vCTB cell fusion

Cell fusion is a rare event in human cells that has been reported in physiological events such as fecundation [57], placental formation [58], muscle differentiation [59] and bone maintenance, remodelling and repair [60]. Additionally, in pathophysiological circumstances such as viral entry [61] and cancer [62], cell–cell fusion also takes place. Although certain common features have been described in cell fusion events, each type of cell fusion has its own mechanism.

36 Previous studies have identified proteins that are implicated in vCTB cell fusion and a trophoblastic fusion mechanism has been proposed and accepted by the research community.

The vCTBs fuse upon implantation of the blastocyst into the endometrium to create a multinucleated cell layerthe STB (Figure 12). However, underlying vCTBs continue to fuse with the STB during pregnancy, allowing renewal of the syncytia [63]. In this way, the expression of fusogenic proteins must be stable over time and cell fusion should be tightly controlled. The expression of cell–cell adhesion proteins, such as E-cadherin, is increased in vCTBs prior to cell fusion [64]. Moreover, gap junction proteins such as connexin 43 [65], and tight junction proteins such as zona occludens 1 (ZO-1) [66], are expressed in vCTBs allowing functional inter-trophoblastic communication and favouring cell fusion.

Nevertheless, the most well-studied proteins that are essential for correct cell fusion are the syncytins and their associated receptors [67].

Figure 12. Villous cytotrophoblast fusion and syncytiotrophoblast formation. On the left, a schematic view of a chorionic villous showing the localisation of the different cell types: syncytiotrophoblast (STB), villous cytotrophoblast (vCTB) and extravillous cytotrophoblast (evCTB). On the right, an in-vitro fusion model of vCTB, which leads to formation of the STB. The trophoblast was stained after 24 h and 72 h of culture for desmoplakin (magenta) and nuclei (blue). Scale bar represents 15 µm. Adapted from [68].

37 The syncytins are class I envelope human endogenous retroviruses (HERVs) that were acquired by humans from retroviruses [69-71]. Syncytin-1 (HERV-W) was inserted into the human genome 19–28 million years ago [69,70,72], while syncytin-2 (HERV-FDR) was inserted 40 million years ago [71]. The fusion mechanisms of these proteins are very similar to vesicle–lysosome-facilitated fusion, which is achieved by target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) and vesicular SNARE (v-SNARE) [73]. In fact, they form bundles of alpha helices, resulting in intracellular membrane apposition and fusion, and their engineered expression in the surface cell membrane promotes cell–cell fusion [73-75]. The syncytin receptors are the alanine-, serine- and cysteine-selective transporter 1 or 2 (ASCT1/2) receptor for syncytin-1 [76] and the major facilitator superfamily domain-containing 2 (MFSD2) receptor for syncytin-2 [77].

The viral origin of the syncytin proteins facilitated the understanding of their mechanism in vCTB cell fusion since it was largely studied in the context of virus–cell fusion. The syncytins in vCTBs bind to their receptors and achieve membrane fusion in a pH-independent manner, just as their homologues do in viruses [78,79]. Fortunately, the structure of syncytin-1 and the syncytin-syncytin-1-dependent fusion mechanism have been very well characterised.

As expected from its retroviral envelope protein origin, syncytin-1 is a glycoprotein composed of two units: a surface unit (SU) and a transmembrane (TM) unit. At the same time, the SU is subdivided into a receptor-binding domain (RBD), a CΦΦC (¹⁸⁶⁻CX2C⁻¹⁸⁹) motif, a furin cleavage site (³¹⁴⁻RNKR⁻³¹⁷) and six N-glycosylation sites. The TM unit is also subdivided into different domains, including a fusion peptide (FP), two heptad repeats (HR1 and HR2), a ³⁹⁷⁻Cx6C⁻⁴⁰⁷ domain, a TM anchorage domain (tm), a cytosolic tail (cyt) and one N-glycosylation site. To become a fully functional protein, post-transcriptional

38 modifications need to take place in the endoplasmic reticulum (ER). In fact, the SU and the TM unit are translated together as one unit; however, the SU contains a furin cleavage site that can be cleaved by the furin-convertase enzyme in the ER, separating both subunits.

Subsequently, a disulphide bond between the ³⁹⁷⁻Cx6C⁻⁴⁰⁷ domain of the TM unit and a ¹⁸⁶⁻CX2C⁻¹⁸⁹ motif of the SU is formed (Figure 13) [78-81]. Finally, syncytin-1 forms a functional protein when it exists as a trimer. Trimerisation takes place in the ER, while prior maturation takes place in the Golgi apparatus (GA), and the protein is transported to the cell membrane where it can trigger cell–cell fusion [79].

Figure 13. Schematic representation of the structure of syncytin-1. The signal peptide domain is represented in light blue. The surface unit (SU) is represented in yellow and contains a receptor binding domain (RBD;

SDGGGX2DX2R) and a CXXC motif. The transmembrane unit (TM) contains a fusion peptide and is represented in red, heptad repeats 1 (HR1) are represented in purple, heptad repeats 2 (HR2) are represented in pink, a transmembrane domain is represented in black, an intracytoplasmic domain is represented in green and a CX6CC domain. Between the SU and the TM unit is a furin cleavage site (RNKR) represented in light red.

The Y indicates N-glycosylation sites and numbers indicate the amino acid position. A disulphide bond is formed between the CXXC motif of the SU and the CX6CC domain of the TM unit. Taken from [82].

The mechanism in charge of approaching the cell membranes and accomplish cell–cell fusion begins when syncytin-1 recognises ASCT-1 or ASCT-2 in the target membrane. The RBD of syncytin-1 recognises the receptor located in the target membrane and the first conformational change in the trimer takes place. More concretely, the SU and the TM unit

39 dissociate from each other by breakage of the disulphide bond. The structural modification causes a projection of syncytin-1 fusion peptide towards the top of the protein, allowing it to interact with the target membrane and insert itself into the target membrane [78,79]. The connection of both cell membranes through syncytin-1 generates folding of the HR2 domain, which interacts with the HR1 domain. This interaction reverses the direction of the cell membrane and brings both membranes into close proximity, reducing the free energy needed to overcome the merging barrier (Figure 14) [78,79,82].

Figure 14. Schematic representation of syncytin-1-dependent cell fusion. a. Resting stage. b. RBD (yellow) binding to the hASCT2 receptor (light green). c. Disulphide bond breaking and removing of SU domains, producing a conformational change in syncytin-1 protein leading to insertion of the fusion peptide (red) into the cell membrane. d. Assembly of HR2 (pink) and HR1 (purple). e. Final stage with the membranes in close proximity and initiation of membrane bending. Taken from [82].

Membrane merging is a multistep process where different forces and energies take part. First, the close proximity of cell membranes and contact of opposing outer cell membranes cause

40 dehydration of the contact site. This dehydration reduces hydration repulsion between the outer leaflets of the membranes, leading to fusion of the outer leaflets or hemifusion [78,79,83]. Nevertheless, the proximity of the cell membranes and the reduction in hydration are not sufficient for hemifusion to take place. It has been reported that negatively charged phospholipids, such as phosphatidylserine, need to be located in the outer membrane.

Phosphatidylserine is actively held at the cytosolic side of the cell membrane and internal signals need to be unleashed to achieve the phosphatidylserine flip. [78,79,83-86] The localisation of phosphatidylserine at the outer leaflet of the membrane reduces the energy needed for the cell membranes to fuse, facilitating the process. For successful cell fusion, partial hemifusion or the fusion stalk need to radially expand. The expansion allows the inner leaflets to fuse as well, completing the merge of the vCTB cell membranes and the opening of a pore by which the cell content can mix [79].

As previously mentioned, other factors distinct from the syncytins and their receptors have also been implicated in cell fusion. Some of these factors are the phosphatidylserine flip [87], cadherin-11 [88], caspase-8 [89], CD98 [90], a disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) [91], connexin-43 [65], ZO-1 [66] and glucose-regulated protein 78 (GRP78) [92,93]. Concretely, reduced expression of GRP78 by siRNA leads to a decrease in cell fusion, suggesting that GRP78 plays a role in trophoblastic cell fusion [93].

However, the mechanism of cell fusion linked to GRP78 remains to be investigated.

Importantly, the cell fusion process not only involves merging of the cell membranes and cell content mixing, but several modifications need to take place for the fused cells to act as a new entity. Cessation of the cell cycle takes place during vCTB fusion and STB formation since the newly form cell layer is a terminally differentiated tissue [94]. The STBs do not

41 perform cell cycle divisions. Instead, the material that needs to be eliminated from the cell, including apoptotic nuclei, is released in the form of syncytial knots into the maternal blood flow [95]. In exchange, new mononuclear vCTBs fuse and incorporate into the STB to expand during pregnancy and maintain a constant STB density at the mature state [63].

Moreover, the recently fused cells need to perform cytoskeletal rearrangements to organise the cytoskeleton of the STB according to the new cell necessities. It was previously reported that caspase-8 activation is involved in cytoskeletal rearrangement of vCTBs during syncytialisation. Additionally, caspase-8 is implicated in the phosphatidylserine flip that occurs during STB formation [89]. The occurrence of the recently mentioned events leads to fusion of vCTBs; however, several changes need to take place in the newly formed cell for it to become a mature and functional STB.