In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.

Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

No description available.

No description available.