuolar membrane to the cell surface upon fusion of the contractile vacuole with the plasma membrane (Fig. 3B) (Sesaki et al. 1997; Sriskanthadevan et al. 2007). DdCAD-1 molecules that fail to bind to the lumenal surface are eventually secreted into the medium. Since DdCAD-1 is a homophilic binding CAM, cell–cell adhesiveness can be modulated by the binding of the secreted protein to the surface-associated DdCAD-1 (Siu et al. 1997).

When cells expressing DdCAD-1-GFP are subjected to confocal microscopy, DdCAD-1 can be observed inside the lumen of contractile vacuoles while calmodulin defines the contractile vacuole network and the cytoplasmic surface of the vacuoles (Fig. 3C). Time-lapse microscopy shows transient appearance of DdCAD-1 in vesicular structures inside contractile vacuoles (Sriskanthadevan et al. 2009). DdCAD-1 docked on contractile vacuoles is mobilized to fill membrane invaginations, which are then pinched off to become vesicles inside the lumen (Fig. 3D). DdCAD-1 is released upon the burst of these vesicles in the hypotonic environment of the lumen. Mutational analysis shows that the import of DdCAD-1 depends on its integrity and proper conformation. In many ways, the export of DdCAD-1 via contractile vacuole is similar to the budding of vesicles in yeast vacuoles (Muller et al. 2000) or the formation of multivesicular bodies in murine macrophages (Qu et al. 2007).

Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

Cells at the onset of the aggregation stage become more elongated and the surface is covered with many filopodial structures. DdCAD-1 is the first CAM detected at the onset of development and it is enriched on filopodia (Fig. 4A). Initial contacts between neighboring cells are mediated by the interdigitation of filopodia between adjacent cells. DdCAD-1 becomes especially prominent on the periphery of cell streams to facilitate the recruitment of neighboring cells (Sesaki & Siu 1996). Once initial contacts are made, cells retract their filopodia while the contact surfaces begin to expand and merge, giving rise to an extensive contact zone between adjacent cells. Intriguingly, DdCAD-1 is present in cell–cell contacts only transiently during cell streaming and its redistribution from the contact regions may reflect the dynamic nature of cell aggregation at this stage when cells must constantly break and remake cell–cell contacts. As csA/gp80 moves into the cell–cell contact regions, DdCAD-1 begin to disappear from the contact surfaces of most cells (Sesaki & Siu 1996). How DdCAD-1 is downregulated from the cell surface remains unclear. Since the cellular level of DdCAD-1 remains more or less constant throughout the aggregation phase of development (Yang et al. 1997), it is conceivable that membrane-bound DdCAD-1 is internalized and not degraded by proteases while stable contacts are being formed. This pattern of DdCAD-1 redistribution suggests that, in addition to cell–cell adhesion, DdCAD-1 may play other intracellular roles during Dictyostelium morphogenesis.

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Figure 4.  Spatial expression of cell–cell adhesion molecules (CAMs) during development. (A) Immunolocalization of DdCAD-1 on cells at the aggregation stage. DdCAD-1 is enriched on filopodia and lamellopodia (a), which are involved in making initial contacts between cells at the aggregation stage (b). Bars, 5 μm. (Reprinted from Sesaki & Siu 1996.) (B) Co-localization of csA/gp80 and sterols in cell–cell contact regions. Cells at the aggregation stage were fixed and incubated with filipin showing an abundance of sterol at cell–cell contacts (arrowheads) (a). Double staining using (b) filipin and (c) anti-csA/gp80 mAb shows co-localization of csA/gp80 and sterols in cell–cell contact regions (arrowheads). Bars, 5 μm. (Reprinted from Harris et al. 2001a.) (C) Maximum projections of optical section series of aggregates at the mound stage. Development was carried out on agar until the mound stage. Mounds were fixed and stained with anti-TgrC1 antibody for confocal microscopy. Serial optical sections were taken at 1 μm steps (a) or 2 μm steps (b). Panel (a) shows the top view of a mound, where the TgrC1-positive cells were present primarily in the periphery of the lower half of the mound. Panel (b) shows the side view of another mound where the TgrC1-positive cells had moved to the apical region of the mound. Bar, 25 μm.

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The accumulation of csA/gp80 in cell–cell contact regions becomes prominent during cell streaming. An interesting feature of the cell stream is that csA/gp80 is present in both end-to-end contacts and side-to-side contacts. However, morphological studies have revealed major differences in these two types of cell–cell contacts. Small filopodia-like protrusions frequently decorate the end-to-end contacts, whereas side-to-side contacts occur between smooth apposing surfaces (Harris & Siu 2002). Additionally, end-to-end contacts are characterized by an abundance of cofilin (Harris & Siu 2002), indicative of the involvement of dynamic actin cytoskeleton re-organization in the constant “breaking” and “re-making” of cell–cell contacts at both anterior and posterior ends of the migrating cell.

An interesting feature of csA/gp80 is that its hydrophobic C-terminal region is replaced by a ceramide-containing lipid glycan anchor (Yoshida et al. 2006), which facilitates localization in lipid rafts (Harris et al. 2001a). The clustering of csA/gp80 in rafts promotes cis-oligomerization, which in turn may stabilize the raft domains (Harris et al. 2001b). Coalescence of rafts can facilitate the rapid assembly of csA/gp80 clusters in the plasma membrane and promote the establishment of high avidity trans-interactions of csA/gp80 molecules between cells. The presence of csA/gp80 dimers and oligomers on the cell surface has been confirmed by chemical crosslinking and antibody-induced antigen capping studies. Large adhesion complexes with raft-like properties, together with an abundance of sterols, can be visualized in cell–cell contacts (Fig. 4B), and they can be isolated as a Triton-insoluble floating membrane fraction or low-density membrane fragments after sonication of the plasma membrane. Interestingly, studies of this assembly process in ponticulin-null cells suggest that it can occur without contribution from the actin cytoskeleton (Harris et al. 2003). Since DdCAD-1 has not been detected in lipid rafts (Harris et al. 2001a), the disappearance of DdCAD-1 from contact regions may promote the formation of homogeneous sheets of csA/gp80, which, in turn, may facilitate the side-to-side gliding motion between cells during streaming (Harris & Siu 2002).

CAM involvement during slug formation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

The developmental phenotypes of CAM knockout mutants have revealed major roles of CAMs in morphogenesis. The tgrC1 cells are arrested at the loose aggregate stage (Dynes et al. 1994). Cell differentiation is blocked and components of the extracellular matrix and post-aggregation proteins are not synthesized (Wang et al. 2000). However, cells continue to rotate within the cell mass. In the absence of the slime sheath, the shearing force of the rotational movement leads to the shedding of the csA/gp80 adhesion complexes that have been holding them together, resulting in the dissolution of the aggregates into single amoeboid cells (Liansheng Hou and Chi-Hung Siu, unpubl. data, 2009).

Although transcription of tgrC1 is initiated in the mid-aggregation stage, only background levels of TgrC1 are detected. TgrC1 begins to accumulate rapidly in the peripheral cell layer of loose aggregates (Wang et al. 2000). The peripheral cells migrate to the apex of the mound and eventually differentiate into prestalk cells (Fig. 4C). Furthermore, TgrC1 is cleaved at the juxtamembrane region to release a 145 kDa extracellular fragment, which associates with the extracellular matrix to become part of the slime sheath (Wang et al. 2000). Therefore, the slime sheath may provide a favorable substrate for the migration of the peripheral cells. Even though all cells contain a moderate level of TgrC1 later at the slug stage, cells in the anterior region contain a much higher level of TgrC1 (Dynes et al. 1994; Wang et al. 2000). The differential adhesive environment created by TgrC1, coupled with chemotactic migration, may account for the sorting out of cells and the establishment of the anterior-posterior pattern in slugs (Siu et al. 1983; Palsson 2008).

In addition to cell–cell adhesive interactions, TgrC1-mediated adhesion triggers signaling events that regulate cell-type specification and differentiation. Recent studies show the involvement of the comC and tgrD1 (previously known as lagD) genes in the tgrC1 signaling pathway (Kibler et al. 2003a,b). When developed with wild-type cells in chimeras, knockout strains of comC, tgrD1, and tgrC1 (which are defective in spore formation) are able to sporulate. However, pairwise chimeras of these three mutant strains fail to form viable spores, suggesting that all three genes function in the same signaling pathway, with tgrC1 being the terminal node of this signaling network.

CAMs and cell type proportioning

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

In addition to the Ca2+-dependent cell–cell adhesion, DdCAD-1 plays a role in cell sorting and cell differentiation. Disruption of the cadA gene leads to defects in morphogenesis, although cadA cells are capable of completing the developmental cycle and form fruiting bodies (Wong et al. 2002). Cell sorting is defective among null cells and most slugs show abnormal prespore-prestalk patterns. Cell-type proportion is stringently regulated during development, with ∼20% of the cells forming prestalk cells. However, the cadA-null cells produce taller stalks and smaller sori and the spore yield is reduced by ∼50%, with a corresponding increase in prestalk cells (Wong et al. 2002). The phenotype of knockout cells implicates the involvement of DdCAD-1 in both cell sorting and cell type proportioning.

Since DdCAD-1 redistributes from the contact regions of cells and becomes internalized in cell aggregates, how it may influence cell sorting at a later stage becomes an enigmatic issue. Close examination of cells expressing DdCAD-1-GFP reveals a novel spatial distribution pattern of DdCAD-1 in migrating slugs. Although DdCAD-1 disappears from the cell surface of most cells soon after their entry into the post-aggregation phase, it is retained in the cell–cell contact regions of a small subset of cells located in the tip region of the slug (Sriskanthadevan et al. 2011). Significantly, normal cell sorting is restored when cadA cells incubated with recombinant DdCAD-1, which also becomes internalized in posterior cells but remains in the contact regions of anterior cells. Therefore, the migration of peripheral cells and the morphogenesis of the slug tip at the mound stage may require the coordinated participation of both DdCAD-1 and TgrC1.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

Adhesion molecules are of fundamental importance in the regulation of pattern formation and morphogenesis in multicellular organisms. Comparative genomics has yielded critical insights into the evolutionary origin of cell adhesion and the emergence of multicellularity (Harwood & Coates 2004; Abedin & King 2010). Genetic knockouts of CAMs and ectopic expression of GFP fusion CAM proteins provide powerful tools for the dissection of CAM participation in many cellular and developmental processes besides cell–cell adhesion. Further analysis of the spatiotemporal expression CAMs should lead to a better understanding of the mechanisms that regulate CAM expression and the diverse roles of CAMs during development.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References

Work carried out in our laboratory was supported by an Operating Grant (FRN-6140) from the Canadian Institutes of Health Research. Shrivani Sriskanthadevan and Jun Wang were supported in part by an Ontario Graduate Scholarship; Gong Chen is supported in part by a University of Toronto Open Scholarship; and Chunxia Yang is the recipient of an International Scholarship from the China Scholarship Council.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Regulation of temporal expression of CAMs
  5. Unique structural features of DdCAD-1
  6. DdCAD-1 is targeted for surface presentation by an unconventional protein transport pathway
  7. Coordinated appearance of DdCAD-1 and csA/gp80 during cell aggregation
  8. CAM involvement during slug formation
  9. CAMs and cell type proportioning
  10. Concluding remarks
  11. Acknowledgments
  12. References
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