Impairment of the actin-dependent transport due to FF-stimulation As previously indicated the function of a correctly folded and mature protein depends on its

correct transport and sorting. Therefore, several proteins initially use microtubules in their intracellular transport (GILBERT et al. 1991; ACHLER et al. 1989) and are delivered to the cell surface via actin filaments (JACOB et al. 2003). An example of this family of proteins is SI, whose transport is substantially hampered in FF-treated COS-1 cells as has been demonstrated by a set of different experimental approaches. SI is known to associate with sphingolipid and cholesterol enriched DRMs (JACOB and NAIM 2001), which serve as a sorting platform to the apical membrane (ALFALAH et al. 1999). Moreover, a putative link between DRMs or membrane rafts and the actin cytoskeleton has been described to be meditated through adapter proteins (BRDICKOVA et al. 2001), which would support the actin dependent transport of SI and therefore explain its impaired trafficking. In contrast, the trafficking of LPH is not affected. This conforms with the results of JACOB and co-workers (2003) who demonstrate that LPH follows an actin-independent pathway that presumably implicates microtubules alone, whose function is apparently not influenced by FF.

COS-1 cells provide a suitable model to assess the transport behavior of intestinal epithelial glycoproteins, although these cells are not epithelial. In fact, sorting pathways at the TGN occur as has been previously shown for segregating SI and LPH (JACOB and NAIM 2001) and also in other non-epithelial cell lines like fibroblasts (CHO, 3T3, BHK) (RUSTOM et al.

2002; PAROLINI et al. 1999; MUSCH et al. 1996; YOSHIMORI et al. 1996).

Unlike COS-1 cells, Caco-2 cells endogenously express a set of intestinal brush border glycoproteins that follow different transport routes to the BBM. Here, an altered actin cytoskeleton also impairs the apical transport of SI and, to a lesser extent, ApN, but since the amounts of both proteins in P1 decrease as well, it does neither induce a missorting to the basolateral membranes nor a stack in the intracellular compartments. KERSTING et al.

(2004) were able to demonstrate that SI is reduced at the BBM in Crohn´s disease, an inflammatory bowel disease (IBD), but it increases in the cytosol. This is in line with a down-regulation of SI in human intestinal epithelial cells by interleukin-6 (ZIAMBARAS et al.

1996) which is enhanced in IBD (ATREYA et al. 2000). Additionally, down-regulation of villin using the anti-sense RNA affects the brush border assembly in Caco-2 cells leading to

DISCUSSION

98

cytosolic distribution may therefore explain the disposition of the proteins. In addition to its impaired trafficking to the apical membrane resulting from an altered cytoskeletal actin, the strong decrease in the BBM expression of SI could be due to a loss of a particular amount of SI that has already reached the BBM. DANIELSON (1995) found that SI also resides in DRMs in the BBM and TYSKA and MOOSEKER (2004) suggest that an interaction of the actin-based motor protein myosin IA with those DRMs is responsible for the retention of SI in the BBM, since a loss of SI is concomitant with low levels of myosin IA. This interaction would also provide a link from SI to the underlying cytoskeleton. TYSKA and MOOSEKER (2004) presume that in the absence of a cytoskeletal linkage, SI may be more susceptible to removal from the cell via the continuous apical membrane shedding that occurs at microvillar tips in Caco-2 cells. In contrast to the latter model, JACOB et al. (2003) reported that SI-containing apical vesicles contain myosin IA that triggers the apical transport along actin filaments. This is in line with observations of FATH et al. (1994), who proposed an active role of myosin IA in the apical targeting of Golgi-derived vesicles. As mentioned before, myosin IA is also a component of the 110kD-CM protein complex that connects the actin derived core bundle of the microvilli with the BBM. The disruption of this complex by, for instance, ATP leads to a dissociation of actin from the BBM. Altogether, these observations suggest that myosin IA could be one of the actin-associated proteins that is also affected by FF-stimulation. The affected trafficking pattern of ApN compared to SI presumably results from a transcytotic pathway (see Fig. 5-2) that this protein partially follows to the apical membrane (MATTER et al. 1990a) and that does probably not require the actin cytoskeleton.

In fact, microtubules have been described to be crucial in the basolateral to apical transcytosis (Fig. 5-2) (LEUNG et al. 2000; BREITFELD et al. 1990; HUNZIKER et al. 1990). This may another fact that explains why the cell surface levels of DPPIV that is targeted to the apical membrane both directly and via transcytosis (see Fig. 5-1) (LE BIVIC et al. 1990; MATTER et al. 1990a), are partially reduced at low FF concentrations, while they are increased at higher FF concentrations. At high FF concentrations, an elevation of α-tubulin in the intracellular membrane fraction P1 was observed. The fact that Caco-2 cells represent a polarized cell line with an apical and a basolateral domain imposes an entirely different challenge compared to non-polarized COS-1 cells. One essential process in preserving this

cellular polarity is the delivery of newly synthesized proteins and lipids to specific sites at the cell surface (JACOB and NAIM 2001),

Figure 5-2: Cytoskeletal tracks and transport routes in polarized epithelial cells

In polarized cells, proteins follow different pathways to reach either the apical or the basolateral membrane domain. Proteins like SI follow a direct pathway to the apical surface that implicates the actin cytoskeleton. In contrast, the transport of LPH, which is also directly transported to the apical membrane, occurs in an actin independent manner that rather implicates microtubules. ApN and DPPIV represent proteins that follow both a direct and an indirect route to the apical membrane. Here, the indirect transport occurs via transcytosis from the basolateral to the apical membrane that possibly requires microtubules.

Source: www.nature.com/.../fig_tab/nature01602_F5.html (modified)

which includes transcytosis of certain proteins. These events are maintained by a sophisticated sorting process within the TGN that implicates specific signals for the apical and basolateral domains (GRIFFITHS and SIMONS 1986), and an organized post-TGN protein traffic that requires coordinated interactions between different cytoskeletal systems (MAYS et al. 1994; FATH et al. 1993). Therefore, the discriminative sorting signals of ApN and DPPIV may lead to differences in their appearance at the BBM, although both of them

DISCUSSION

100

its association with microdomains containing cholesterol and sphingolipids (ALFALAH et al.

2002), the sorting of ApN implicates neither O-linked nor N-linked glycans and is driven most likely by carbohydrate-independent mechanisms (NAIM et al. 1999). Although the apical sorting of DPPIV has more in common with the sorting of SI than with the one of ApN, the impaired transport to the BBM after FF-treatment of the latter one shows more similarities to that of SI. However, transport studies in Caco-2 with microtubule reducing agents revealed a transport competence of ApN to the BBM of approximately 80% of the newly synthesized protein, whereas DPPIV is rather resident at the basolateral membrane (GILBERT et al.

1991). The high rate of apically transported ApN to the BBM in the absence of microtubules suggests a direct route that implicates actin, whereas only a small amount of ApN (approximately 10%) follows a transcytotic pathway along microtubules. In contrast, the transport of DPPIV is apparently more hampered by disruption of the microtubule system (GILBERT et al. 1991). Together with the present results on FF, it can be presumed that DPPIV follows a direct route along actin to a certain extent, which would explain the decrease after treatment with 5mg and 25mg FF. On the contrary, the main transport of DPPIV presumably occurs through the basolateral membrane via microtubules, which in turn leads to the increase of DPPIV in the BBM fraction concomitant with elevated amounts of α-tubulin in cells treated with 100mg FF.

In comparison to SI, ApN and DPPIV, the targeting of LPH remains unaffected by stimulation with gliadin peptides, as previously demonstrated in COS-1 cells. Although it has been shown here that the BBM-expression of LPH is not affected by FF-treatment which presumably results from its actin independent transport to the apical membrane several studies reveal a certain correlation between CD and lactose intolerance (RADLOVIC et al. 2009). For example, OJETTI et al. (2005) found a high prevalence of CD in patients with lactose intolerance. Lactose intolerance is the most common intestinal disorder associated with an absence or drastically reduced levels of intestinal LPH (NAIM 2001), which opposed to the results of this study. The generally low expression level of LPH in Caco-2 cells consequently leads to the creation of an over-expressing hybrid clone. Although no evidence could be found that the over-expression has altered the behavior of LPH or the Caco-2 cells per se, it cannot be excluded that the nature of LPH in enterocytes differs from that of Caco-2 cells.

Contrarily, the decrease in the levels of LPH in enterocytes of patients with CD that possibly

trigger lactose intolerance may also be derived from mucosal lesions or villous atrophy (PRASAD et al. 2008) which often results in a consecutive malabsorption syndrome (CICLITIRA and MOODIE 2003; SOLLID 2002; MARSH 1992).

However, the specific effect of FF on the targeting of a specific population of proteins and the discriminatory role of actin is supported by the observation that OVA, which has been frequently utilized as a negative control in the analysis of pathomechanisms of CD (SHIDRAWI et al. 1995), does neither induce changes in the actin cytoskeleton nor in the transport competence of SI. Since OVA-stimulation has no impact on the enzymatic activity levels of brush border SI, the dramatic reduction in its activity levels after FF-stimulation is directly associated with an altered trafficking and could partially contribute to the onset of carbohydrate malabsorption in patients with CD.

5.4 FF treatment hampers endocytic events and subsequent membrane