title: Actin and microtubule regulation of trans-Golgi network architecture, and copper-dependent protein transport to the cell surface.
abstract: The Menkes disease ATPase (MNK) is a copper transporter that localizes to the mammalian trans-Golgi network (TGN) and shows substantial co-localization wih a ubiquitous TGN resident protein and marker, TGN46. We tested our hypothesis that these two TGN residents and integral membrane proteins are localized to biochemically distinct TGN sub-compartments using constitutively active mutant proteins and drugs that disrupt membrane traffic, lumenal pH and the cellular cytoskeleton. The pH-disrupting agent, monensin, causes MNK to be more diffusely distributed with partial separation of staining patterns for these two TGN residents. Expression of a constitutively active Rho-kinase (ROCK-KIN), which causes formation of juxta-nuclear astral actin arrays, also effects separation of MNK and TGN46 staining patterns. Treatment of ROCK-KIN expressing cells with latrunculin B, an actin-depolymerizing agent, causes complete overlap of MNK and TGN46 staining patterns with concomitant disappearance of polymerized actin. When microtubules are depolymerized in ROCK-KIN expressing cells by nocodazole, both MNK and TGN46 are found in puncate structures throughout the cell. However, a substantial proportion of MNK is still found in a juxta-nuclear location in contrast to TGN46. Actin distribution in these cells reveals that juxta-nuclear MNK is distinct to the astral actin clusters in ROCK-KIN expressing cells where the microtubules were depolymerized. The TGN to cell-surface transport of MNK requires both actin and microtubules networks, whilst the constitutive trafficking of proteins is independent of actin. Taken together, our findings indicate that at least two TGN sub-domains are regulated by separate cytoskeletal dynamics involving actin and tubulin.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, 0X3 7BN Oxford, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Cobbold
authors.forename: Julie
authors.initials: J
authors.lastname: Coventry
authors.forename: Sreenivasan
authors.initials: S
authors.lastname: Ponnambalam
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Monaco
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D006023:Glycoproteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007476:Ionophores / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D054460:rho-Associated Kinases; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D027682:Cation Transport Proteins; D006023:Glycoproteins; D047908:Intracellular Signaling Peptides and Proteins; D007476:Ionophores; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C072237:TGOLN2 protein, human; D013844:Thiazoles; D053778:Thiazolidines; D003300:Copper; D008985:Monensin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases; C037067:latrunculin A