Protein flexibility is required for vesicle tethering at the Golgi
Membrane trafficking involves the collection of cargo into transport vesicles , movement of vesicles along cytoskeletal tracks , and tethering , docking and fusion of vesicles at their target membranes . Tethering is the process by which partner membranes are brought together into close proximity to permit their subsequent fusion ( Pfeffer , 1999; Sztul and Lupashin , 2006; Yu and Hughson , 2010 ) . Two classes of transport vesicle tethers have been described to date: multi-subunit complexes such as the Exocyst , COG , Dsl1 and TRAPP complexes ( Bröcker et al . , 2010 ) , and larger , dimeric coiled-coil containing proteins such as p115 and EEA1 ( Sztul and Lupashin , 2006; Munro , 2011 ) . Both classes share the ability to bind to Rab GTPases , SNARE proteins and vesicle coat complexes , suggesting that they play important roles in coordinating molecular events important for target recognition and membrane fusion ( Short et al . , 2005; Cai et al . , 2007; Sinka et al . , 2008; Hayes et al . , 2009; Hughson and Reinisch , 2010 ) . Golgins are long , Golgi-associated proteins that contain a high proportion of sequences that are predicted to form coiled coils ( Munro , 2011; Short et al . , 2005 ) . They are anchored to the Golgi via their C-termini , and models suggest that they protrude long distances , relying on the relative rigidity of the coiled coil structure to provide a meshwork that can capture vesicles in the vicinity of the Golgi ( Yu and Hughson , 2010; Munro , 2011 ) . GCC185 is a Golgin required for the transport of mannose 6-phosphate receptors ( MPRs ) from late endosomes to the trans Golgi network ( Reddy , 2006; Derby et al . , 2007 ) . Cells depleted of GCC185 accumulate MPRs in Rab9 GTPase- and AP-1-decorated transport carriers ( Hayes et al . , 2009; Reddy , 2006; Brown et al . , 2011 ) , providing an assay for GCC185 tethering capacity . GCC185 is likely anchored on the TGN surface by cooperative binding of its C-terminus to both Rab6 and Arl1 GTPases ( Burguete et al . , 2008 ) . We ( Reddy , 2006; Burguete et al . , 2008 ) and others ( Derby , 2004 ) have shown that GCC185 binds Arl1 rather weakly , but Rab6A binding greatly enhances Arl1 interaction ( Burguete et al . , 2008 ) . Arl1’s role in anchoring GCC185 was questioned because it seemed not to be required if siRNA-depleted by 80% ( Houghton et al . , 2009 ) ; in fact , 90% depletion of Arl1 is required to displace GCC185 from membranes and to detect its role in Golgi localization of the GCC185 C-terminus ( Burguete et al . , 2008 ) . To date , we have little ( if any ) information , whether Golgins are actually as long as predicted , whether they are relatively rigid , or even where or how they bind transport vesicles . Moreover , once they bind vesicles , what happens next ? Many coiled-coil tethers contain putative hinges , located between predicted rod-like , coiled-coil domains ( Barr and Short , 2003; Cai et al . , 2007; Lupas et al . , 1991; Munro , 2011; Pfeffer , 1999 ) . Do these regions function as hinges ? We have taken a multidisciplinary approach to investigate the mechanism of vesicle tethering by a trans Golgi network ( TGN ) -associated Golgin , GCC185 . Our data provide novel insight into the tethering process and suggest that the TGN Golgin , GCC185 , uses an unexpected mechanism to accomplish vesicle tethering .
Current models posit that a tethering protein should capture vesicles at one end and be anchored to the target membrane at the other end . For GCC185 , the N-terminus would therefore be predicted to bind vesicles . We used differential centrifugation to isolate a crude vesicle fraction carrying the cargoes , GFP-Rab9 ( Figure 7B ) and endogenous cation-independent MPR ( CI-MPR; Figure 7C ) and Rab9 ( Figure 7D ) proteins . We then compared the ability of GCC185 N-terminal residues 1-358 or ( similarly sized ) internal residues 1032–1331 ( Figure 7A ) to bind these vesicles after immobilization on glutathione-Sepharose . Figures 7B-D show that vesicles were indeed captured by the N-terminal 1–358 fragment but not by the internal fragment of similar length . Interestingly , the 1–358 sequences tethered most effectively when the obligate GST dimer was located at the fragment’s C-terminus ( 1–358-GST ) rather than the N-terminus ( GST-1–358; Figure 7C ) . Specificity control experiments showed that neither early endosome ( EEA1 ) nor endoplasmic reticulum ( calnexin ) markers were captured by the constructs tested ( Figure 7C ) . N-terminal sequences are essential for GCC185 function in cells ( Hayes et al . , 2009 ) , consistent with their ability to bind transport vesicles in vitro . 10 . 7554/eLife . 12790 . 013Figure 7 . GCC185 N-terminal domain tethers cargo-containing vesicles . ( A ) Coomassie blue stained SDS-PAGE of purified His-1–358-GST , GST-1–358 and GST-1032–1331 . ( B ) Binding of GFP-Rab9-vesicles to the indicated , immobilized GCC185 constructs . Top panel , anti-GFP immunoblot; bottom panel , Ponceau S stain . ( C , D ) Binding of endogenous CI-MPR or Rab9-vesicles , respectively , to the indicated constructs as in B . Top panels , C , anti-CI-MPR , anti-EEA1 and anti-calnexin blots; ( D ) anti-Rab9A blot; ( C , D ) bottom panels , Ponceau S staining . Mobility of marker proteins is indicated at left in Kd . A representative example from at least 3 independent experiments ( in duplicate ) is shown for GFP-Rab9- and endogenous CI-MPR-vesicle capture; 2 independent experiments ( in duplicate ) were performed for endogenous Rab9-vesicle capture . DOI: http://dx . doi . org/10 . 7554/eLife . 12790 . 013 The functional significance of N-terminal splaying detected in 80% of purified GCC185 molecules ( Figure 2 , 3 ) is confirmed by the finding that the fragment capable of splaying at the N-terminus ( 1–358-GST ) showed highest tethering activity ( Figure 7C , D ) . Although we cannot rule out completely , the possibility of GST-steric hindrance for vesicle binding when present at the N-terminus , these data suggest that free N-termini represent both the active and most abundant form of this tethering protein . These experiments show that GCC185 N-terminal sequences can bind cargo—essential for a bona fide tethering protein . Moreover , this represents the first direct demonstration of in vitro vesicle tethering by a TGN localized , GRIP-domain containing tethering protein .
We have shown , for the first time , that flexibility in a tethering protein is required for its functionality in cells , both in supporting the receipt of transport vesicles at the Golgi and maintaining Golgi ribbon structure . We have shown that transport vesicles can bind to GCC185’s N-terminus , and seem to prefer to bind to splayed ends of this dimeric , coiled coil tether . The presence of multiple Rab-binding sites along the length of individual Golgin proteins ( Sinka et al . , 2008; Hayes et al . , 2009; Short et al . , 2001 ) has led others to suggest that vesicles may hop along individual tethers ( Barr and Short , 2003 ) or be captured by Golgin ‘tentacles’ within a potential Golgin meshwork towards the target membrane ( Munro , 2011 ) . Such a model considers tethers as rigid , extended rods ( Figure 8 , left ) . Our data favor a model whereby GCC185 is C-terminally anchored to the TGN through Rab6 and Arl1 GTPases ( Burguete et al . , 2008 ) and captures Rab9-decorated transport vesicles ( Hayes et al . , 2009; Reddy , 2006 ) by binding them at the N-terminus and/or near the hinge region ( Brown et al . , 2011 ) ; importantly , the central bubble gives significant flexibility to the protein and would allow it to collapse onto the Golgi surface , bringing vesicles close to the trans Golgi target membrane ( Figure 8 ) . GCC185 can bind multiple Rab GTPases along its length ( Hayes et al . , 2009 ) , as well as syntaxin16 , Arl1 and Arl4 GTPases ( Burguete et al . , 2008; Ganley et al . , 2008; Lin et al . , 2011 ) . GCC185 also binds to CLASP proteins that catalyze microtubule polymerization from the Golgi surface ( Efimov et al . , 2007 ) . These additional interactions will all pull the tether towards the Golgi surface , bringing N-terminally bound vesicles closer to the Golgi surface for productive vesicle docking . 10 . 7554/eLife . 12790 . 014Figure 8 . Collapse model for vesicle tethering at the TGN . Previous models based on GMAP-210 have led to a model that tethers extend from the Golgi as highly rigid rods that may bend in the middle ( 'Rigid hinge' ) . Data presented here show that GCC185 is shorter than originally predicted ( 145 nm vs . >204 nm ) , has splayed ends that can capture vesicles , and an unusual central bubble and floppiness as detected by atomic force microscopy . These features lead us to propose a flexible collapse model whereby the tether may collapse onto the Golgi surface , possibly facilitated by Rab GTPases and other proteins that are localized there . Note that vesicles may also ( or alternately ) bind near the bubble , and engage both the AP1 and Rab9 binding sites there , in addition to GCC185’s N-terminal arms . DOI: http://dx . doi . org/10 . 7554/eLife . 12790 . 014 We have shown here that the functionally essential , GCC185 N-terminus has the capacity to bind to transport vesicles in vitro . N-terminal vesicle binding is shared with GMAP-210 , a Golgin localized to the cis-Golgi ( Sato et al . , 2015 ) ; for other Golgins , it is presumed due to binding sites for various coat proteins . In previous work ( Brown et al . , 2011 ) , we showed that GCC185 residues 938–1032 comprise an AP-1 binding site that is also needed for GCC185 vesicle tethering ( blue bar in Figure 1A ) ; the transport vesicles are AP-1 decorated , thus this was presumed to represent a possible vesicle binding site . This AP-1 binding site is located immediately downstream of the regions deleted in the △hinge protein studied here ( 751-805 , 890939 ) ( Figure 1A ) . Moreover , an apparently dispensible , Rab9 GTPase binding site is located directly between the two deletions made to generate the △hinge mutant ( orange bar , Figure 1A ) . Binding of Rab9 to that site could influence the ability of that part of the protein to form a bubble , and could influence the efficiency of tethering on a time scale finer than that assayed here and previously . Both Rab9 and AP-1 are present on the transport vesicles captured , but we do not yet know the precise molecular interaction underlying the ability of the N-terminal domain to bind vesicles . That a splayed N-terminus is utilized suggests that the tether gains efficiency by avidity , and can reach out to 'hug' the vesicle . How the various binding sites along GCC185 coordinate interactions with the vesicle and the target membrane to accomplish vesicle tethering represents an important area for future work . Given the presence of AP-1 and Rab9 binding sites adjacent to the central bubble ( Figure 1A ) and the additional capacity of the splayed N-terminus to capture transport vesicles , it is also possible that rather than being held near GCC185’s N-terminus , the vesicle could bind primarily near the bubble while still being enwrapped by the N-terminal arms . The model in Figure 8 is drawn to scale and assumes that the transport vesicles are ~50 nm in diameter ( Barbero , 2002 ) ; they need not be larger to be able to occupy both potential vesicle binding domains . The AP-1 binding site ( 939–1 , 031 ) is essential for GCC185’s role in vesicle tethering but not Golgi ribbon maintenance ( Brown et al . , 2011 ) , and must be considered in any model of GCC185 vesicle tethering . Like GCC185 , the other human GRIP domain Golgins are also predicted to contain a high proportion of coiled coil sequences . Golgin-245 shares with GCC185 two very obvious breaks in the coiled coil that may also form a central bubble; its absolute N-terminus is also likely splayed . The shorter Golgin-97 and GCC88 proteins are likely about half as long as GCC185 by sequence alone; Golgin-97’s N-terminus is likely splayed , and GCC88 may very well also contain a central bubble . Finally , a domain between GCC185 residues 1332 and 1438 is required for maintenance of Golgi ribbon structure but not transport vesicle tethering ( Brown et al . , 2011 ) . This observation highlights the fact that workers in this field have oversimplified thinking about how tethers function—simple engagement of N- and C-termini is not sufficient for both vesicle capture and Golgi ribbon stabilization . A recent paper studying Golgi tethers ( Wong and Munro , 2014 ) concluded that unlike other TGN tethers , GCC185 could not tether vesicles upon re-localization to mitochondria . The extent of Golgi membrane mobilization seen in that study was far greater than the small pool size of the much rarer , Rab9-containing , AP-1 positive , mannose phosphate receptor transport vesicle intermediates—that are only visualized in cells upon depletion of tethering factors ( Reddy , 2006; Brown et al . , 2011 ) . It is very possible that that study assayed the ability of TGN tethers to recruit/assemble TGN ribbons , rather than capture rare transport vesicle intermediates . Since ribbon assembly and transport vesicle capture are driven by separate domains of GCC185 , perhaps GCC185 is more potent in vesicle tethering rather than Golgi ribbon assembly . A remaining mystery is why depletion of any one of the GRIP domain-containing Golgins leads to Golgi fragmentation . Whatever the explanation , the floppy appearance of purified GCC185 , when deposited on mica in the absence of fixative , and the proximity of GCC185’s N- and C-termini on the Golgi in cells , argue strongly against this protein behaving like a pipe cleaner , protruding on the surface of the trans Golgi network in cells . Instead , tether collapse represents an appealing , alternative model .
cDNAs encoding GCC185 full-length and fragments were prepared by ligating the full-length sequence and sequence encoding residues 1–358 , 394–751 , 1–889 , 890–1684 into EGFP-C1 with a FLAG tag ( DYKDDDDK ) inserted downstream of GFP flanked by the GGATCC linker ( AS ) for purification purposes . The hinge mutant constructs were prepared by introducing GAGCTC ( EL ) at residue 751 and 805 , and CTCGAG ( LE ) at 890 and 939 to remove the original sequence . Sequential digestion with SacI and XhoI restriction enzymes ( New England Biolabs , Ipswich , MA ) followed by re-ligation of the plasmid or ligating GGS repeats to the cleaved site created the △hinge ( 1–750-EL-806-889-LE-940-1684 ) and ( GSS ) n hinge constructs ( 1–750-EL- ( GSS ) 13-EL-806-889-LE- ( GSS ) 11-LE-940–1684 ) , respectively . The HA-fusion construct was prepared by inserting the HA tag sequence ( YPYDVPDYA ) at residue 805 flanked by linker nucleotide sequence GAGCTC ( EL ) using the same strategy ( 1–804-EL- YPYDVPDYA-EL-807–1684 ) . cDNAs encoding rescue plasmids were prepared with 8 silent mutations in the siRNA-targeted region and ligated into pcDNA3 . 1 ( + ) ( Invitrogen , Grand Island , NY ) modified with an N-terminal Myc tag as described ( Hayes et al . , 2009 ) . Bacterial expression plasmids were prepared by ligating PCR products encoding residues 1–358 or 1032–1331 into pGEX4T-1 ( GE Healthcare , Pittsburgh , PA ) ( GST-1–358 , GST-1032-1331 ) or using the pQE60 vector ( Qiagen , Hilden , Germany ) with a 6X His tag upstream and a GST tag downstream of residues 1–358 ( His-1–358-GST ) . Residues 1342–1684 were inserted into the 6X-His fusion vector pET28a ( Stratagene , La Jolla , CA ) . HeLa cells and HEK293T cells were cultured at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium ( GIBCO , Life Technologies , Grand Island , NY ) supplemented with 7 . 5% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin . Transfection with siRNA and rescue plasmids was performed as described ( Brown et al . , 2011 ) . In brief , cells were transfected with GCC185 siRNA targeting the sequence 5′-GGAGTTGGAA CAATCACAT-3′ using Oligofectamine ( Invitrogen ) according to the manufacturer . For rescue experiments , depletion was for 72 hr; Myc -tagged rescue constructs resistant to the siRNA were transfected 24 hr after initial siRNA treatment using FuGENE6 ( Roche , Indianapolis , IN ) . For GFP-Rab9 membrane tethering assay , GCC185-depleted 293T cells grown to 50% confluency were transfected with GFP-Rab9 construct construct using polyethylenimine ( Polysciences Inc . , Warrington , PA ) for 24 hr before the extraction of membranes . Endogenous vesicle tethering assays were preformed with non-depleted 293T cells grown to 80–90% confluency . For proximity ligation assays , HeLa cells grown to 50% confluency were transfected with GFP-fusion constructs for 24 hr before fixation . Cell fixation , staining and mounting in Mowiol were performed as described ( Hayes et al . , 2009 ) . In brief , cells were split onto 22 x 22-mm coverslips in a 6-well plate 48 hr before fixation . Cells were washed twice in PBS and fixed for 20 min in 3 . 7% formaldehyde in 200 mM Hepes , pH 7 . 4 . Fixed cells were then washed twice in PBS and permeabilized for 5 min with 0 . 2% Triton X-100 in PBS followed by two washes with PBS and a 15-min blocking with 1% BSA in PBS . Monoclonal mouse anti-cation-independent MPR antibody ( 2G11; Lombardi et al . , 1993 ) and chicken anti-Myc ( 1:1000 , Bethyl Laboratories , Inc . , Montgomery , TX ) primary antibodies , and Alexa Fluor 488 goat anti-mouse ( 1:2000 ) and Alexa Fluor 555 goat anti-chicken ( 1:2000 ) secondary antibodies ( Invitrogen ) were used . Micrographs were acquired as previously described ( Brown et al . , 2011 ) . In brief , images were acquired using a microscope ( Eclipse 80i; Nikon , Tokyo , Japan ) fitted with a 60x/NA 1 . 4 plan apochromat objective lens , a Sedat Quad filter set ( Chroma Technology Corp . , Bellows Falls , VT ) and a charge-coupled device camera ( CoolSnapHQ; Photometrics , Tucson , AZ ) at room temperature . Wavelength selection was performed using a controller ( Lambda 10–3; Sutter Instrument , Novato , CA ) . Z sections were acquired with a Z axis drive ( MFC-2000; Applied Scientific Instrumentation , Eugene , OR ) at 0 . 2-mm steps . All instrumentation was controlled by MetaMorph imaging software ( Molecular Devices , Sunnyvale , CA ) . Image deconvolution was performed using a theoretical point spread function with softWoRx ( v . 4 . 1 . 0; Applied Precision Inc . , Issaquah , WA ) . The deconvolution micrographs displayed in Figure 5 represent projections of three to five central slices of the z stack acquired . Two individuals used blinded datasets to score rescue experiment images . The Duolink in situ proximity ligation assay ( PLA , Olink Bioscience , Uppsala , Sweden ) was performed according to the manufacturer . Briefly , HeLa cells transfected with GFP-GCC185 or GFP-GCC185-△hinge were washed , fixed and blocked . Rabbit anti-GCC185 C-terminus ( 1:500 , Reddy , 2006 ) , mouse anti-GCC185-1-358 N-terminus ( 1:500 ) , mouse anti-GM130 ( 1:500; BD Biosciences , San Jose , CA ) , mouse anti-Golgin 245 ( 1:500; BD Bioscience ) and mouse anti-GFP ( clones 7 . 1 and 13 . 1; 1:100; Roche ) primary antibodies were used . After 1 hr primary antibody incubation at room temperature , the PLA probe anti-mouse Plus and anti-rabbit Minus were diluted in PBS containing 1% BSA and added to cells for 1 hr at 37°C in a humidified chamber . All subsequent steps were performed at 37°C in a humidified chamber . Cells were then incubated with oligonucleotides complementary to the proximity probe and T4 DNA ligase for 30 min . Rolling-circle amplification was initiated by addition of Phi29 DNA polymerase and Texas red-labeled nucleotide probe . After 100 min , cells were washed and mounted and imaged using an Axiophot2 epifluorescence microscope fitted with a Plan-Neofluar 40x/0 . 75 objective ( Carl Zeiss MicroImaging , Thornwood , NY ) , and a digital CCD camera ORCA-R2 C10600 ( Hamamatsu , Hamamatsu City , Japan ) . All microscope instrumentation was controlled by AxioVision4 . 7 . 2 software ( Carl Zeiss MicroImaging ) . Individual cells ( objects ) were identified , and proximity ligation and GFP signals were quantified using CellProfiler ( Broad Institute , Cambridge , MA; www . cellprofiler . org ) . Expression and purification of GST-tagged constructs were as described ( Brown et al . , 2011 ) . Constructs were transformed into BL21 ( DE3 ) RIPL competent cells and bacterial cultures were induced at OD600 = 0 . 5-0 . 7 with 0 . 25 mM IPTG for 3 hr at 30°C . Cells were resuspended in 50 mM Hepes , pH7 . 4 , 250 mM NaCl , 10% ( v/v ) glycerol and protease inhibitors and lysed by two passes through an EmulsiFlex-C5 apparatus ( Avestin , Ottawa , ON ) at >10 , 000 lb/in2 followed by centrifugation at 13 , 000 rpm for 30 min in a FiberLite F15 ( 8x50c ) rotor ( Thermo Scientific ) . Clarified lysates containing GST-tagged proteins ( His-1-358-GST , GST-1-358 and GST-1032-1331 ) were incubated with glutathione Sepharose resin ( GE Healthcare ) for 1 hr at 4°C . Bound GST fusion proteins were eluted in buffer containing 20 mM glutathione . GST-1-358 and GST-1032-1331 were loaded onto PD MiniTrap G-25 ( GE Healthcare ) to remove glutathione . His-1-358-GST protein was further incubated with Ni-NTA agarose ( Qiagen ) for 2 hr at 4°C in buffer containing 20 mM imidazole and eluted with 300 mM imidazole after 3 washes . Same amount of purified His-1–358-GST , GST-1–358 and GST-1032–1331 were immobilized on glutathione-Sepharose resin ( 3–5 mg protein/ml resin ) in 50 mM Hepes pH7 . 4 , 200 mM NaCl , 0 . 5 mM DTT and 10% glycerol . Resins were washed and equilibrated in 50 mM Hepes pH7 . 4 , 150 mM NaCl , 5mM MgCl2 , 0 . 5 mM DTT and 50 µM GTP for subsequent membrane capturing assays . His-tagged 1342–1684 was incubated with His-Pur Ni-NTA resin ( Thermo Scientific ) for 2 hr at 4°C and eluted with buffer containing 200 mM imidazole after 3 washes . Eluted protein was further purified by gel filtration on Superdex 75 ( GE Healthcare ) in 50 mM Tris , pH7 . 4 250 mM NaCl and 10% ( v/v ) glycerol . GFP-FLAG-GCC185 constructs for AFM were produced by transfection of HEK293F mammalian suspension cells grown in FreeStyle 293 expression medium ( GIBCO , Life Technologies ) with the indicated constructs for 22 hr . Cells were lysed in 50 mM Tris , pH 7 . 4 , 250 mM NaCl , 0 . 5% Triton X-100 , 10% glycerol , 1 mM EDTA , 5 mM sodium orthovanadate and protease inhibitors . Lysates were clarified by centrifugation at 13 , 000 rpm for 30 min in a FiberLite F15 ( 8x50c ) rotor ( Thermo Scientific , Waltham , MA ) . Supernatants were incubated with anti-FLAG M2 affinity gel ( Sigma Aldrich , St . Louis , MO ) for 4 hr at 4°C , followed by washes with buffer containing 1 M NaCl and two final washes in 50 mM Tris , pH7 . 4 , 250 mM NaCl and 10% glycerol . Bound proteins were eluted with 0 . 1 mg/ml 3X FLAG peptide ( Sigma-Aldrich ) . Eluted proteins were further purified on a Sepharose 4B ( Sigma-Aldrich ) column . Anti-HA antibody labeling of the 805-HA construct was performed after FLAG peptide elution ( Sigma-Aldrich ) . Excess anti-HA antibody ( Abcam , Cambridge , MA ) was added at 4°C for >2 hr . Excess antibodies were removed using Sepharose 4B . At least three transfected 10-cm dishes ( 80–90% confluency ) were harvested for vesicle isolation . Cells were washed with PBS and incubated in hypotonic buffer ( 10 mM Hepes pH7 . 4 ) for 10 min , scraped immediately into resuspension buffer ( 10 mM Hepes , pH7 . 4 , 150 mM NaCl , 5 mM MgCl2 + protease inhibitors ) and further lysed by passage through a 25-G needle . Lysed cells were spun at 800 g for 5 min at 4°C to remove nuclei and cell debris . The supernatant was spun at 135 , 000 g for 15 min at 4°C in TLA100 . 2 rotor . The membrane pellet was resuspended in 50 mM Hepes pH7 . 4 , 150 mM NaCl , 5 mM MgCl2 , 0 . 5 mM DTT and 50 μM GTP . The crude suspension was further spun at 8 , 000 g for 5 min at 4°C to pellet large membranes; the supernatant was used as a crude vesicle fraction . Fifteen microliters GST-tagged protein-bound glutathione Sepharose resin was incubated with 100 μl of crude vesicle membranes ( 5–20 μg of GFP-Rab9-transfected cell or 50 μg non-transfected cell membrane ) for 45 min at room temperature with rotation . Samples were washed with 100 column volumes of buffer and eluted with buffer containing 20 mM glutathione or sample buffer without reducing agent for CI-MPR immunoblot detection . Captured components were detected by immunoblotting with chicken anti-GFP ( 1:1000 , Life Technologies ) , rabbit anti-CI-MPR ( 1:1000 ) mouse anti-EEA1 ( 1:1000 , BD Biosciences ) , goat anti-Calnexin ( C-20 , 1:1000 , Santa Cruz Biotechnology , Dallas , TX ) and the corresponding horseradish peroxidase ( HRP ) -conjugated secondary antibodies: rabbit anti-chicken HRP ( 1:1500 , Promega , Madison , WI ) , goat anti-rabbit HRP ( 1:3000 , BioRad , Hercules , CA ) , goat anti-mouse HRP ( 1:2000 , BioRad ) and fluorescently-labeled bovine anti-goat DyLight 649 ( 1:2000 , Jackson ImmunoResearch Laboratories , West Grove , PA ) . HRP antibodies were detected using ECL plus ( ThermoFisher ) in conjunction with film and Versadoc . For immobilization , 25 μl purified sample ( ~1 nM ) in 50 mM Tris , pH 7 . 4 , 200 mM NaCl , 5 mM MgCl2 , 0 . 5 mM DTT and 10% glycerol was directly placed onto freshly cleaved mica . After 8 minutes , the mica surface was gently rinsed with 2 ml buffer followed by 2 ml of ddH20 to remove unbound proteins and dried by soft argon flow . Imaging was done in air with a Park100 AFM ( Park Systems , Santa Clara , CA ) operated in non-contact mode . We used ultra-sharp diamond-like carbon probes ( NSG10 DLC , ~2–5 nm radius of curvature at the tip ( K-Tek Nanotechnology , Wilsonville , OR ) driven near resonance at ~200 kHz . To minimize both sample and tip damage during scanning , the set point was set to the largest possible value ( largest average tip-sample distance ) that allowed good surface tracking . Raster scans ( 1 . 2 μm X 1 . 2 μm ) were performed at a resolution of 512x512 pixels with a scan rate of 1Hz . For presentation and analysis , images were first processed using a custom Matlab script ( Source code 1 provided ) to remove sample slant ( flattening ) by row and column-wise median subtraction . Individual molecules of each type were selected manually based on the following criteria: the whole object should be within the captured field without any part ( s ) being cropped by the borders , easily identified as isolated , individual , non-aggregated object with no overlap ( s ) with any other objects , and traceable/measurable . The number of selected molecules usually covered 40–70% of all the objects in the field , depending on the distribution of molecules and local quality of an image . To quantify the conformation of each molecule , a selected molecule was segmented into maximally 8 sections based on apparent structural features . The length of each segment ( X1 to X8 ) was measured with ImageJ by setting the measuring scale with the known image spatial size and outlining the molecule contour with the segmented line tool . Statistics- Minimum sample sizes were determined assuming 5% standard error and >95% confidence level; P values and their methods of determination are indicated in the legends .
The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets . GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes . Although predicted to be relatively rigid and highly extended , we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle . Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi . In physiological buffers without fixatives , atomic force microscopy reveals that GCC185 is shorter than predicted , and its flexibility is due to a central bubble that represents local unwinding of specific sequences . Moreover , 85% of the N-termini are splayed , and the splayed N-terminus can capture transport vesicles in vitro . These unexpected features support a model in which GCC185 collapses onto the Golgi surface , perhaps by binding to Rab GTPases , to mediate vesicle tethering .
Some cells release molecules , such as hormones and neurotransmitters , to signal to other cells and influence how they work . As part of the release process , these molecules are packaged into small , balloon-like structures called vesicles . Such vesicles move around within cells and are able to find the right place to release their contents to the outside . A cellular compartment called the Golgi complex helps to prepare proteins for release from the cell . Vesicles can bind to tethering proteins on the surface of the Golgi , but it was not clear how these proteins are able to capture the correct kind of vesicle . The prediction was that the proteins are rigid , shaped like pipe cleaners that stick out from the Golgi as a meshwork that traps vesicles . Cheung et al . isolated a specific Golgi tethering protein ( called GCC185 ) from cultured human cells and used a technique called atomic force microscopy to visualize its structure . This revealed that this protein is not rod-like; it is instead rather floppy , and has two arms at one end that may ‘hug’ the incoming vesicle . Cheung et al . showed that this protein needs its middle portion to be floppy to work correctly . This changes the way we think about how vesicles are able to find their corresponding targets on different compartments inside cells . Further experiments are now needed to answer a number of questions . What does the tether look like when actually bound to a vesicle ? What happens after the vesicle binds – how does the tether let go ? What other components are needed for vesicle capture and release ?