Class II Arfs require a brefeldin-A-sensitive factor for Golgi association.

Arf proteins are small Ras-family GTPases which recruit clathrin and COPI coats to Golgi membranes and regulate components of the membrane trafficking machinery. It is believed membrane association and activity of Arfs is coupled to GTP binding, with GTP hydrolysis required for vesicle uncoating. In humans, four Arf proteins (Arf1, Arf3, Arf4 and Arf5) are Golgi-associated. Conflicting reports have suggested that HA-GFP-tagged Class II ARFs (Arf4 and Arf5) are recruited to membrane independently of the brefeldin A sensitive exchange factor GBF1, suggesting regulation fundamentally different from the Class I Arfs (Arf1, Arf3), or alternately that the GTPase cycle of GFP-tagged Class II Arfs is similar to other Arfs. We show that these results depend on the fluorescent tag, with Arf4-HA-GFP tag resistant to brefeldin, but Arf4-GFP acting similarly to Arf1-GFP in brefeldin-sensitivity and photobleach assays. Arf4-HA-GFP could be partially reverted to the behavior of Arf4-GFP by mutation of two aspartic acids in the HA tag to alanine. Our results, which indicate a high sensitivity of Arf4 to tagging, can explain the discrepancies between previous studies. We discuss the implications of this study for future work with tagged Arfs.

ARFGAP2 and ARFGAP3 are essential for COPI coat assembly on the Golgi membrane of living cells.

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.

New Method for Quantitation of Lipid Droplet Volume From Light Microscopic Images With an Application to Determination of PAT Protein Density on the Droplet Surface.

Determination of lipid droplet (LD) volume has depended on direct measurement of the diameter of individual LDs, which is not possible when LDs are small or closely apposed. To overcome this problem, we describe a new method in which a volume-fluorescence relationship is determined from automated analysis of calibration samples containing well-resolved LDs. This relationship is then used to estimate total cellular droplet volume in experimental samples, where the LDs need not be individually resolved, or to determine the volumes of individual LDs. We describe quantitatively the effects of various factors, including image noise, LD crowding, and variation in LD composition on the accuracy of this method. We then demonstrate this method by utilizing it to address a scientifically interesting question, to determine the density of green fluorescent protein (GFP)-tagged Perilipin-Adipocyte-Tail (PAT) proteins on the LD surface. We find that PAT proteins cover only a minority of the LD surface, consistent with models in which they primarily serve as scaffolds for binding of regulatory proteins and enzymes, but inconsistent with models in which their major function is to sterically block access to the droplet surface.

New automated single-cell technique for segmentation and quantitation of lipid droplets.

Lipid droplets are the major organelle for intracellular storage of triglycerides and cholesterol esters. Various methods have been attempted for automated quantitation of fluorescently stained lipid droplets using either thresholding or watershed methods. We find that thresholding methods deal poorly with clusters of lipid droplets, whereas watershed methods require a smoothing step that must be optimized to remove image noise. We describe here a novel three-stage hybrid method for automated segmentation and quantitation of lipid droplets. In this method, objects are initially identified by thresholding. They are then tested for circularity to distinguish single lipid droplets from clusters. Clusters are subjected to a secondary watershed segmentation. We provide a characterization of this method in simulated images. Additionally, we apply this method to images of fixed cells containing stained lipid droplets and GFP-tagged proteins to provide a proof-of-principle that this method can be used for colocalization studies. The circularity measure can additionally prove useful for the identification of inappropriate segmentation in an automated way; for example, of non-cellular material. We will make the programs and source code available to the community under the Gnu Public License. We believe this technique will be of interest to cell biologists for light microscopic studies of lipid droplet biology.

Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1.

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addition, loss of Arf x GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf x GTP, rather than the formation of an Arf x GDP x BFA x GBF1 complex.

Scyl1, mutated in a recessive form of spinocerebellar neurodegeneration, regulates COPI-mediated retrograde traffic.

Scy1-like 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, was recently identified as the gene product altered in muscle-deficient mice, which suffer from motor neuron degeneration and cerebellar atrophy. To determine the function of Scyl1, we have now used a mass spectrometry-based screen to search for Scyl1-binding partners and identified components of coatomer I (COPI) coats. The interaction was confirmed in pull-down assays, and Scyl1 co-immunoprecipitates with betaCOP from brain lysates. Interestingly, and unique for a non-transmembrane domain protein, Scyl1 binds COPI coats using a C-terminal RKLD-COO(-) sequence, similar to the KKXX-COO(-) COPI-binding motif found in transmembrane endoplasmic reticulum (ER) proteins. Scyl1 co-localizes with betaCOP and is localized, in an Arf1-independent manner, to the ER-Golgi intermediate compartment and the cis-Golgi, sites of COPI-mediated membrane budding. The localization and binding properties of Scyl1 strongly suggest a function in COPI transport, and inhibitory RNA-mediated knock down of the protein disrupts COPI-mediated retrograde traffic of the KDEL receptor to the ER without affecting anterograde traffic from the ER. Our data demonstrate a function for Scyl1 as an accessory factor in COPI trafficking and suggest for the first time that alterations in the COPI pathway result in neurodegenerative disease.

Rab18 and Rab43 have key roles in ER-Golgi trafficking.

Rabs and Arfs/Arls are Ras-related small GTPases of particular relevance to membrane trafficking. It is thought that these proteins regulate specific pathways through interactions with coat, motor, tether and SNARE proteins. We screened a comprehensive list of Arf/Arl/Rab proteins, previously identified on purified Golgi membranes by a proteomics approach (37 in total), for Golgi or intra-Golgi localization, dominant-negative and overexpression phenotypes. Further analysis of two of these proteins, Rab18 and Rab43, strongly indicated roles in ER-Golgi trafficking. Rab43-T32N redistributed Golgi elements to ER exit sites without blocking trafficking of the secretory marker VSVG-GFP from ER to cell surface. Wild-type Rab43 redistributes the p150(Glued) subunit of dynactin, consistent with a specific role in regulating association of pre-Golgi intermediates with microtubules. Overexpression of wild-type GFP-Rab18 or incubation with any of three siRNAs directed against Rab18 severely disrupts the Golgi complex and reduces secretion of VSVG. Rab18 mutants specifically enhance retrograde Golgi-ER transport of the COPI-independent cargo beta-1,4-galactosyltransferase (Galtase)-YFP but not the COPI-dependent cargo p58-YFP from the Golgi to ER in a photobleach assay. Rab18-S22N also potentiated brefeldin-A-induced ER-Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway.