Signaling at the Golgi Apparatus During Cell Migration and Implication for Cancer Cell Metastasis

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Signaling at the Golgi Apparatus During Cell Migration and Implication for Cancer Cell

Metastasis

Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.)

Presented by Valentina Millarte

At the

Faculty of Sciences Department of Biology University of Konstanz

Date of the oral examination: 03.07.2015 First referee: PD Dr. Hesso Farhan Second referee: Professor Daniel F. Legler

Konstanz, 2015

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-296696

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ABSTRACT

Cell migration is a complex cellular event involved in tissue morphogenesis and repair.

Therefore, understanding the regulation of cell migration is of fundamental biomedical importance. Mounting evidence suggests a prominent role of the secretory pathway in cell migration. Disruption of Golgi integrity and failure of the Golgi to re-orient towards the leading edge in migrating cells have been shown to block directional cell movement. However, the link between the morphology of the Golgi apparatus and directional cell migration has so far not been analysed using a systematic approach. We performed an RNAi screen to identify proteins that regulate both, cell motility and Golgi structure. Our data uncover a strong connection between Golgi morphology and directional motility with almost 70% of knockdowns that affect the Golgi also inhibiting migration. Among the hits of our screen we focused on PLCγ1. Silencing PLCγ1 led to a significant reduction of the Golgi volume, together with a 50% inhibition of cell migration. Notably, knockdown of PLCγ1 did not inhibited the directionality of cell movement, but rather the speed of migration. Previous research has already described PLCγ1 as a regulator of cell migration that is activated downstream of several growth factor receptors leading to reorganization of the actin cytoskeleton. The role of PLCγ1 in cell motility is further underscored by the observation that the expression of this lipase is elevated in cancer cells, and it correlates with high migration power and invasiveness. The rationale for focusing on PLCγ1 is that no evidence has so far linked this protein to the secretory pathway. We show that PLCγ1 acts in early stages of anterograde secretory trafficking. Depletion of PLCγ1 not only leads to a reduction of Golgi volume, but also to a lower number of endoplasmic reticulum exits sites (ERES), and peripheral ERGIC structures. Mechanistically, we show that the effects of PLCγ1 on the secretory pathway are dependent on it interaction with the tethering factor p115. Moreover, the effects of PLCγ1 on cell motility are also dependent on the interaction with p115. Strikingly, all the phenotypes observed upon PLCγ1 knockdown were unrelated to the catalytic activity of this phospholipase. To the best of my knowledge, this is the first demonstration of an effect of PLCγ1 that is independent from enzymatic activity and I therefore propose that I have identified a new scaffolding function for this protein. Another important finding from my work is that this is the first mechanistic link between pre-Golgi secretory trafficking and cell migration.

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ZUSAMMENFASSUNG

Die Zellmigration ist ein komplexer zellulärer Prozess, welcher von Bedeutung für die Morphogenese und Reparatur von Geweben ist. Daher ist es wichtig die Regulation der Zellmigration besser zu verstehen. Eine Reihe von Hinweisen deutet auf eine prominente Rolle des sekretorischen Apparates in der Regulation der Zellmigration. Zum Bespiel führt eine Störung der Integrität des Golgi-Apparates zu einer Hemmung der gerichteten, polarisierten Zellmigration. Trotz weitegehender Akzeptanz der Tatsache dass der Golgi für die Zellmigration wichtig ist, fehlt uns sein systematisches Verständnis wie dieser Zusammenhang reguliert wird.

Wir haben einen RNAi Screen durchgeführt um nach Regulatoren zu suchen, welche sowohl die Zellmigration als auch die Golgi Struktur regulieren. Unsere Daten zeigen einen starken Zusammenhang zwischen der Golgi-morphologie und der Zellmigration, da 70% der Hits welche die Zellmigration störten auch eine Störung der Golgi-morphologie aufwiesen. Unter den Hits haben wir uns genauer mit PLCγ1 beschäftigt. Die Depletion von PLCγ1 führte zu einer signifikanten Reduktion des Golgi-volumens und einer 50%igen Hemmung der Zellmigration.

Beachtenswert ist, dass PLCγ1 eher die Geschwindigkeit der Zellmigration als ihre Richtung beeinflusste. Frühere Arbeiten konnten zeigen dass PLCγ1 ein Bestandteil von Signalkaskaden ist, welche zur Regulation des Aktin-zytoskelettes führen und somit die Zellmigration auch beeinflussen. Weiterhin wird die Rolle von PLCγ1 in der Zellmigration dadurch untermauert dass diese Phospholipase in verschiedenen Tumoren erhöht ist. Wir haben uns dennoch mit PLCγ1 beschäftigt da bisher keine Daten existieren welche dieses Protein mit dem sekretorischen Apparat verbinden. In meiner Arbeit konnte ich zeigen dass PLCγ1 an einem führen Punkt der Sekretion ansetzt und den anterograden Transport zwischen dem endoplasmatischen Retikulum (ER) und dem Golgi reguliert. Die Depletion von PLCγ1 führt nicht nur zu einer Reduktion des Golgi-Volumens, sondern auch zu einer niedrigeren Anzahl der ER exits sites (ERES), und des peripheren ERGIC. Mechanistisch zeigen meine Daten dass der Effekt von PLCγ1 auf die Sekretion von dessen Interaktion mit dem Vesikel-Bindungsfaktor p115 ist. Die Effekte von PLCγ1 auf die Zellmigration waren auch von der Interaktion mit p115 abhängig.

Interessanterweise, waren alle beobachteten Effekte von PLCγ1 vollkommen unabhängig von dessen katalytischer Aktivität. Nach bestem Wissen ist das die erste Beschreibung von Effekten des PLCγ1 die unabhängig von dessen katalytischer Aktivität sind. Daher zeigt meine Arbeit die Identifikation einer sogenannten Scaffolding (Gerüst) Funktion des PLCγ1. Ein weiterer

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wichtiger Aspekt meiner Arbeit ist dass es die erste Beschreibung einer Rolle des pre-Golgi Transportes in der Zellmigration darstellt.

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INTRODUCTION

1. Introduction to the secretory pathway

The secretory pathway in eukaryotic cells is used to send proteins and lipids from the endoplasmic reticulum (ER) to the plasma membrane and membrane-bound organelles and to release material outside the cell. There are two types of secretion: constitutive and regulated.

Constitutive secretion is the default pathway and is used primarily to replenish material at the plasma membrane and internal organelles. Regulated secretion proceeds basically like the constitutive section, except that the journey of the protein terminates in secretory vesicles, which store secreted material until a signal triggers fusion with the plasma membrane. What allows cargoes to take the route of the regulated secretion is the recognition of particular sorting sequences within their structure (Pollard T. D., Easrnshaw W.C., et al. Cell Biology. Second edition. Saunders Elsevier. Cellular Organelle and Membrane Trafficking. p. 313-314).

The existence of the secretory pathway was shown for the first time between the 1940 and the 1950 by the George Palade and his team in pancreatic acinar cells by labeling newly synthesized proteins with radioactive amino acids. Under autoradiography, the newly synthesized proteins where detected in different membrane-bound compartments, indicating the newly synthesized proteins move between these compartments and some proteins finally end up being secreted to the extracellular milieu. (Palade and Porter, 1954; Palade, 1975)

Some years later, the knowledge of the secretory pathway became broader with the discovery of the ER exit sites (ERES), or transitional ER, where newly synthesized protein are packaged into COPII vesicle (Farquhar and Palade, 1981; Bannykh et al, 1996), and of the ER-to-Golgi intermediate compartment (ERGIC) (Griffiths et al, 1994, Schekman 1992; Appenzeller-Herzog and Hauri, 2006).

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2. The endoplasmic Reticulum

2.1 Structure and functions

The endoplasmic reticulum (ER) is the largest organelle of eukaryotic cells. It consist of a network of continuous membrane-enclosed tubules and sacs (cisternae) that extends from the nuclear envelop throughout the cytoplasm. We distinguish two different types of ER performing different functions within the cell, the rough ER (RER) and the smooth ER (SER). The rough ER is covered by ribosomes on its cytosolic surface and is the place where protein synthesis, takes place. The smooth ER is free of ribosomes and is involved in steroid synthesis (mainly in endocrine cell), and drug metabolism (mainly in hepatocytes). The ER lumen is enriched in chaperones, oxidoreductases and isomerases, enzymes that are important to ensure proper folding and assembly of newly synthesized proteins, and in enzymes regulating post-translational modifications of the newly synthesized proteins. The major example of ER enzyme involved in post-translational modification is represented by the oligosaccharyltransferases, which catalyzes protein N-glycosylation (Nilsson and Heijne, 1993; Braakman and Hebert, 2013).

In case proteins are misfolded, luminal chaperones are able to “correct” the structure of misfolded proteins but in case of failure, incorrectly folded proteins are either sent back to the cytoplasm where they undergo degradation, or accumulate in the ER at high levels leading to ER stress and unfolded protein response (UPR). Activation of the UPR triggers the expression of a wide set of genes such as chaperones, vesicles coats, membrane fusion machinery. The goal of the UPR is to help the cell cope with the stress due to accumulation of misfolded proteins.

The ER lumen also represents the major Ca2+ storage within the cell. The ER membranes are rich in Ca2+ pumps, responsible for regulation of the release and uptake of calcium ions in response to cellular signals.

Another function of the ER membranes is to form the inner and the outer nuclear envelope; the inner nuclear envelope contains proteins specialized to interact with chromatin, while the outer envelope allows the movement of molecules between the nucleus and the ER.

All proteins are synthesized in the cytoplasm and the ones destined for ER translocation contain a hydrophobic signal sequence or a transmembrane signal segment. The signal sequence is an N-

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terminal leader sequence consisting of 15-30 amino acids containing and hydrophobic core of at least 6 residues. The transmembrane signal sequence contains instead a hydrophobic region of 16-25 aa. Newly synthesized proteins tagged with one of these sequences are transferred to ER membranes via a cotranslational pathway or posttranslational one. In the first case the translocation occurs concurrently with the protein synthesis by membrane-bound ribosomes. In case of the posttranslational pathway, protein translocation to the ER occurs only after the protein has been fully synthesized in the cytosol.

Once the peptide sequence has been grown to about 150 residues the signal sequence is removed by a signal peptidase in the ER lumen. The remaining cleaved peptide (cargo), which is normally destined to other cell compartments, is next folded and leaves the ER within vesicles, at the level of ER exit sites (ERES).

2.2 ERES and COPII vesicles

The work of Palade showed that in mammalian cells translation of proteins in the ER was followed by their entry in ribosome-free ER-sub domains before being transported to the Golgi (Palade and Porter, 1954; Palade, 1975; Cooper G.M. The Cell: A molecular Approach. 2nd Edition. Sunderland (MA). Sinauer Associates 2000; The Golgi Apparatus). Mammalian cells are characterized by several hundred of these ER-sub domains, also named transitional ER or ER exit sites (ERES), spread through the cytoplasm and mainly clustering at the perinuclear region (Hong and Tang, 1993; Shaywitz et al, 1995; Tang et al, 2000). Studies performed by time-lapse imaging showed that these small structure, with diameter of approximatively 0.5-2 µm, are immobile or mainly move in short distance (Stephens et al, 2000; Hammond and Glick, 2000;

Gupta et al, 2008). The number of ERES in mammalian cells varies within the cell cycle. They have been shown to proliferate during interphase and to be in G2 almost in double number than in G1 (Hammond and Glick, 2000; Stephens, 2003). SEC16A is a major constituent of the ERES (Connerly et al 2005; Ivan et al, 2008) that was first identified in yeast in a screen for regulators of the secretory pathway (Novick et al, 1980; Novick et al 1981). Further studies in Drosophila and mammalian cells attributed to SEC16A a major role in ERES organization. In both mammals and flies, siRNA depletion of SEC16A led to a drastic reduction of the ERES number and to inhibition of ER transport (Watson et al, 2006; Bhattacharyya and Glick, 2007; Iinuma et al,

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2007; Tillmann et al, 2015; Ivan et al, 2008). Moreover, two recent works described SEC16A as a linking platform between mitogenic signaling, proliferation and secretion. Experiments performed with siRNA knockdown or overexpression of SEC16A showed indeed that proliferation and number of ERES are phenomena strictly dependent on the cellular level of SEC16. Particularly, the number of ERES increases upon SEC16 overexpression, together with a higher rate of cell proliferation, while siSEC16 depletion leads to the opposite effects.

Furthermore, also ERK2-dependent phosphorylation of SEC16A leads to an increase of the number of exit sites and positively influences cell proliferation. (Farhan et al, 2010; Tillmann et al, 2014).

At the level of ER exit sites, secretory proteins are packed into COPII-coated vesicles (60-80nm diameter). The assembly of the COPII coat around the secretory cargos is initiated by the recruitment of the small GTPase SAR1. The GDP/GTP exchange promoted by the guanine exchange factor SEC12 mediates to activation of SAR1, event that in turn lead to exposure of its N-terminal amphipathic region which dips into the ER membrane (Lee et al, 2005; Bielli et al, 2005). This step is crucial for ER membrane deformation (Lee et al, 2005). An important role for the activated SAR1 is the recruitment of the SEC24-SEC23 heterodimers, the inner COPII coat (Yoshihisa et al, 1993). The inner COPII coat, and especially SEC24, is responsible for binding cargo proteins exiting the ER (Mossessova et al, 2003; Miller et al, 2002; Miller et al, 2003). The following step consists in the complete assembly of the COPII coat via the recruitment of the outer COPII coat, formed by of SEC13-SEC31 heterotetramer (Lederkremer et al, 2001).

Importantly, the recruitment of the outer coat results in the activation of SEC23 which is the GAP (GTPase-activating protein) for GTP hydrolysis on SAR1 (Yoshihisa et al, 1993; Bi et al, 2007).

Hydrolysis of GTP by SAR1 assures COPII vesicles scission from the ER and COPII-coat disassembly (Oka and Nakano, 1994). Experiments conducted in vitro, showed that purified SEC23-SEC24, SEC13-SEC31 and SAR1 are the minimal components required for reconstitution of COPII vesicles (Matsuoka et al, 1998) and that SEC12 is needed for GTP- dependent budding of these vesicles (Futai et al, 2004). Anyway, in vivo many more factors have been showed to play important roles in the regulation of the COPII-coat assembly, and of COPII vesicle scission from the ER membranes. In mammals for example, several kinases act as modulator of COPII budding (Lee and Linstedt, 2000). To mention some examples, SEC24C/D phosphorylation by the serine/threonine kinase AKT positive regulate the binding between

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SEC24 and SEC23, thus allowing the assembly of the COPII coat (Sharpe et al, 2011). CK2 is another kinase which has recently been shown to have an important role in ER export. SEC31 phosphorylation by CK2, diminishes the affinity for SEC31 and SEC23. Since SEC23 is the COPII component responsible for the inactivation of SAR1 and since the activity of SEC23 towards SAR1 is tenfold enhanced by the interaction with SEC31, depletion of CK2 results in the inhibition of the COPII coat assembly (Antonny et al, 2001; Koreishi et al, 2013). PCTAIRE is a kinase also necessary for ER export, but its role is not understood. It interacts with SEC23 although it does not mediate its phosphorylation and its kinase activity is not required for ERES regulation (Palmer et al, 2005).

Cargo export from the ER and regulation of ERES are thus complex events which involve the interplay of a wide variety of signaling. Although at present there is a lot known about the mechanisms involved in COPII-coat assembly and vesicle budding at the ERES, many further studies are still required to gain a complete insight into these fundamental processes.

2.3 Vesicle Budding, SNAREs and Tethering factors

The budding of transport vesicles and the selective incorporation of cargo into the forming vesicles are both mediated by protein coats. The coats are involved in cargo selection as they recognize sorting signals tagging transmembrane cargo proteins. Once the coats recognize specific sorting signals, they deform flat membrane patches into round buds, reaction followed by the release of the coated vesicles (Bonifacino and Lippincott-Schwartz 2003; Bonifacino and Glick, 2004). Clathrin coats participate in the vesicular transport of cargo destined to the plasma membrane, endosomes and TGN. Non-clathrin coats are instead involved in vesicular transport within the early secretory pathway. The COPII coat, which mediates transport from the ER to the ERGIC (and from the ER to the Golgi in yeast, where the ERGIC is absent), and has been already described in the previous paragraph, belongs to this category. Another kind of coat is COPI, that like the COPII-coat also consists of a complex of different coatomers organized in an inner and an outer-coat (Szul and Sztul, 2011). COPI-coated vesicles are involved in intra-Golgi transport and in retrograde vesicular traffic from the Golgi to the ER through the ERGIC (Waters et al, 1991; Barlowe et al, 1994; Letourneur et al, 1994; Bonifacino et al, 2004). A deeper description

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of the COPI-coat and of its role in the secretory pathway will be thematic of the chapter 4

“Coatomer and COPI”.

2.4 Vesicle Tethering and Fusion

SNAREs proteins: after tethering to the membrane of the acceptor compartment, the vesicle needs to undergo fusion in order to deliver its content to the next station along the secretory route. The first factor discovered to be important in this step was the N-ethylmaleimide-Sensitive Factor (NSF), existing in cytosolic and membrane bound form (Glick and Rothman, 1987; Block et ak, 1988 Malhotra et al, 1988). NSF is the mammalian orthologue of the yeast Sec18p, a factor that was shown to play a role in ER-to-Golgi trafficking (Wilson et al, 1989; Eakle et al, 1988).

NSF belongs to the family of ATPases associated with diverse cellular activities (AAA family) and as many members of this family, NSF forms homo-hexamers. In order to bind membranes, NSF requires a peripheral membrane proteins that was called soluble NSF association protein (SNAP, also known as Sec17p in yeast). In mammals, three isoforms of SNAP exist, α-, β-, and γ-SNAP and while α-SNAP and γ-SNAP are ubiquitous, β-SNAP appears to be brain-specific (Clary et al, 1990; Zhao et al, 2012). With respect to the early secretory pathway, α-SNAP appears to be more important since γ-SNAP was shown to have only a weak activity to regulate Golgi transport (Clary et al, 1990). SNAP is itself recruited to membranes via proteins initially called SNAP receptors (SNAREs). SNAREs are now recognized to be the minimal machinery required for membrane fusion not only in the secretory pathway (Weber et al, 1998) but also in the endosomal system (Ohya et al, 2009). SNAREs are divided into two classes: those on the vesicles (v-SNAREs) and those located on the target membrane (t-SNAREs). Newer nomenclatures propose to divide them, into Arginine (R)-SNAREs and Glutamine (Q)-SNAREs (Fasshauer et al, 1998), but I will use the classical terminology that is based on SNARE localization. Typically, v-SNAREs are similar to synaptobrevin, while t-SNAREs are either syntaxin-like or SNAP-25-like. Syntaxins and synaptobrevins are c-terminally anchored membrane proteins, while SNAP-25 binds membrane via a covalently linked palmitate residue lying in the central part of the protein (Bonifacino et al, 2004). SNAREs contain a heptad repeat motifs consisting of 60-70 amino acids which originate coiled-coil protein structures (Bock et al, 2001). Pairing of the v- and t-SNAREs results in the formation of the trans-SNARE complex which involves the formation of long, parallel four-helix bundle; in this bundle, one α-helix is provided by the v-SNARE associated with the vesicular membrane and three helices from the t-

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SNAREs on the target compartment (Poirier et al, 1998; Sutton et al, 1998; Sztul and Lupashin 2006). In each SNARE complex, three glutamines and one arginine form a central ionic layer in the hydrophobic core of the four-helix bundle (Sutton et al., 1998). After the fusion reaction between a coated vesicle and the acceptor membrane, the trans-SNARE complex persists and become a cis-SNARE complex in the fused membrane. At this point, α-SNAP can bind to the SNARE complex and recruit NSF from the cytosol, which in turn hydrolyses ATP leading to dissociation of the SNARE complex (Rice and Brunger, 1999; Mayer at al, 1996; Yu et al, 1999).

Thus, the role of α-SNAP and NSF is not to promote fusion reactions but have instead an important role in SNAREs recycling.

Tethering factors: the tethering factors described up to now are classified in two major categories, the coiled-coil tethers and the multisubunit tethering complexes. Together with their role in bridging membranes, they are thought to be central regulators of SNARE complex assembly (Shorter et al, 2002; Sapperstein 1996), cargo selection (Morsomme and Rietzman 2002; Roti et al, 2002), coat assembly/disassembly and signaling events (Preisinger et al, 2004; Hicks and Machamer, 2005).

Among the coiled-coil tethers (see Review Sztul and Lupashin, 2006), the most widely described are p115, GM130 and Giantin. p115 is a parallel homodimer with a long tail consisting in four coiled-coil domains and two globular heads (Sapperstein et al, 1996). The four coiled-coil regions are separated by proline-rich “hinge” that facilitate rotation relative to the polypeptide backbone (Yamakawa et al, 1996; Sztul and Lupashin, 2006).

p115 is a peripheral membrane protein localizing at the level of ERGIC and at the cis where it binds GM130 (Waters et al, 1992; Nelson et al, 1998; Nakamura et al, 1997; Seemann et al, 2000) and at the medial Golgi, where it binds Giantin (Linstedt et al, 1993; Sonnichsen et al, 1998). Uso1p, the yeast homolog of p115, has been shown to promote ER-to-Golgi SNARE complexes, and thus to act as vesicle docking (Sapperstein et al, 1996; Nakamura et al, 1997).

Studies in yeast showed that Uso1p promotes ER-to-Golgi transport (Nakajima et al, 1991), mediates COPII vesicle tethering at the Golgi (Barlowe, 1997) and regulates sorting of cargoes into COPII vesicles (Morsomme et al, 2003; Morsomme et Riezman, 2002). In mammalian cells, the first studies on p115 assigned it a role in COPI-vesicle mediated intra-Golgi transport (Waters et al, 1992; Clary and Rothman 1990; Sapperstein et all 1996). This evidence was strongly supported first by the detection of p115 on COPI vesicles (Malsam et al, 2005) and later by the

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identification of the interaction between p115 and the β-COP subunits of the COPI-coat (Guo et al, 2008). In vivo studies on mammalian cells demonstrated also a key role for p115 at the pre- Golgi stage of vesicle traffic. In these studies, depletion of p115 from cells led to a strong Golgi fragmentation into ministacks, ant to the inhibition of ER-to-Golgi secretion of VSVG (vesicular stomatitis virus glycoprotein) at the ERES level while secretion of soluble proteins was not inhibited but rather delayed (Alvarez et al, 1999; Puthenveedu and Linstedt, 2004; Sohda et al, 2005; Sohda et al, 2007; Grabski et al, 2012). Protein trafficking from the ER to the Golgi is thus much more dependent on p115 then secretion of soluble protein. The function of p115 in ER-to- Golgi traffic is to tether ERES-derived COPII vesicle to the Golgi membrane and allow vesicle- membrane/Golgi membrane heterotypic fusion (Allan et al, 2000; Alvarez et al, 2001). Moreover, in vitro studies showed p115 also to be strictly required for homotypic fusion of COPII vesicle to generate larger structures, which some experts hypothesized to be ERGIC structures.

Unfortunately, up to now there are no studies able to prove or discard this hypothesis and what we have is only a speculation about the model mechanism of action of p115 in the ERGIC biogenesis. This model will be discussed in details in the chapter “ERGIC: biogenesis and functions”.

GM130 is an extended rod-like protein with 6 coiled-coil domains which localizes to cis-Golgi cisternae, by binding the Golgi structural protein GRASP65 (Nakamura, 1995; Barr, 1998).

Likely p115, also GM130 has been shown to be strictly necessary for cargo trafficking in in vivo experiments. The addition of anti-GM130 antibodies to semi intact cells in fact interferes with VSVG delivery at the Golgi (Alvarez et al, 2001). Moreover, as already described in the previous paragraph, GM130 is the binding partner of p115 at the cis-Golgi, thus allowing COPII vesicle (in yeast) or ERGIC targeting/fusion with the Golgi membranes (Moyer et al, 2001).

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The following table gives a general overview about other coiled-coil tethering factors.

Name in mammals

Name in

yeast Localization Function References

p115 (VDP;

TAP9) uso1 ERGIC, Golgi

tethering/fusion of COPII- coatedvesicles

Nakajima et al,1991; Sappertstein et al, 1996; Sapperstein et al, 1995; Allan et al 2000; Yamakawa et al, 1996; Linstedt et al, 2000; Nakamura et al, 1997; Brandon et al, 2003; Shorter

et al, 2002; Lupashin et al, 1996; Alvarez et all, 2001

GM130 (GOLGA2;

Golgin-95)

cis-Golgi cisternae

Binding partner of p115 at the cis-

Golgi. COPII vesicles (yeast) or ERGIC fusion with

the cis-Golgi

Nakamura et al, 1995; Barr, 1999; Barr et al, 1997; Barr et al, 1998; Moyer et al, 2001; Nakamura et al, 1997; Preisinger et

al, 2004

GIANTIN (GOLGB1;

CCP372)

cisternalrims of the medial Golgi

Tether for COPI vesiclesduringrecy cling from distal to

proximal Golgi cisternae

Sonnichsen et al, 1999; Misumi et al, 2001; Lesa et al, 2000;

Barr, 1999; Linstedt et al, 1993

Golgin-45

(BLZF1; JeM-1)

cis and medial Golgi

Experimetal data suggest a role in COPII tetheringat

the Golgi.

Mechanismnotkno

wn Short et al, 2001; Barr and Short, 2003

Golgin-84

(GOLGA5)

Golgi integralprotein

Experimetal data suggest a role in COPII tetheringat

the Golgi.

Mechanismnotkno wn

Diao et al, 2003; Satoh et al, 2003; Malsam et al, 2005;

Bacom et al 1999

Golgin-97

(GOLGA1) TGN

Tought to be involved in tethering events

from the endosomes to the

TGN Barr, 1998; Luke et al, 2003; Lu and Hong, 2003

Golgin-245 (GOLGA4;

p230; tGolgin-

1) Imh1 TGN

Tought to be involved in tethering events

from the endosomes to the

TGN

Barr,1998; Luand Hong, 2003; Panic et al, 2003; Gleeson et al, 1996

GRASP55 (GORASP2;

GOLPH6; GRS2)

medial Golgi cisternae

Stacking of Golgi cisternae

Short et al, 2001; Kuo et al, 2000; Shorter et al, 1999; Barr et al, 2001; Jesch et al, 2001

GRASP65 (GORASP1;

p65; GOLPH5)

cis and medial Golgi cisternae

Stacking of cis and medial Golgi cisternae. Tough to partecipate in

COPII

vesicletethering Barr et al, 1997; Barr et al, 1998; Marra et al, 2001

Scheme1: Coiled-coil tethering factors.

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The multisubunit tethering complexes have also been shown to interact with small GTPases and SNAREs.

COG (Conserved Oligomeric Golgi) is a complex of eight subunits (COG1-8) (Loh and Hong, 2004; Suvorova et al, 2001; Suvorova et al, 2002; Ungar et al, 2002) which physically interact with the COPI coat and intra-Golgi SNAREs. Thus, the COG complex is believed to act as tethering factor for COPI vesicle during retrograde trafficking (Suvorova et al, 2002). Moreover, experiments in yeast suggest an additional role of COG in ER-to-Golgi anterograde trafficking (Morsomme and Riezman, 2002; VanRheenen et al, 1999; Wuestehube et al, 1996). Functional studies identified the subcomplexes COG1-4 and COG5-8; the current hypothesis is that the pairing of the two subcomplexes might ensure correct membrane tethering (Loh and Hong, 2004).

The Golgi Associated retrograde protein (GARP) complex has been well studied in yeast model and recently identified in mammalian cells. It localizes at the late Golgi/TGN and consists in 4 subunits: Vps51p, Vps52p, Vps53p, Vps54p. The role of this complex is to tether endosome- derived and lysosomal-derived vesicles to the TGN. Mutations in the subunits in fact lead to mislocalization of late Golgi membrane proteins (Conibear et al, 2003; Conibear and Stevens 2000; Liewen et al, 2005).

Bet5p, Trs20p, Bet3p, Trs23p, Trs33p, Trs31p and Trs85p are the subunits constituting the TRAPPI (transport protein particle) complex. The laboratory of Ferro-Novick showed in 2000 that COPII vesicle specific bind Golgi-associated TRAPPI, without the requirement of other tethering factors, in vitro COPII vesicle reconstitution assays (Barrowman et al, 2000). The TRAPPI complex has been shown to act as GEF for the yeast homolog RAB1 (Ypt1p). (Wang et al, 2000). This finding evidence support the model that TRAPPI might act as Golgi receptor for COPII vesicle and activate Ypt1p, which in turn then recruit other tethering factors (Barrowman et al, 2000). Recent studies in mammalian cells fully support this model. In fact, transport assays in semi-intact cells interestingly showed that ER-to-Golgi trafficking of cargo requires COPII/BET3/RAB1/α-SNAP/GS28 SNARE sequential interaction (Loh et al, 2005).

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The TRAPPII complex consist in the same seven subunits of the TRAPI complex plus three additional ones: Trs65p, Trs120p, Trs130p (Sacher et al, 2001). This complex is thought to be essential for intra-Golgi trafficking. Also TRAPII acts as a GEF, in this case for the yeast trans- Golgi protein Ypt31/32 (Jones et al, 2000).

2.5 Recruitment of the tethers to the membrane

ARL-mediated recruitment. Active GTP-bound forms of ARL facilitates recruitment of protein containing the GRIP domain (typical of golgin 97 and golgin 254) at the TGN. Studies on golgin- 245 showed that the GRIP domain homodimerizes and bind ARL1-GTPs (Lu and Hong, 2003;

Luke at al, 2003; Panic et al, 2003; Wu et al, 2004). In yeast ARL1 is recruited to membranes by active ARL3, thus a cascade of ARLs seems to be required for membrane association of GRIP- containing golgins. This seems to be also the case in mammalian cells. Membrane association of the human golgin-97 in yeast needs indeed Arl1p-GTP and Arl3p-GTP (Setty et al, 2003).

RAB-mediated recruitment. RABs cycle between membrane-associated GTP-bound form and GDP-bound cytosolic state. GTP-bound RABs are important for the recruitment of specific tethering factor at the membrane (Allan et al, 2000). The specific role of RAB1 in the recruitment of the tether p115 has been already discussed above. Anyway, RABs do not look like to act alone in tethers recruitment; p115 is indeed still able to target membranes upon overexpression of dominant negative inactive RAB1 (Alvarez et al, 2003). Furthermore, Satoh and collegues found that although golgin-84 and GRASP65-anchored GM130 are recruited to membrane independently on RAB1, they are however bound to RAB1 (Satoh et al, 2003). These observation together rise the hypothesis that RABs might not be involved in tethers recruitment but in the stabilization of the tethering; another hypothesis suggests RABs as regulators of the interaction between tethering factors and SNAREs (Sztul and Lupashin, 2006).

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19 Scheme 2: Model for tethering-fusion during membrane traffic

2.6 Membrane dynamics of the tethering factors

Some tethers have been shown to cycle between membrane-associated and cytosolic states.

GRASPs and p115 follow this rule, while the TRAPP complex seems to be tightly bound to membranes (Barrowman et al, 2000). In 2006 the laboratory of Elizabeth Stzul described the cycle of GFP-tagged p115 between Golgi membranes and cytosol by measuring the on kinetics (cytosol-to-Golgi) and off-kinetics (Golgi-to-cytosol) of p115-GFP via FRAP (fluorescence recovering after photobleaching) and FLIP (fluorescence loss in photobleaching) respectively.

FLIP experiments showed a rapid membrane/cytosol exchange of Golgi-localized p115-GFP after photobleaching at the cell periphery (t1/2 ∼20 s). Likewise, also the on-kinetic of p115-GFP measured as fluorescence recovery after photobleaching at the Golgi, resulted to occur rapidly,

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with a t1/2 ∼13 s) (Brandon et al, 2006). Interestingly, p115 recruitment at the Golgi was found to not require GM130 and Giantin, since p115 mutants missing the binding sites for GM130 and Giantin exhibited similar dynamics as the wild type protein. The study identified instead RAB1 and SNAREs as modulators of p115 on-kinetic to the Golgi membranes. Indeed, in cells overexpressing the inactive RAB1-N121I, p115-GFP exchange from the cytoplasm to the Golgi measured by FRAP, occurred more rapidly than in control cells (t1/2 ∼8 s versus t1/2 ∼13 s in control cells). The opposite effect was instead observed upon expression of dominant negative mutant of NSF. NSF is a chaperone-like protein involved in SNARE complex disassembly and overexpression of its ATPase-deficient NSF-E329Q mutant has been shown to prevent the binding between endogenous NSF and SNAREs with consequent inhibition of SNARE disassembly (Rice and Brunger, 1999; Mayer at al, 1996; Yu et al, 1999). FRAP experiment upon NSF-E329Q expression registered significantly slower on-kinetic of p115-GFP to the Golgi membrane (t1/2 ∼21 s). The data provided from this work strongly suggested that p115 dynamics on the membrane is regulated not only by RAB1 but also provided by unassembled SNAREs (Brandon et al, 2006).

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3. The ERGIC

3.1 History and Structure

The ERGIC (ER-to-Golgi Intermediate Compartment), also called IC (Intermediate Compartment), VTC (Vesicular-Tubular Clusters), or TCs (tubovesicular complexes), is the compartment of the secretory pathway most recently identified and very little is still known about its biogenesis and functions within the secretory pathway. However, some studies assign to the ERGIC the role of sorting place of cargos direct to the Golgi (anterograde traffic) or back to the ER (retrograde traffic) (Muniz et al, 2000; Fiedler and Simons 1994; Itin et al 1996; Ben-Tekaya et al, 2005; Appenzeller-Herzog et al, 2004). At present, the general view is that the ER and the ERGIC are two physically distinct organelles with different luminal environments and that newly synthesised protein move from the ER to the ERGIC in COPII coated vesicles and tubules (see for rewiev, Appenzeller-Herzog et Hauri, 2006). However, there is also a second point of view which believes the ERGIC in direct continuity with the rough ER in mammal cells as in yeast (Griffiths et al, 1994, Schekman 1992). What it also unclear is whether this organelle is stable or transiently formed by fusion of COPII-coated vesicles budding from the ER (Appenzeller-Herzog et al, 2006; Bannykh et al 1996; Hauri et al, 2000; Tomas et al, 2010; Ben Tekaya et al, 2005; Xu and Hay, 2004). The very first evidence for the possible existence of an intermediate compartment between the ER and the Golgi was provided only in 1984 by Saraste and Kuismanen. Their studies of the movement of newly synthesized SFV (Semliki Forest Virus) viral proteins showed that at 15°C these accumulated in tubulovesicular structures in close vicinity of the ER exit sites but distinct from the ER. The SFV membrane proteins were then able to move from these pre-Golgi elements to the cis-Golgi when returned at 37°C (Saraste and Kuismanen, 1984). This intermediate structure from the ER and the Golgi was designed as a distinct compartment few years later, with the identification of stable markers specific for this organelle. Among these markers ERGIC53, RAB2 and RAB1A are the most abundant (Schweizer et al, 1988, Schweizer et al, 1991; Saraste et al 1987; Chavrier et al, 1990; Tisdale et al, 1992); their identification was of great importance since enabled the purification of the ERGIC membrane and thus studies on its molecular composition (Schweizer et al, 1988; Breuza et al, 2004).

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Fractionation experiments showed that the ERGIC has a different biochemical composition from the Golgi and the ER (Schweizer et al, 1990; Breuza et al, 2004). According to their biological functions, the ERGIC proteins are classified in three groups: chaperones, cargo receptors, and proteins involved in membrane trafficking. ERGIC53 is the cargo transport receptor for glycoproteins (Appenzeller et al, 1999; Appenzeller-Herzog et al, 2004) and the most abundant protein detectable in ERGIC fractions. It is a type-I membrane protein with a cytoplasmic domain containing binding sites for COPI and COPII proteins (Wendeler et al 2007; Nufer et al, 2002) and thus it is capable of moving bi-directionally between the ERGIC and the ER. Other ERGIC proteins belonging to the category of cargo receptors are the lecitin VIP36, and the p24 family members p24A Gp25L2, Tmp21 and p28. Like ERGIC53, they also bind the COPI and COPII coats (Blum et al 1999; Fiedler et al, 1996; Dominguez et al, 1998; Breuza et al, 2004) and recycle between the ER and the ERGIC (Fiedeler et al, 1994; Itin et al, 1996; Klumperman et al 1998; Kappeler et al, 1997; Appenzeller et al, 1999; Fullekrug et al, 1999). The chaperones that were found to localize to the ERGIC fraction include ER enzymes, such as protein disulfide isomerase and BIP, and Golgi enzymes, such as nucleobinding (CULNAC) 1 and 2 and CBP1/Hsp47 (Satoh et al, 1996; Lin et al, 1998; Breuza et al 2004)

The majority of the studies on the ERGIC compartment mainly investigate the role of ERGIC proteins acting as cargo receptors or involved in membrane traffic. RAB1A, RAB1B and RAB2 were found to localize to the ERGIC and to be involved in membrane traffic (Tisdale et al 1992).

RAB GTPases are well known as regulators of membrane traffic and to be involved in conferring organelle identity. Inhibition of RAB2 was shown to strongly interfere with assembly of pre- Golgi intermediates. In fact, at 15°C, a condition that usually results in larger ERGIC structures, the presence of an inhibitory RAB2 peptide results in ERGIC structures that are significantly reduced in size compared to control cells. Since members of the RAB family are well known to play a role in docking of vesicles and fusion events within the secretory/endocytic pathway, it is likely that RAB2 has a critical function in ERGIC biogenesis (Tisdale et al, 1996). RAB1 is also used as ERGIC marker and it is known to promote the recruitment of p115 tethering factor to COPII vesicles (Allan et al, 2000; Szul and Sztul, 2011). Tethering proteins are fundamental to initiate the binding between carrier and target membrane, mechanism followed by the SNAREs- mediated membrane fusion (Cao and Barlowe, 2000). For example, Bernard et al in 2000, showed that the mutant RAB1-N124I blocks the recruitment of p115 to COPII vesicles resulting

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in the inhibition of fusion with the Golgi membrane. Since this result refers to in-vitro reaction and since it is known that in eukaryotic cells COPII vesicles fuse to the ERGIC, we could speculate that in vivo RAB1 inactivation interferes with docking and fusion of COPII vesicles at the ERGIC level. Moreover, also p115 itself localizes to the ERGIC structures (Alvarez et al, 1999). The role of p115 in ERGIC biogenesis might be to facilitate tethering of COPII vesicles to each other, the formation of trans-SNARE complexes and finally fusion of COPII vesicles which generates the ERGIC (Sztul and Lupashin, 2009).

3.2 COPII-vesicle homotypic fusion: a model for ERGIC biogenesis

Fusion between COPII vesicles was shown for the first time in the laboratory of Hay, in 2004.

Secretory radiolabeled VSVG (VSVG*)-vesicle and unlabeled VSVG-myc-tagged-vesicle, were obtained from permeabilized NRK cells. The two populations of secretory vesicles were coincubated under optimal transport conditions and later VSVG-myc-containing vesicles were immunoisolated from the reaction. The next step, the exposure by autoradiography of the immunoisolated VSVG-myc-vesicles revealed positivity for the radiolabeled VSVG, showing that VSVG*-vesicles co-isolated with VSVG-myc-vesicles. This interesting finding was indicative of tethering between VSVG-containing vesicles. In order to assess whether the co- immunoisolated vesicles were not only tethered but also fused together, the same work investigated the nature of the isolated VSVG. According to the work of Zagouras and Rose in 1993, VSVG exists in form of monomers and trimers (Zagouras and Rose, 1993). After immunoisolation, vesicles were sedimented with a speed able to separate VSVG monomer from VSVG trimers and the latter analyzed for the presence of heterotrimers. The detection of VSVG heterotrimers (containing at least one subunit of VSVG-myc and one subunit of VSVG*) on the isolated vesicles clearly showed that the co-isolated COPII vesicles have not only tethered but also fused together (Xu and Hay, 2004)

Interestingly, release/isolation of vesicles was strongly inhibited by addition of GDP-locked SAR1T39N and stimulated by the wild type SAR1, indicating that the released VSVG-vesicles were COPII-coated. In further experiments, the coisolated vesicles, and VSV-G* -expressing NRK cells were subjected to digestion with endoglycosidase H (endo-H) and analyzed by autoradiography. Strikingly, whereas VSV-G* retained within semi-intact cells gained endo-H

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resistance within 60 min of incubation, the VSV-G* co-isolated with VSVG-myc-vesicles remained completely sensitive to endo-H. Thus, the fusion intermediates are likely to represent pre-Golgi intermediates that lack cis-Golgi enzymes (Xu and Hay, 2004). This suggests that the ERGIC biogenesis might occur by homotypic fusion of COPII vesicles. COPII-vesicle homotypic fusion also depends on the presence of α-SNAP SNAREs. The dominant-negative α-SNAP L294A inhibited both vesicle co-isolation and heterotrimer formation, with a more potent and complete inhibition of the latter. Moreover, antibodies against syntaxin 5, also inhibited VSVG heterotrimer formation, thus suggesting that syntaxin 5, as well as α-SNAP, is required for the homotypic fusion of COPII vesicles to form early VTCs. Significant inhibition of co-isolation under these conditions was anyway surprising since SNAREs are not known to function in tethering. One potential explanation is that SNARE complexes may be required to recruit or activate the tethering machinery (Xu and Hay, 2004). In line with this hypothesis, it has been observed the RAB1-dependent recruitment of p115 into cis-SNARE complexes (Allan et al, 2000; Shorter et al, 2002). Also the recruitment of p115 to COPII vesicles is mediated by small GTPase RAB1 (Allan et al, 2000; Moyer et al, 2001; Beard et al, 2005). This mimics the activity of the yeast homologue, Ypt1p, which recruits Uso1p (p115) to membranes and promotes tethering events during ER to Golgi and intra-Golgi transport (Barlowe, 1997; Cao et al, 1998;

Lupashin et al, 1996; Cao and Barlowe, 2000). In cell-free assays, the recruitment of p115 on COPII vesicles has been shown to be temperature-dependent, SAR1-dependent and inhibited upon addition RAB-GDI or RAB1-N124I in the reaction. Both, RAB-GDI and RAB1-N124I are well known to interfere with the normal RAB1-GTPase cycle and inhibit ER-to-Golgi transport (Tisdale et al, 1992; Pind et al, 1994; Peter et al, 1994);

Summing up all these findings together, the model proposed for the ERGIC biogenesis strictly requires the RAB1-dependent recruitment of p115 onto ER-derived COPII vesicles. On the level of COPII vesicles, p115 acts then as tethering factor allowing COPII vesicles homotypic fusion, process that according to this model corresponds to the ERGIC biogenesis. The subsequent interaction between p115 with GM130 and SNAREs at the cis-Golgi membranes would then allow docking and fusion of the intermediate compartment with the Golgi, leading to the release of cargos in the Golgi apparatus. Anyway, p115 is not the only protein allowing vesicle tether and COPII homotypic fusion. In vitro studies showed in fact that BET3, subunit of the tethering factor TRAPPI, binds with SEC23 on the COPII coat and this interaction has been suggested to

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promote both homotypic fusion of COPII-coated vesicles and fusion with the cis-Golgi (Cai et al, 2007).

3.3 Regulators of the ERGIC structures

The lack of information about the ERGIC is due to difficulties in the study of this instable compartment. The subcellular distribution of ERGIC53 is for example sensitive to temperature manipulation. When exposed for long time at the low temperature of 15°C, condition that blocks in the ERGIC ER-derived cargos, the ERGIC markers, like ERGIC53, accumulate in rounded cytoplasmic structures significantly bigger than the one observed at the normal temperature of 37°C. Shorter exposure (30-45min) at 15°C leads instead to tubulation of the ERGIC as consequence of the release of the COPI coat from the membranes to the cytoplasm (Martinez- Alonso et al, 2005; Tomas et al, 2010) and redistribution of ERGIC53 in the Golgi (Lippincott- Schwartz et al, 1990; Schweizer et al, 1990; Saraste and Svensson, 1991). The ERGIC tubules formed by short exposure to 15°C are long, transient (no longer detectable after 1h exposure at this low temperature) and dynamic, since able move in long distance (Tomas et al, 2010).

Morphologically similar transient ERGIC tubules are also observed upon treatment with BrefeldinA, although shorter than the tubular structures formed by short exposure at 15°C (8µm versus 24µm in length). (Lippincott-Schwartz et al, 1990; Tomas et al, 2010). Also in this case tubulation of the ERGIC appears to be consequent to inhibition of the COPI-coat assembly (Donaldson et al, 1990; Scheel et al, 1997). Kinesin is thought to link COPI-coated ERGIC to the microtubules and to modulate retrograde traffic of recycling proteins from the Golgi to the ER (Lippincott-Schwartz et al, 1995). Kinesin depletion leads in fact not only to inhibition of retrograde traffic (due to COPI detachment) but also to ERGIC tubulation. Dynein is another motor protein associated to the ERGIC membranes (Chen et al, 2005), preferentially in non- coated areas (Tomas et al, 2010), regulator of the ERGIC-to-Golgi anterograde traffic. The model proposed by Tomas and colleagues suggests that kinesin and dynein might associate with the ERGIC clusters and originate the equilibrium of forces that in necessary to maintain the ERGIC static and vesicular. Inhibition of retrograde trafficking through COPI detachment from the ERGIC (following to short exposure at 15°C or BFA treatment) might block kinesin at the ERGIC vesicles and break the equilibrium of forces in favor of the dynein-mediated anterograde

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traffic. As consequence, ERGIC tubules might originate from the ERGIC cluster and be able to move towards the cis-Golgi (Tomas et al, 2010).

ARF1 and ARF4 are known regulators of COPI recruitment in the early secretory pathway (Volpicelli-Daley et al, 2005) and brefeldin A is a drug known to prevent ARF activation by nucleotide exchange (Klausner et al, 1992; Renault et al, 2003). Interestingly, while inhibition of ARF activation by BFA results in transient and short lived ERGIC tubules, double siRNA knockdown of ARF1 and ARF4 leads to formation of long lived microtubule-dependent ERGIC53 positive tubules. In this case, tubulation of the ERGIC has been linked to hyperactivity of phospholipaseA2G6-A (PLA2G6-A) at the ERGIC, which influences local changes in lipid composition. Treatment of double knockdown cells with the PLA2G6-A inhibitor BEL in fact inhibits the number of ERGIC tubules and the phenotype is restored by subsequent treatment with LPC, product of PLA2G6-A activity (Ben-Tekaya et al, 2010). PLA2G6-A-dependent tubules have been shown to connect ERGIC cluster together and to act as efficient routs for cargo movement to the ERGIC clusters, which are still functional as sorting station of cargos (Ben- Tekaya et al, 2010). According to the model proposed by Ben-Tekaya and colleagues, under normal conditions the discontinuity of ERGIC cluster is due to the inhibitory effect of ARF1 and ARF4 on PLA2G6-A. Strikingly, the effect of ARF1 and ARF4 on PLA2G6-A has been proposed to be independent on their GTPase activity (Ben-Tekaya et al, 2010). Interestingly, another member of the PLA2 alpha family, the Ca²⁺-dependent cytosolic PLA2 (cPLA2α), has been described to regulate the formation of intercisternal tubules at the Golgi, which are likely to play a role during intra-Golgi traffic. Transport through the Golgi cisternae leads indeed to rapid recruitment of the cytosolic cPLA2α on the Golgi membranes and interfering with cPLA2α recruitment not only inhibit intracistrenal tubules formation but also intra-Golgi trafficking (San Pietro et al, 2009). ARFs are thus involved in the regulation of both COPI-coat assembly and of enzymes involved in the lipid metabolism, and inhibition of both processes leads to ERGIC tubulation. Since ERGIC tubules are carriers within cargo movements occur rapidly and efficiently (Ben-Tekaya et al, 2010), it has been speculated that formation of tubules might be an alternative strategy adopted by cells in case of cargo excess (Simpson et al 2006; Trucco et al, 2004; Tomas et al, 2010).

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27 3.4 The ERGIC in the secretory pathway

Very little is known about signaling at the ERGIC. The small GTPase RAB2 has been shown to promote recruitment of the complex aPKC/GAPDH (atypical protein kinace C and glyceraldehyde-3-phosphate dehydrogenase) at the ERGIC level and that injection of antibody against GAPDH led to inhibition of ER-to Golgi traffic (Tisdale 2000, 2001, Tisdale et al, 2003).

Thus, RAB2 appears to be a key regulator of transport from the ERGIC. This hypothesis is in agreement with previous findings which showed arrest of VSVGts045 transport at the level of pre-Golgi compartments upon overexpression of RAB2 GTPase-deficient mutant (Tisdale 1999;

Tillmann et al, 2013).

Since we do not yet understand the function of the ERGIC in the secretory pathway and its regulation by signaling, also the fate of secretory cargos in the ERGIC remains poorly understood. However, few studies exist that investigated the fate of an ERGIC53 cargo.

ERGIC53 is a lectin that is involved in the transport of glycoproteins and one of its cargo is procathepsin Z. In vivo experiments showed that the association between ERGIC53 and procathepsin Z is pH-dependent. Already in the 80’s, several works showed the existence of a pH gradient along the secretory pathway, whit a progressive acidification from the ER (7.4) to the Golgi (6.4 at the TGN) (Schmidt and Moore, 1995; Hutton, 1982; Orci et al, 1986; Orci et al, 1987; Barasch et al, 1988; Urbe et al, 1997; Wu et al, 2001; Machen et al, 2003; Appenzeller- Herzog et al, 2004) and this gradient is thought to be essential for the sorting of secretory proteins into the regulated secretory pathway (Chanat and Huttner, 1991; Arvan and Castle, 1998). The ERGIC is thus the first acidic organelle of the secretory pathway and its pH, lower than the in the ER, has been suggested to promote the dissociation of cargo-receptor complexes (Appenzeller- Herzog et al, 2004). The binding between ERGIC53 and procathepsin Z has been studied in cells subjected to patch-clamp. This approach showed that acidification of the cellular pH to pH 6.5 and 6 inhibited the association between cargo and receptor. Contrarily, pH neutralization by chloroquine had an inhibitory effect on the dissociation of procathepsin Z from ERGIC53 (Appenzeller-Herzog et al, 2004). This finding strongly support the idea that the ERGIC is a distinct compartment from the ER and the Golgi, with distinct feature and functions. In fact, a very important conclusion of the work of Appenzeller-Herzog and collegues is that the neutral pH of the ER allows the interaction between cargo proteins and their receptors, while the acidic pH of the ERGIC promotes their dissociation. Dissociation of cargoes from their receptor is

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needed in order to proceed through the secretory pathway and to finally reach the Golgi (Appenzeller-Herzog et al, 2004). The same study further showed that the pH sensitivity of ERGIC53 depends on the calcium levels. In in vitro binding assays, lowering the calcium concentration resulted in fact in a faster pH-induced cargo dissociation from ERGIC53 (Appenzeller-Herzog et al, 2004). Interestingly, also the interaction between KDEL-bearing proteins and the KDEL receptor has been shown to be pH sensitive but in the case, in vitro cargo- receptor biding is favoured by acidic pH and lower at pH 7.4 (Scheel and Pelham, 1996). The opposite effect of the pH on the KDEL receptor is not surprising if considering that it normally has an ERGIC/cis-Golgi localization and that binding with the KDEL ligand is required for the COPI-dependent retrograde transport of the receptor to the ER (Tang et al, 1995; Appenzeller- Herzog et Hauri, 2006).

Once at the ERGIC, how cargos move from the ERGIC to the Golgi is still unknown because no carries have been found to bud from the ERGIC and fuse with the cis-Golgi. A hypothesis suggests that the ERGIC structures themselves move towards the Golgi and fuse with the Golgi membrane (Sztul and Lupashin, 2009). Moreover, since the ERGIC is predominantly COPI- coated, it is thought that tethering of ERGIC-β-COP proteins to the Golgi involves p115 (Guo et al, 2008; Sztul and Lupashin, 2009). This view of the ERGIC is supported by the fact that fluorescent tagged COPI-coated tubovesicular complexes (TCs), were shown to move towards the Golgi shuttling anterograde cargos (Shima et al, 1999). However, some other works suggest that cargo traffic between ERGIC and Golgi occur within a tubular and vesicular elements present between the ERGIC clusters and the cis-Golgi compartment. This was shown for the anterograde traffic of soluble GFP (Blum et al, 2000) and VSVG-GFP and for retrograde transport of ERGIC53-GFP (Ben-Tekaya et al, 2005), by time lapse microscopy.

Contrarily to the lack of insights into the role of the ERGIC in the anterograde traffic towards the Golgi, its function during COPI-mediated recycling of proteins back to the ER has been much better investigated.

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4. COPI coat

Backward movement of TCs to the ER is responsible for sorting of ER recycling proteins. (Scales et al, 1997; Shima et al, 1999; Stephens et al, 2000). Many works showed that COPI-coated vesicles are major mediators of intra-Golgi and retrograde Golgi-ER traffic (Letourneur et al,1994; Shima et al, 1999; Spang and Schekman, 1998; Orci et al, 2000), and the current widely accepted model describes the formation of COPI carriers at the ERGIC level and from the cis- Golgi cisternae (Szul and Sztul, 2011). The COPI coat consists of a heptameric complex (or coatomer), organized in in an inner and outer-coat. The α, β´ and ε tetrameric subcomplex constitutes the outer layer of the COPI coat, while the inner layer is formed by the γ, δ, ζ, and β trimeric complex. Studies conducted by electron microscopy showed the existence of two structurally and functionally types of COPI vesicles. COPI vesicles with light content and 11nm coat are found at the cis-Golgi and between the cis-Golgi and the ER; they are thought to regulate protein recycle from the cis-Golgi to the ER. COPI vesicles with a dark content and a 18nm coat localize instead close to the medial and trans-Golgi; therefore they are suggest to play a role in retrograde traffic within the Golgi apparatus (Donohoe et al, 2007; Orci et al, 2000). The distinction between COPI vesicle destined to the ER and COPI vesicles involved in intra-Golgi retrograde traffic has been further confirmed by in-vitro reconstitution of COPI vesicle budding.

In this assay, ER-destined vesicles were showed to contain p24 family members, while the ones destined to intra-Golgi traffic contained GOS28 SNARE and mannosidaseII (Lanoix et al, 2001;

Malsam et al, 2005). The recruitment of the COPI-coat to the donor membranes involves class I and class II of the small GTPases ARFs, ARF4 in case of recruitment to the ERGIC (Chun et al, 2008). ARFs have been shown to recruit COPI coatomers by direct interaction with the β, subunit (Eugster et al, 2000), and their activity is strictly dependent on the exchange factor GBF1. siRNA depletion of GBF1 or inactivation with specific inhibitors leads in fact to dissociation of the COPI coat from membranes (Saenz et al, 2009; Manolea et al, 2008; Szul et al, 2007). GBF1 colocalizes with COPI at the ERGIC and at the Golgi level, where it seems to be recruited via interaction with RAB1B (Monetta et al, 2007). Sorting of type I transmembrane proteins for recycling to the ER is driven by recognition of two highly conserved amino acid motifs (WXXW/Y/F and KKXX), by the α, β´, γ and δ subunits of the COPI coat (Cosson and Letourneur, 1995). Type II transmembrane proteins, like all Golgi glycosyl-transferases, have a distinct semi-conserved sequence (F/L-L/V/I-X-X-RK) which is instead recognized by the linker

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protein Vps74 that bins the β and γ subunits of the COPI coatomer (Schmitz et al, 2008; Tu et al, 2008). Not only transmembrane proteins but also soluble proteins containing the KDEL COOH- motif are recycled back to ER into COPI vesicles. This motif is recognized by the KDEL receptor, a seven-span trasmembrane protein which constantly recycles between Golgi and ER, and directly binds COPI coatomers (Townsley et al, 1993). Also fusion of COPI-vesicles with the acceptor membrane requires the involvement of tethering factors; as already described in the paragraph “tethering factors”, tethering of COPI-vesicles to the ER is mediated by the Dsl1 complex in yeast (Reilly et al, 2001; VanRheenen et al, 2001) and by the Dsl1-like complex in mammals, which interacts with the ER-localized SNARE syntaxin-18 (Hirose et al, 2004).

Signaling at the Golgi Apparatus During Cell Migration and Implication for Cancer Cell Metastasis