Traffic 2009; 10: 1034–1046 © 2009 John Wiley & Sons A/S

doi: 10.1111/j.1600-0854.2009.00930.x

Fluorescence Lifetime Imaging of Interactions betweenGolgi Tethering Factors and Small GTPases in Plants

Anne Osterrieder1, Claudine M. Carvalho1,2,

Maita Latijnhouwers3,4, Jorunn Nergaard

Johansen1,5, Christopher Stubbs6, Stanley

Botchway6 and Chris Hawes1,*

1School of Life Sciences, Oxford Brookes University,Headington, Oxford, OX3 0BP, UK2Current address: Department of Plant Pathology,Federal University of Vicosa, 36570000 Vicosa-MG, Brazil3Current address: Department of Mathematics andNatural Science, University of Stavanger, N-4036Stavanger, Norway4Plant Pathology Programme, Scottish Crop ResearchInstitute, Invergowrie, Dundee DD2 5DA, UK5Current address: Institute for Cancer Research,Department of Immunology, Rikshospitalet-Radiumhospitalet Medical Centre, Montebello, 0310Oslo, Norway6Central Laser Facility, STFC Harwell Science InnovationCampus, Didcot, Oxon. OX11 0QX, UK∗Corresponding author: Chris Hawes,chawes@brookes.ac.uk

Peripheral tethering factors bind to small GTPases in

order to obtain their correct location within the Golgi

apparatus. Using fluorescence resonance energy transfer

(FRET) and fluorescence lifetime imaging microscopy

(FLIM) we visualized interactions between Arabidopsis

homologues of tethering factors and small GTPases at

the Golgi stacks in planta. Co-expression of the coiled-

coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA

element modulatory factor (TMF)] and the putative

post-Golgi tethering factor AtVPS52 fused to green

fluorescent protein (GFP) with mRFP (monomeric red

fluorescent protein) fusions to the small GTPases AtRab-

H1b, AtRab-H1c and AtARL1 resulted in reduced GFP

lifetimes compared to the control proteins. Interestingly,

we observed differences in GFP quenching between

the different protein combinations as well as selective

quenching of GFP-AtVPS52-labelled structures. The data

presented here indicate that the FRET-FLIM technique

should prove invaluable in assessing protein interactions

in living plant cells at the organelle level.

Key words: Flim, FRET, GFP, Golgi, small GTPase,

tethering factor

Received 20 January 2009, revised and accepted for pub-

lication 21 April 2009, uncorrected manuscript published

online 24 April 2009, published online 20 May 2009

The Golgi apparatus plays a central role in the plantendomembrane system, as it is not only involved inthe processing of protein cargo received from the

endoplasmic reticulum (ER) but also in the synthesisof complex cell wall polysaccharides (1). Plant cells cancontain up to hundreds of discrete Golgi stacks that aremobile in an actin-dependent manner with the surfaceof the ER (2–5). It is not known how Golgi stacksmanage to keep their structural integrity while movingthrough the cytoplasm, but the existence of a plantGolgi matrix has been proposed after electron microscopyrevealed the existence of intercisternal elements and aribosome-excluding zone around the stacks (6,7). Golgimatrix proteins could also be involved in tethering Golgistacks to the ER surface (8).

The golgins, a Golgi-localized protein family with longcoiled-coil domains, are ideal candidate proteins for beingpart of a putative Golgi matrix, as in animal cells golginshave been implied in formation and maintenance ofGolgi stack structure (9–11). One example is the Golgireassembly stacking proteins (GRASPs) that are involvedin stacking of Golgi cisternae (12,13). Many golgins, suchas the cis-Golgi localized p115 and GM130, have multipletethering functions and can act in multiprotein tetheringcomplexes with other golgins, small GTPases and SNAREproteins (reviewed in 14). Examples are the p115-giantin-GM130-Rab1 tether and the golgin-84-CASP (CCAAT-displacement protein alternatively spliced product) tether,which are found on coat protein I (COPI) membranesand could define different subpopulations of COPIvesicles (15). Homologues of golgins have been identifiedin Arabidopsis and localized to the Golgi apparatus, buttheir function remains largely unknown (16–22), althoughthe Arabidopsis homologue of p115 has been suggestedto play a role in tethering the Golgi stack to the ER (23).

Whereas some golgins are targeted to the Golgi stackvia their C-terminal transmembrane domains, peripheralgolgins obtain Golgi localization by interaction with othergolgins or small regulatory GTPases. One example isthe human peripheral coiled-coil protein TATA elementmodulatory factor (TMF) that binds to Rab6 (24) andwas displaced from the Golgi stack upon RNAi-mediateddepletion of the Rab protein (25). Other peripheral golginscontain a conserved C-terminal sequence consisting of∼42 amino acids. This domain was originally identifiedin four mammalian golgins and named GRIP (golgin-97,RanBP2a, Imh1p, and p230/golgin-245) domain accordingto the starting letters of those proteins (26,27). The GRIPdomain is highly conserved in eukaryotes and is requiredfor targeting of proteins to the trans-Golgi or trans Golginetwork (TGN) through interaction with the ARL1 (ARF-like) GTPases (28,29).

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Although the organization of the plant Golgi apparatusdiffers significantly from that of yeast or animal cells (30),some basic structural mechanisms appear to be con-served, as several interactions between golgins and reg-ulatory proteins have been identified in different species.For example, the interaction between human TMF andthree isoforms of Rab6 (24) was also observed in yeastbetween the TMF homologue Sgm1p and the Rab6 homo-logue YPT6 (31) and in Arabidopsis between the homo-logues of Rab6, AtRab-H1b/c (32) and the homologue ofTMF, GC5 (golgin candidate 5, 22). GRIP domain proteinsappear to interact with the small GTPase ARL1 in yeastand animals (29,33,34) as well as in plants (19,21). AtGRIPshows high homology to the mammalian golgin-97, whichcontains a conserved C-terminal GRIP domain that inter-acts with Rab6 and is necessary and sufficient to targetthe protein to the trans-Golgi in mammalian cells (35).

Coiled-coil proteins have not only been implied in transportpathways between the ER and the Golgi but also in traf-ficking between the TGN and post-Golgi compartments.In yeast, the multisubunit tethering complex GARP (Golgi-associated retrograde protein) is involved in retrogradetransport from endosomes to the TGN (36,37). Thiscomplex consists of the four subunits Vps51/52/53/54p,each containing coiled-coil domains (36), and has beenshown to interact with Ypt6, the yeast homologue ofmammalian Rab6 (38). Similarly, it has been shownthat Vps52 interacts with Rab6 in mammalian cells (39).In Arabidopsis, a gene termed pok (poky pollen tube)identified in a mutant screen shares significant homologywith the yeast Vps52p gene (40). POK/AtVPS52 appearsto be part of a large protein complex and located tothe Golgi, pre-vacuolar compartment and an unidentifiedpost-Golgi compartment (41), but nothing is known todate about possible interactions between POK and theArabidopsis homologues RabH1b and RabH1c.

Characterizing the interactions between golgins andregulatory or other structural proteins provides a first steptowards a better understanding of their putative role withinthe Golgi stack. In vitro techniques such as yeast two-hybrid screens and pull down assays (reviewed in 42,43)can provide a general indication whether two proteins havethe potential to interact with each other. Such techniquescan, however, be compromised by the occurrence of falsepositive results and the artificial environment in which thebinding takes place. Proteins interacting in vitro may neverdo so in vivo because they might be spatially separatedin different cellular compartments (44). Therefore, it ispreferable that in vitro results are confirmed using in vivoapproaches.

A range of techniques are available to study pro-tein–protein interactions in vivo using fluorescencemicroscopy. In the bimolecular fluorescence comple-mentation (BiFC) assay, a fluorophore is split into twonon-functional halves fused to two proteins of inter-est and interaction between the proteins restores the

fluorescent signal (45,46). Analysis of FRET (Forster or flu-orescence resonance energy transfer) between a donorand an acceptor fluorophore fused to two proteins ofinterest is more commonly used. FRET occurs under theconditions of a sufficiently large spectral overlap betweendonor emission and acceptor absorption spectrum, afavourable orientation of fluorophores to each other in aproximity of 1–10 nm (47). Interacting proteins are closeenough to allow non-radiative energy transfer from thedonor to the acceptor fluorophore, leading to quenchingof donor fluorescence and an increase in acceptor fluo-rescence. Besides spectral bleed-through, other problemsof this technique include the dependence on fluorophoreexpression levels and unintended photobleaching duringmeasurements (48).

A method that combines FRET with fluorescencelifetime imaging (FLIM) has certain advantages overconventional FRET measurements (49–52). A fluorophorehas a characteristic lifetime that remains unaffected byfluorophore concentration levels or excitation intensity,and spectral bleed-through does not constitute a problemas only the donor lifetime is measured (47). Thefluorophore lifetime can be influenced by changes intemperature, environment, calcium ion concentrationand the occurrence of FRET (51). Quenching of thedonor fluorophore lifetime indicates interaction with theacceptor fluorophore and can be measured directly.FRET-FLIM has been successfully used in planta tostudy protein–protein interactions in protoplasts ofcowpea (53–55), petunia (56), tobacco (57), maize (58)and Arabidopsis (59,60) as well as in barley (61) andtobacco leaf epidermal cells (62,63). Whereas FRET-FLIMhas been used successfully on animal cells to visualizeinteractions between Rab GTPases and Rab bindingproteins (64,65), in plants no such in vivo analyses ofRab or golgin protein interactions have been performedso far to our knowledge.

In this study we aimed to confirm in planta interactionsbetween several plant Golgi tethering factors and smallGTPases that to date only had been performed in vitro, byanalysing the fluorescence lifetime (FLIM) after FRET bytwo-photon excitation (66). In this system, fluorophoresare excited by the absorption of multiphotons in the near-infrared wavelength, which allows deeper penetrationof tissues and leads to less cell damage. Absorption isconfined to a narrow focussed plane and lacks out-of-focus excitation, therefore resulting in higher resolutionand further reduction of photo damage (67).

Results

AtRab-H1b but not AtRab-H1c interacts with AtGRIP

in vitro

To investigate if the small GTPases AtRab-H1b and AtRab-H1c would interact with the plant homologue of golgin-97in vitro, we analysed the interaction between AtGRIP and

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both AtRab-H1b and AtRab-H1c in vitro using the yeasttwo-hybrid system (68). Wild-type AtRab-H1b and AtRab-H1c were fused at their N-termini with the activationdomain of the yeast transcription factor GAL4. AtGRIPwas fused to the C-terminus of the GAL4-binding domain.Prey and bait constructs were sequentially transformedinto yeast and the transformants were plated on selectionplates. Only the GAL4BD-GRIP and GAL4AD-AtRab-H1b

produced colonies and tested positive for β-galactosidaseactivity, indicating that these two fusion proteins interactin yeast (Figure 1A). No interaction was detected betweenthe AtGRIP and AtRab-H1c in this assay (Figure 1B).

To examine whether the interaction between AtRab-H1b

and AtGRIP was dependent on the state of GTP binding,the GDP-locked mutant AtRab-H1b[T23N], the GTP-lockedmutant AtRab-H1b[Q68L] and the dominant-negativemutant AtRab-H1b[N122I] with reduced GDP/GTP affin-ity (32) were each fused to the C-terminus of the GAL4-activation domain. Again, the prey and bait constructswere sequentially transformed into yeast and plated on theselection medium (Figure 1C–E). Only GAL4AD-AtRab-H1b[Q68L] and GALBD-GRIP showed a positive inter-action (Figure 1D). Neither AtRab-H1b[T23N] nor AtRab-H1b[N122I] interacted with AtGRIP (Figure 1C,E).

Figure 1: AtRab-H1b, but not AtRab-H1c, interacts with AtGRIP in vitro. A–E) Yeast two-hybrid analysis. Yeast transformantswere plated on selection plates. The presence of two interacting partners allows yeast to grow in the absence of histidineand adenine. GAL4BD, Gal4-binding domain and GAL4AD, Gal4-activation domain. A) GAL4BD-AtGRIP + GAL4AD-AtRab-H1b,(B) GAL4BD-AtGRIP + GAL4AD-AtRab-H1c, (C) GAL4BD-AtGRIP + GDP-locked mutant GAL4AD-AtRab-H1b[T23N], (D) GAL4BD-AtGRIP + GTP-locked mutant GAL4AD-AtRab-H1b[Q68L] and (E) GAL4BD-AtGRIP + dominant-negative mutant GAL4AD-AtRab-H1b[N122I]. F–H) Confocal images of tobacco leaf epidermal cells transiently transformed with fluorescent fusion constructs of AtGRIPand AtRab-H1b wild-type and mutants. All constructs were transformed at an OD600 of 0.03 and imaged 2–3 days after inoculation.F) Co-expression of YFP-AtRab-H1b[wt] (magenta channel) and AtGRIP-GFP (green channel) shows that the construct co-located (mergedimage). G) Co-expression of the GTP-locked mutant YFP-AtRab-H1b[Q68L] (magenta channel) and AtGRIP-GFP (green channel) showsthat the constructs co-locate (merged image). H) Co-expression of the dominant-negative mutant YFP-AtRab-H1b[N122I] (magentachannel) with AtGRIP-GFP (green channel) results in aggregation of the two constructs (merged image). All scale bars = 10 μm.

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When transiently expressed in tobacco leaf epidermalcells, YFP-AtRab-H1b (magenta channel) co-located withAtGRIP-green fluorescent protein (GFP) (Figure 1F, greenchannel). Because interaction between AtRab-H1b andAtGRIP was dependent on the nucleotide-binding statusof AtRab-H1b, AtGRIP was co-expressed with yellowfluorescent protein (YFP)-AtRab-H1b[T23N], YFP-AtRab-H1b[Q68L] and YFP-AtRab-H1b[N122I] in tobacco leaves(Figure 1H–I). The YFP-AtRab-H1b[Q68L] signal showedoverlap with AtGRIP-GFP (Figure 1G) similar to thatobserved for YFP-AtRab-H1b[wt]. However, when AtGRIPwas co-expressed with YFP-AtRab-H1b[N122I] (Figure 1H)and YFP-AtRab-H1b[T23N] aggregates containing bothfusion proteins were formed.

Peripheral plant coiled-coil proteins co-locate with

small GTPases in tobacco leaf epidermal cells

Figure 2 shows confocal images of the fluorescentfusion proteins used in this study, transiently expressedin tobacco leaf epidermal cells after transfection ofleaves with agrobacterium suspension cultures. The trans-Golgi coiled-coil proteins AtGRIP-GFP (Figure 2A–D, greenchannel) and GFP-cGC5 (henceforth referred to as GFP-cTMF, Figure 2E,F, green channel) were observed in thecytoplasm and on punctate structures that previously havebeen shown to co-locate with a Golgi marker (19,22).AtGRIP-GFP and mRFP-AtARL1 signals co-located in thecytoplasm and at larger punctate structures resemblingGolgi bodies as reported by Latijnhouwers et al. (19,Figure 2A). AtGRIP-GFP additionally labelled smaller punc-tate structures (Figure 2A, arrowheads). mRFP-AtRab-H1b

was observed in the cytoplasm and punctate structures,co-locating partially with AtGRIP-GFP (Figure 2B) andfully with GFP-cTMF (Figure 2E, magenta channel). Thesame localization was observed for mRFP-AtRab-Hc co-expressed with AtGRIP-GFP (Figure 2C, magenta channel)or GFP-cTMF (Figure 2F, magenta channel). AtGRIP-GFPlocated to the same structures as AtCASP-mRFP but withonly partial overlap confirming their predicted locationto cis- and trans-faces of Golgi stacks (Figure 2D). GFP-AtVPS52 labelled punctate structures (Figure 2G,H, greenchannel), some of which partially co-located with mRFP-AtRab-H1b (Figure 2G, magenta channel, arrowheads inmerged channel) or mRFP-AtRab-H1c (Figure 2H, magentachannel, arrowheads in merged channel).

Spatial proximity of GFP and mRFP does not affect

the lifetime of GFP

To establish the lifetime of GFP in tobacco leaf epidermalcells, we first measured the lifetime of the Golgi markerSTtmd-GFP (2) expressed in stable tobacco G41 plants.To stop movement of Golgi bodies during data collection,all leaf samples in this study were incubated in 25 μmlatrunculin B to depolymerize the actin cytoskeleton (4).Data was collected from 84 Golgi bodies in three samplesand the lifetime values ranged between 2.4 and 3.1 nswith an average of 2.72 ns (Figure 3A, Table 1). To excludethe possibility of FRET occurrence between GFP andmRFP caused by close spatial proximity in the same

organelle, we transfected wild-type tobacco leaf epidermalcells with plasmids encoding ST-GFP and ST-mRFP (18),which both target to the medial and trans-Golgi. Onesample with 20 Golgi bodies provided sufficient data foranalysis. The lifetime distribution ranged between 2.5 and3 ns (Figure 3A) with an average of 2.74 ns (Figure 4) andwas not found to be statistically different from the controllifetime distribution (Student’s t-test p > 0.05, Table 1).This result shows that the pure spatial proximity of thetwo fluorophores does not lead to a reduced lifetimecompared to GFP expressed alone.

To determine the lifetime of AtGRIP-GFP, GFP-cTMFand GFP-AtVPS52, we expressed each fluorescent fusiontransiently in wild-type tobacco leaf epidermal cells. Thelifetime of AtGRIP-GFP ranged between 2.2 to 2.6 ns(Figure 3B) and an average of 2.49 ns (Figure 4). Datawere collected from three samples with 38 Golgi bodies.This value was slightly lower than the lifetime of theST-GFP control. Figure 5A,B shows a representative cellexpressing AtGRIP-GFP and the corresponding false-coloured lifetime map respectively with green coloursrepresenting lifetime values around 2 ns and blue shadeshigher lifetimes of around 3 ns. A representative decaycurve for a single point within a Golgi body with alifetime of 2.54 ns is shown in Figure 5C. The χ2 valueof 1 indicates an optimal single exponential fit. Toconfirm that spatial proximity of fluorophores in the samecompartment, but with the fluorophores exposed onthe cytoplasmic face of the Golgi, does not affect thelifetime, AtGRIP-GFP was co-expressed with the cis-Golgimatrix protein mRFP-AtCASP, which is targeted to theopposite end of the Golgi stack. Data from three sampleswith 35 Golgi bodies were analysed with lifetime valuesfrom 2.2 to 2.6 ns (Figure 3B) and an average of 2.42 ns(Figure 4). No reduction in the lifetime of AtGRIP-GFPwas detected (Student’s t-test p > 0.05, Table 1). Thelifetime distribution of GFP-cTMF obtained from threesamples with 42 Golgi bodies ranged between 2.2 and2.9 ns (Figure 3D), averaging 2.44 ns (Figure 4). For GFP-AtVPS52, data was analysed from three samples with 25Golgi bodies and the lifetime distribution ranged between2.3 and 2.7 ns (Figure 3E), averaging 2.42 ns (Figure 4).

AtGRIP-GFP interacts with small regulatory GTPases

in planta

We have previously demonstrated the interactionbetween AtGRIP and AtARL1 in vitro in plants (19,21).Here we used this protein combination as positive con-trol. AtGRIP-GFP was co-expressed with mRFP-AtARL1(Figure 5D) and three samples with 37 Golgi bodieswere analysed. Compared to the non-quenched GFP con-trol (Figure 5A,B), clear quenching of AtGRIP-GFP wasobserved and a change of Golgi body colour could bevisualized in the false-coloured lifetime map (Figure 5E).Figure 5F shows a representative decay curve for a singlepoint analysis with a reduced lifetime of 2.08 ns and a χ2

of 1.05, representing a good single exponential fit. Thelifetime distribution was shifted towards a lower range

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Figure 2: The subcellular localization of

coiled-coil and small regulatory proteins.

Confocal images showing GFP (green) andmRFP (magenta) fusion proteins co-expressedin tobacco leaf epidermal cells 3 days after trans-fection with agrobacterium suspension cultures.AtGRIP-GFP, GFP-cTMF, mRFP-CASP and GFP-VPS52 were infiltrated at OD600 = 0.1, mRFP-Rab-H1b/c and mRFP-AtARL1 at OD600 = 0.05.A) AtGRIP-GFP + mRFP-AtARL1. B) AtGRIP-GFP+ mRFP-Rab-H1b. C) AtGRIP-GFP + mRFP-Rab-H1c. D) AtGRIP-GFP + mRFP-AtCASP. E)GFP-cTMF + mRFP-AtRab-H1b. F) GFP-cTMF+ mRFP-AtRab-H1c. Arrowheads indicate co-loalization between GFP-VPS52 and mRFP-AtRab-H1b. G) GFP-VPS52 + mRFP-AtRab-H1b.H) GFP-VPS52 + mRFP-AtRab-H1c. Arrowheadsindicate co-localization between GFP-VPS52 andmRFP-AtRab-H1c. Scale bars, 10 μm.

between 1.8 and 2.4 ns (Figure 3B), averaging 2.14 ns(Figure 4) and indicating a strong interaction (Student’s t-test p < 0.05, Table 1). The low number of higher lifetimevalues can be explained by the presence of large brightaggregates labelled by both proteins, which do not showreduced lifetime values (Figure 5E, arrowhead).

Co-expression of AtGRIP-GFP and mRFP-AtRab-H1b alsoresulted in quenching of AtGRIP-GFP. Data were collectedfrom five samples with 76 Golgi bodies and the

average lifetime was 2.17 ns (Figure 4) with lifetimevalues ranging from 1.8 to 2.7 ns (Figure 3C), indicatingprotein interaction (Student’s t-test p < 0.05, Table 1).Figure 5G,H shows two cells, the left one expressingboth constructs (arrowhead), whereas the right cell in theupper corner only expressed AtGRIP-GFP (cell markedwith asterisk in Figure 5H). Golgi bodies in the cell withboth constructs showed a reduced lifetime, whereasthose labelled by AtGRIP-GFP had a lifetime between2.3 and 2.6 ns.

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Figure 3: Lifetime distributions of GFP constructs with and without their putative binding partners. To determine the lifetimeof GFP expressed with or without putative binding partners in tobacco leaf epidermal cells, fluorescence lifetime measurements wereperformed 2–4 days after agrobacterium-mediated infiltration. Lifetimes for individual Golgi bodies were obtained on a pixel base. Thisfigure shows the percentage of the total analysed number of Golgi bodies with their corresponding lifetime values on a range from 1.8to 3.1 ns and compares the lifetime distribution of control proteins against protein combinations. A) The lifetime values of the Golgimarker ST-GFP ranged between 2.4 and 3.1 ns (n = 84). Although ST-GFP and ST-mRFP both target to the medial/trans-Golgi apparatus,no quenching was observed and the lifetime values ranged between 2.5 and 3 ns (n = 20). B) The lifetime values of AtGRIP-GFP rangedbetween 2.2 and 2.6 ns (n = 38). Co-expression of AtGRIP-GFP with mRFP-CASP, both being located in the Golgi stack, resulted in alifetime distribution between 2.2 and 2.6 ns (n = 35). The lifetime distribution of the positive control AtGRIP-GFP co-expressed withmRFP-AtARL1 ranged between 1.8 and 2.4 ns (n = 76). C) Compared to the lifetime distribution of the control AtGRIP-GFP (B), uponco-expression with mRFP-AtRab-H1b lifetimes between 1.8 and 2.7 ns were observed (n = 84). The lifetimes of AtGRIP-GFP expressedwith mRFP-AtRab-H1c ranged between 2 and 2.5 ns (n = 27). D) GFP-cTMF had a lifetime distribution between 2.2 and 2.9 ns (n = 42).When expressed with mRFP-AtRab-H1b, the lifetime ranged between 2.1 and 2.7 ns (n = 41), whereas upon co-expression withmRFP-AtRab-H1c values between 1.8 and 2.6 ns were observed (n = 17). E) The lifetime of GFP-VPS52 ranged between 2.3 and 2.7 ns(n = 25). Co-expression of GFP-VPS52 with mRFP-AtRab-H1b resulted in a lifetime range between 1.7 and 2.6 ns (n = 123). The lifetimedistribution for GFP-VPS52 expressed with mRFP-AtRab-H1c ranged between 1.9 and 2.6 ns (n = 75).

Similarly, expression of AtGRIP-GFP with mRFP-AtRab-H1c resulted in a reduced lifetime with values between2.0 and 2.5 ns (Figure 3C), averaging 2.19 ns (Figure 4).This result was statistically different to the control lifetimeof AtGRIP-GFP (Student’s t-test p < 0.05, Table 1). Datawere collected from three samples with 27 Golgi bodies.Compared to the interaction of AtGRIP with AtRab-H1b,no values below 2.0 ns were observed for AtGRIP-GFPexpressed with mRFP-AtRab-H1c, which might indicatea weaker interaction between mRFP-AtRab-H1c andAtGRIP-GFP.

GFP-cTMF interacts with mRFP-AtRab-H1c and may

interact with mRFP-AtRab-H1b

GFP-cTMF was expressed together with mRFP-AtRab-H1b and data were collected from three samples with41 Golgi bodies. The majority of lifetime values werebetween 2.3 and 2.7 ns (Figure 3D) with an average of2.44 ns (Figure 4), but lower lifetimes of 2.1 or 2.2 nswere observed as well. This indicates that the two proteinsmight interact very weakly or only rarely (Student’s t-testp < 0.05, Table 1). When GFP-cTMF was co-expressedwith mRFP-AtRab-H1c, the lifetime distribution wasshifted towards 2.2–2.4 ns with several lower values

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Table 1: Average lifetime values of GFP control and GFP–mRFP interaction experiments

Donor Acceptor Average life time (ns) and SD n p (two-tailed Student’s t-test)

ST-GFP – 2.72 ± 0.14 84 –ST-GFP ST-mRFP 2.74 ± 0.11 20 0.4002AtGRIP-GFP – 2.49 ± 0.12 38 –AtGRIP-GFP mRFP-AtCASP 2.42 ± 0.13 35 0.0077AtGRIP-GFP ARL1-mRFP 2.14 ± 0.17 76 1.14 × 10−22

AtGRIP-GFP mRFP-AtRab-H1b 2.17 ± 0.2 84 1.31 × 10−16

AtGRIP-GFP mRFP-AtRab-H1c 2.19 ± 0.13 27 6.16 × 10−15

GFP-cTMF – 2.57 ± 0.17 42 –GFP-cTMF mRFP-AtRab-H1b 2.44 ± 0.14 41 0.0001GFP-cTMF mRFP-AtRab-H1c 2.3 ± 0.21 17 6.37 × 10−08

GFP-VPS52 – 2.42 ± 0.11 25 –GFP-VPS52 mRFP-AtRab-H1b 2.3 ± 0.17 123 0.0005GFP-VPS52 mRFP-AtRab-H1c 2.37 ± 0.17 75 0.1122

Average lifetimes of GFP fusion proteins expressed alone or together with mRFP-tagged putative interaction partners and correspondingstandard deviations (SD) and significance values.

between 1.8 and 2.0 ns (Figure 3D), averaging 2.3 ns(Figure 4). Data was collected from one sample with17 Golgi bodies and the appearance of lower lifetimesindicates that interaction took place (Student’s t-testp < 0.05, Table 1).

GFP-AtVPS52 is quenched selectively by

mRFP-AtRabH1b/c

GFP-AtVPS52 was co-expressed with mRFP-AtRab-H1b

(Figure 5I) and 10 samples with 123 GFP-AtVPS52-labelled structures were analysed. Lifetimes between

Figure 4: Average lifetime values of control proteins and combinations. The average lifetime values were calculated fromthe lifetime distributions of the GFP fusion proteins both expressed alone and in combination with their putative binding partners.Compared with AtGRIP-GFP (2.49 ns), the average lifetimes values were reduced upon co-expression with mRFP-AtARL1 (2.14 ns),mRFP-AtRab-H1b (2.17 ns) and mRFP-AtRab-H1c (2.19 ns). Looking at the average lifetime values solely, only a slight reduction ofaverage lifetime values was observed when comparing GFP-VPS52 alone (2.42 ns) to the combinations with mRFP-AtRab-H1b (2.3 ns)and mRFP-AtRab-H1c (2.37 ns). In contrast to that, quenching of GFP could clearly be observed looking at the lifetime distributionof individual Golgi bodies (see Figure 3). GFP-cTMF had an average lifetime of 2.47 ns, which was reduced upon co-expressionwith mRFP-Rab-H1b (2.44 ns) or mRFP-AtRab-H1c (2.3 ns). Almost no difference could be detected for ST-GFP (2.72 ns) and ST-GFPco-expressed with ST-mRFP (2.74 ns). Error bars represent standard deviations (see Table 1).

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Figure 5: Interactions between coiled-coil proteins and small regulatory GTPases at the subcellular level. The subcellularlocalizations of interactions between coiled-coil proteins fused to GFP and small regulatory GTPases fused to mRFP were visualizedwith pseudo-coloured lifetime maps generated in SPCImage (Becker & Hickl, Germany). A) Confocal image of a tobacco leaf epidermalcell expressing AtGRIP-GFP alone as control. B) Pseudo-coloured lifetime map of the same cell. Golgi bodies appear in blue, indicatingan unquenched lifetime around 2.5 ns. C) Representative decay curve for a single point analysis of AtGRIP-GFP with a lifetime of 2.54 nsand a χ2 value of 1, indicating an optimal single exponential fit. D) Confocal image of a cell expressing AtGRIP-GFP and mRFP-AtARL1.E) Lifetime map showing quenching of AtGRIP-GFP in punctate structures co-locating with mRFP-AtARL1. The lifetime reduction isreflected in the green colour. Occasionally, large bright aggregates labelled by both proteins were observed, which did not showreduced lifetime values (arrowhead). F) Representative decay curve for a single point analysis of AtGRIP-GFP + mRFP-ARL1 with areduced lifetime of 2.08 ns. The χ2 of 1.05 indicates a single exponential optimal fit. G) Confocal image of two cells, the lower leftexpressing AtGRIP-GFP and mRFP-RabH1b and the upper right only expressing AtGRIP-GFP. H) The lifetime map depicts quenchingof GFP at Golgi bodies in the cell expressing both constructs, whereas Golgi bodies in the cell expressing AtGRIP-GFP show noreduced lifetime (asterisk). I) Confocal image of a cell expressing GFP-VPS52 and mRFP-AtRab-H1b. J) The lifetime map shows thatquenching of GFP-VPS52 does not occur in all, but only occurs in a few punctate structures labelled by GFP-VPS52 and mRFP-AtRab-H1b

(arrowhead). Scale bars, 10 μm.

1.7 and 2.6 ns were observed (Figure 3E), averaging2.3 ns (Figure 4) and indicating an interaction betweenthe two proteins (Student’s t-test p < 0.05, Table 1).Surprisingly, GFP quenching was restricted to a subsetof punctate structures (Figure 5J, arrowheads). A similarresult was observed for co-expression of GFP-AtVPS52with mRFP-AtRab-H1c, where data was collected fromfour samples with 75 GFP-AtVPS52-labelled structures.Here the lifetime values ranged between 1.9 and 2.6 ns(Figure 3E) with an average of 2.37 ns (Figure 4). Althoughthere appears to be no statistically significant differencebetween the control and the protein combination becauseof the wide range of lifetime values (Student’s t-testp > 0.05), quenching of GFP-AtVPS52 clearly indicatesprotein interaction in a subset of structures.

To confirm that the relatively small but significantdifferences in lifetimes between control and interactingpartners occurred due to FRET, we attempted a controlexperiment in which we tried to bleach the mRFPacceptor. In theory, photobleaching of the acceptor shouldprevent non-radiative energy transfer from the donor tothe acceptor and restore the GFP lifetime of the donorto a value similar to the control conditions. In practice,a full reversion to control lifetimes would, however, onlybe achieved after complete bleaching of the acceptor. Inthe protein combinations tested in our study, the mRFPfusion proteins ARL1 and Rab1-Hb/c cycle between Golgibodies and a cytoplasmic pool. To prevent fluorescentrecovery from the cytoplasm, we tried to bleach wholecells expressing AtGRIP-GFP and mRFP-ARL1 using the543 nm laser line but this proved impossible, even after

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continuous scanning at 100% laser strength and very lowresolution (125 × 125 pixels) over several minutes.

Discussion

Interactions between Golgi-localized coiled-coil

proteins and small GTPases can be visualized in

planta

The results from this study demonstrated that interactionsbetween proteins associated at the Golgi apparatus inplants can be confirmed using a FRET-FLIM approach.We have shown that a number of putative matrixproteins interact with small regulatory GTPases. This isin agreement with the data from yeast and mammaliancells where a number of small GTPases of the Rab,YPT (Yeast Protein transport) and ARL and ARF familieshave been suggested to be involved in the recruitment oftethering proteins to Golgi membranes (11). Whether thisis by direct recruitment of tethers, regulating molecularinteraction before membrane fusion or regulating theaccessibility of SNAREs to permit fusion is not clear.Interactions with Rab proteins might not only be requiredfor Golgi localization of coiled-coil proteins but also forcorrect targeting of cargo carriers or cisternae at theGolgi stack. Sinka et al. (69) mapped multiple Rab bindingsites on Drosophila GRIP domain proteins and proposeda model of a ‘tentacular Golgi’, where coiled-coil proteinsare anchored with their C-termini to Golgi membranes andreach out with their N-termini into the cytoplasm to captureand subsequently distribute Rab-containing membraneswithin the polarized Golgi stack.

We have shown that the lifetime of GFP on Golgi-targetedconstructs can be obtained and quenching measuredto assess protein–protein interactions by FRET. Thus,in tobacco leaf epidermal cells we were able to verifyresults from in vitro protein binding assays and study suchprotein interactions in vivo. This permits rapid analysis oftransiently or stably expressed protein combinations inplanta at the organelle level.

We used mGFP6 (70) and mRFP (71) as donor andacceptor pair, as the widely used cyan fluorescent protein(CFP)-YFP combination has several disadvantages (45,63).CFP and YFP are able to form heterodimers (72,73)and therefore exhibit biexponential decay kinetics (74),whereas this problem is avoided using GFP-mRFP dueto the monomeric behaviour of mRFP (71). CFP has alsoa lower extinction coefficient than GFP, meaning that ahigher excitation intensity is needed, resulting in fasterphotobleaching (48). The spectral overlap in emissionspectra between CFP and YFP can also be problematicaland requires the use of narrow band pass filters (52).

GFP lifetimes might be influenced by their subcellular

environment

All lifetime values for all different protein combinationstested are summarized in Table 1. ST-GFP stably

expressed in tobacco plants had an average lifetimeof 2.72 ns. The average lifetime of ST-GFP transientlyco-expressed with ST-mRFP was 2.74 ns. This showsthat detection of protein interaction by FRET-FLIM isspecific, as pure spatial proximity of GFP- and mRFP-tagged constructs targeted to the same compartment didnot lead to GFP quenching and a reduction in lifetime.Similarly, when the trans-Golgi marker AtGRIP-GFP wasexpressed with the cis-Golgi marker mRFP-CASP, noquenching of AtGRIP-GFP was observed, indicating aspredicted that no interaction took place.

The lifetimes of GFP averaged 2.49 ns when fused toAtGRIP, 2.57 ns for cTMF and 2.42 ns for AtVPS52. Thereported lifetime for eGFP was 2.4 ns in Escherichiacoli (75) and in tobacco leaf epidermal cells fused tothe Arabidopsis EB1a (microtubule end binding protein1a, 62). However, to our knowledge no data is availablefor the lifetime of mGFP6 (70), a GFP variant optimizedfor expression in plants and part of the pMDC vectorsused in this study (76). It has been suggested that therefractive index of the environment influences the lifetimeof GFP (77) and this may explain the slight differences inthe lifetimes of our GFP controls.

Protein interactions visualized by FLIM

AtGRIP-GFP had an average lifetime of 2.49 ns, whereasAtGRIP-GFP expressed together with mRFP-AtARL1 had areduced lifetime of 2.14 ns. This combination representeda positive control because the interaction between thetwo proteins has been suggested to be conserved indifferent species (29). This result has also previously beenshown in vitro for the Arabidopsis proteins using a yeasttwo-hybrid assay and affinity chromatography (19) whereit was suggested that recruitment of the GRIP domainprotein to the Golgi was mediated by the ARL1 GTPase.AtGRIP-GFP was also quenched when co-expressed withmRFP-AtRab-H1b with an average lifetime of 2.17 ns ormRFP-AtRab-H1c with an average lifetime of 2.19 ns.However, results from the yeast two-hybrid analysesonly indicated interaction with AtRab-H1b. A possibleexplanation could be that the interaction between AtGRIPand AtRab-H1c is so weak as not to be detectable in theyeast two-hybrid assay. Alternatively, the two proteinsmight not have interacted in vitro due to the artificialenvironment or the in vivo interaction might be temporallyand/or spatially restricted. The data on the GRIP domainprotein indicate that two GTPases may be involved inthe binding of AtGRIP to the trans-Golgi. Such dualGTPase binding to GRIP domain proteins has recentlybeen reported in mammalian cells where it was shownthat Rab6 binding promotes association of ARL1 with theGRIP domain (78).

GFP-cTMF alone had an average lifetime of 2.57 ns. WhenGFP-cTMF was co-expressed with mRFP-AtRab-H1b andmRFP-AtRab-H1c, the average lifetimes were 2.44 and2.3 ns, but in case of GFP-cTMF and mRFP-AtRab-H1b

some Golgi bodies had lifetimes of 2.1 and 2.2 ns, and

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for GFP-cTMF and mRFP-AtRab-H1c significantly lowerlifetimes of 1.8–2.2 ns were observed. These lifetimedistributions suggest that GFP-cTMF might interact morestrongly with AtRab-H1c than with AtRab-H1b. In a yeasttwo-hybrid system and in pulldown experiments, cTMFwas interacting equally strong with AtRab-H1b and AtRab-H1c (22). This could indicate that the proteins have theprincipal capability to bind in vitro, but that the interactionbetween GFP-cTMF and the Rab GTPases might betransient or spatially restricted in planta and generallyweaker between cTMF and AtRab-H1b. Another possibleexplanation could be that the binding site for AtRab-H1c on GFP-cTMF is located closer to its C-terminusthan for AtRab-H1b, thereby resulting in a higher energytransfer from the donor to the acceptor. Ideally, equivalentconcentrations of donor and acceptor would give optimalFRET analysis conditions. However, in practice saturatinglevels of the donor with respect to the acceptor providea good approximation. Thus, although quenching of GFPis not generally dependent on saturating levels of theacceptor (52), it cannot be excluded that the difference inlifetime reduction could be influenced by a small differencein expression levels of mRFP-Rab-H1b and mRFP-AtRab-H1b in tobacco leaf epidermal cells.

Surprisingly, quenching of GFP-AtVPS52 co-expressedwith mRFP-AtRab-H1b/c did not appear homogeneousbut was restricted to a subpopulation of labelledstructures. Insertional pok mutant Arabidopsis linesshowed impaired pollen tube growth (40), but other thanthat not much is known about the function of AtVPS52.VPS52 homologues in yeast and animals have beenimplied in protein sorting at late Golgi compartmentsas part of the GARP complex (36,37,39), and asPOK/AtVPS52 has been shown to locate to post-Golgicompartments in plants (41) it might be involved in similarprocesses (40,41). Possibly, the selective quenching ofGFP-AtVPS52 could indicate transient interaction ofAtVPS52-labelled late Golgi compartments with the Golgiapparatus or might even imply the existence of differentGolgi body subpopulations.

Future perspectives of FRET-FLIM analysis

This study shows that FRET-FLIM analysis provides apowerful tool to confirm interactions between proteinsobserved in vitro as well as to study unknown proteincombinations in planta at the organelle level. Summarizingour observations from in vitro and in planta approaches weconclude that in vitro methods like the yeast two-hybridscreen employed here provide a good first indicationwhether two proteins have the capability to interact. Usingthe FRET-FLIM approach in planta, however, provided uswith valuable additional information. We were not onlyable to distinguish between stronger and weaker GFPquenching implying different strengths of interactionsbut in some cases we also observed selective GFPquenching in a subset of structures, which might beexplained by temporally or spatially restricted interactions.Could those interactions be dependent on the activity of

different Golgi stacks, or might different Golgi bodies havedifferent functions and therefore require different sets ofregulatory proteins at different times? Generally, proteinsmight interact only at certain developmental stages ortime points within the cell or organelles and this couldbe studied using FRET-FLIM. If the resolution of dataanalysis could be increased, it might even be possible todistinguish the exact location of protein interaction withinthe Golgi stack.

Materials and Methods

PlasmidsStandard molecular techniques were used as described in Sambrook andRussell (79). AtVPS52 was amplified from Arabidopsis cDNA with thefollowing primers:

VPS52-AscI (5′-AGG CGC GCC AAA AAT GTC CGA CAT TTC CAT-3′)and VPS52-NotISTOP (5′-GGA AAA AAG CGG CCG CTC AGA AAG TCTTGG AGT-3′). The binary vector pMDC43 (76) was used for fusion to theC-terminus of mGFP5 (80). The polymerase chain reaction (PCR) fragmentwas cloned into pENTR1A-MCS (19) and transferred into pMDC43 usingthe Gateway system following instructions provided by the manufacturer(Invitrogen).

To construct pMDC83-mRFP (for fusion to the N-terminus of mRFP), mRFPwas amplified with mRFP-AscI (5′-AGG CGC GCC TCG AGA TGG CCT CCTCCG AGG ACG TC-3′) and mRFP-SacI (5′-TCC TCC GAG CTC TTA GGCGCC GGT GGA GTG GCG-3′). The PCR fragment was digested with AscIand SacI and used to replace the mGFP5 gene in pMDC84. For pMDC43-mRFP (for fusion to the C-terminus of mRFP), mRFP was amplified withmRFP-KpnI (5′-CGG GGT ACC ATG GCC TCC TCC GAG GAC GTC-3′) andmRFP-AscI (5′-AGG CGC GCC GGC GCC GGT GGA GTG GCG-3′). The PCRfragment was digested with KpnI and AscI and introduced into the KpnIand AscI sites of pMDC32. AtRabH1b, AtRabH1c and AtARL1 were fusedto mRFP by recombination of their respective pENTR clones (19,22) withpMDC83-mRFP or pMDC43-mRFP using the Gateway system.

For yeast two-hybrid analysis, the cDNA encoding the full length of theAtGRIP (At5g66030) was amplified by PCR using the primers Y2H-F-GRIP(5′-TCC AGC ACC ATT CCC GGG GAT GTC CGA AGA CAA G-3′) andY2H-R-GRIP (5′-CCA GCA CAG TTT CCC GGG GTT ATG AAA ACG AGAATC T-3′) containing the SmaI restriction site (underlined) for cloningpurposes. The PCR product was cloned into pGBKT7 bait vector (BDBiosciences Clontech). The AtRab-H1b and the mutants AtRab-H1b[T23N],AtRab-H1b[Q68L] and AtRab-H1b[N122I] were amplified from the plasmidspVKH18-En6-Rab-H1b, pVKH18-En6-Rab-H1b[T23N], pVKH18-En6-Rab-H1b[Q68L] or pVKH18-En6-Rab-H1b[N122I] (32) by PCR using the primersY2H-RabH1.b-F (5′-G TCC AGC ACC ATT CCC GGG ATG GCT CCG GTCTCG GCA-3′ and Y2H-RabH1.b-R (5′-CCA GCA CAG TTT CCC GGG GCTAAC AAG AGC ATC CTC C-3′). The primers Y2HRabH1.c-F (5′-A TCCAGC ACC ATT CCC GGG ATG GCT TCG GTT TCA CCT TTG GCA-3′) andY2HRabH1.c-R (5′-CCA GCA CAG TTT CCC GGG TCA ACA AGA ACAGCC TCC ACC ACC-3′) were designed to amplify AtRab-H1c from theplasmid pVKH18-En6-Rab-H1c. The PCR fragments were cloned in thepGADT7-Rec prey vector, again using SmaI as a restriction site.

Yeast two-hybrid analysisYeast two-hybrid analysis was performed as described recently (22).Yeast strain AH109 was sequentially transformed with pGBKT7-GRIPand pGADT7-Rec-Rab-H1b, pGADT7-Rec-Rab-H1c, pGADT7-Rec-Rab-H1b[T23N], pGADT7-Rec-Rab-H1b[Q68L] or pGADT7-Rec-Rab-H1b[N122I]using a lithium acetate method (81). Colonies were selected in plateslacking histidine, tryptophan, leucine and adenine up to a period of

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10 days. Positive yeast transformants were replated on β-galactosidase(BD Biosciences Clontech) plates to test the expression of the reportergene MEL1.

Expression of fluorescent protein fusions in tobacco

plantsTransient expression of fluorescent protein fusions in tobacco leaveswas performed using agrobacterium-mediated infiltration of lower leafepidermal cells (82). Nicotiana tabacum sp. plants were grown in thegreenhouse at 21◦C, 14 h light, 10 h dark and were used for agrobacteriuminfiltration after 5–6 weeks. Leaf samples were analysed 2–4 days afterinfiltration. Stable ST-GFP tobacco plants were created in our lab asdescribed recently (82).

Latrunculin B treatmentTo inhibit Golgi movement, tobacco leaf samples were treated with theactin-depolymerising agent latrunculin B (4). Latrunculin B (Calbiochem)was dissolved in dimethyl sulphoxide (DMSO) at 1 mm and stored at–20◦C.Leaf samples were incubated in a 25 μm working solution for at least15 min, until Golgi movement had stopped.

Confocal microscopyHigh-resolution confocal images were obtained using an inverted ZeissLSM 510 confocal laser scanning microscope (CLSM) microscope and a100x oil immersion objective. For imaging GFP in combination with mRFP,excitation lines of an argon ion laser of 488 nm and a helium ion laserof 543 nm were used alternately with line switching, using the multitrackfacility of the CLSM. Fluorescence was detected using a 488/543 dichroicbeam splitter, a 505–530 band pass filter for GFP and a 560–615 bandpass filter for mRFP.

FRET-FLIM data acquisitionFRET-FLIM analysis was carried out using a two-photon microscopesetup constructed in the Central Laser Facility of the Rutherford Appletonlaboratory (66). Custom built XY galvanometers (GSI Lumonics) were usedfor the scanning system. Laser light at a wavelength of 920 ± 5 nmwas obtained from titanium sapphire laser (Mira, Coherent) pumped bya frequency doubled vanadate laser (Coherent Lasers) producing 180 fspulses at 75 MHz. The laser beam was focussed to a diffraction limitedspot through a water immersion ultraviolet corrected objective [NikonVC × 60, numerical aperture (NA) 1.2, water immersion]. Specimenswere illuminated at the microscope stage of a modified Nikon TE2000-U.Fluorescence emission was collected without descanning, by-passing thescanning system, and passed through a bandpass filter (BG39, Comar).The scan was operated in the normal mode and line, frame and pixelclock signals were generated and synchronized with an external fastmicrochannel plate photomultiplier tube (Hamamatsu R3809U) used as thedetector. These were linked via a time-correlated single photon counting(TCSPC) PC module SPC830 (Becker and Hickl).

Expression of the fluorescent markers was checked using a two-channelNikon eC1 confocal scanning system coupled to an argon ion 488 nm laserfor GFP excitation and a green HeNe at 543 nm for the mRFP excitation.For FLIM measurements of control constructs, cells with high expressionlevels were preferred. When analysing GFP and mRFP combinations,cells with similar expression levels of both fluorophores were preferredto obtain approximately equal levels of interaction partners within thecell. Fluorescence lifetime imaging micrographs were analysed usingthe SPCImage analysis software (Becker and Hickl, http://www.becker-hickl.com/). Different parameters were varied to obtain the optimal curvefitting for the decay graph for the majority of Golgi bodies, and χ2 valuesover 1.5 indicating poor fitting of the decay data were rejected for furtherdata collection. (An exponential decay fit was considered good when theχ2 value was 1.) As the current software did not permit automatic analysisof whole Golgi bodies, lifetime values were collected on a single pixel basisfrom the centre of individual Golgi bodies and lifetimes were recorded on

a Microsoft Excel worksheet. The collected data values were used togenerate histograms depicting the lifetime distribution of Golgi bodieswithin the samples. Lifetime values of controls and protein combinationswere statistically analysed by performing a two-tailed Student’s t-test inMicrosoft Excel.

Acknowledgments

We would like to thank Mark Curtis for the pMDC vector constructs andRoger Tsien for the mRFP construct. The Biotechnology and BiologicalSciences Research Council are acknowledged for grant funding someof this work. Access to the Central Laser Facility and the multiphotonlaboratory was funded by the Science and Technology Facilities Council.

References

1. Hawes C, Satiat-Jeunemaitre B. The plant Golgi apparatus-going withthe flow. Biochim Biophys Acta 2005;1744:466–480.

2. Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C.Stacks on tracks: the plant Golgi apparatus traffics on an actin/ERnetwork. Plant J 1998;15:441–447.

3. Nebenfuhr A, Gallagher LA, Dunahay TG, Frohlick JA, MazurkiewiczAM, Meehl JB, Staehelin LA. Stop-and-go movements of plant Golgistacks are mediated by the acto-myosin system. Plant Physiol1999;121:1127–1142.

4. Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C.Membrane protein transport between the endoplasmic reticulum andthe Golgi in tobacco leaves is energy dependent but cytoskeletonindependent: evidence from selective photobleaching. Plant Cell2002;14:1293–1309.

5. Runions J, Brach T, Kuhner S, Hawes C. Photoactivation of GFPreveals protein dynamics within the endoplasmic reticulum mem-brane. J Exp Bot 2006;57:43–50.

6. Kristen U. Ultrastructure and a possible function of the intercisternalelements in dictyosomes. Planta 1978;138:29–33.

7. Staehelin LA, Moore I. The plant Golgi apparatus: structure, functionalorganization and trafficking mechanisms. Annu Rev Plant PhysiolPlant Mol Biol 1995;46:261–288.

8. Hawes C, Osterrieder A, Hummel E, Sparkes I. The plant ER-Golgiinterface. Traffic 2008;9:1571–1580.

9. Barr FA, Short B. Golgins in the structure and dynamics of the Golgiapparatus. Curr Opin Cell Biol 2003;15:405–413.

10. Short B, Haas A, Barr FA. Golgins and GTPases, giving identityand structure to the Golgi apparatus. Biochim Biophys Acta2005;1744:383–395.

11. Sztul E, Lupashin V. Role of tethering factors in secretory membranetraffic. Am J Physiol Cell Physiol 2006;290:C11–C26.

12. Barr FA, Puype M, Vandekerckhove J, Warren G. GRASP65, a proteininvolved in the stacking of Golgi cisternae. Cell 1997;91:253–262.

13. Shorter J, Watson R, Giannakou ME, Clarke M, Warren G, Barr FA.GRASP55, a second mammalian GRASP protein involved inthe stacking of Golgi cisternae in a cell-free system. EMBO1999;18:4949–4960.

14. Lupashin V, Sztul E. Golgi tethering factors. Biochim Biophys Acta2005;1744:325–339.

15. Malsam J, Satoh A, Pelletier L, Warren G. Golgin tethers definesubpopulations of COPI vesicles. Science 2005;307:1095–1098.

16. Gilson PR, Vergara CE, Kjer-Nielsen L, Teasdale RD, Bacic A, Glee-son PA. Identification of a Golgi-localised GRIP domain protein fromArabidopsis thaliana. Planta 2004;219:1050–1056.

17. Latijnhouwers M, Hawes C, Carvalho C. Holding it all together?Candidate proteins for the plant Golgi matrix. Curr Opin Plant Biol2005;8:632–639.

1044 Traffic 2009; 10: 1034–1046

Golgi FLIM in Plants

18. Renna L, Hanton SL, Stefano G, Bortolotti L, Misra V, Brandizzi F.Identification and characterization of AtCASP, a plant transmembraneGolgi matrix protein. Plant Mol Biol 2005;58:109–122.

19. Latijnhouwers M, Hawes C, Carvalho C, Oparka K, Gillingham AK,Boevink P. An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1. Plant J 2005;44:459–470.

20. Matheson LA, Hanton SL, Rossi M, Latijnhouwers M, Stefano G,Renna L, Brandizzi F. Multiple roles of ADP-ribosylation factor 1 inplant cells include spatially regulated recruitment of coatomer andelements of the Golgi matrix. Plant Physiol 2007;143:1615–1627.

21. Stefano G, Renna L, Hanton SL, Chatre L, Haas TA, Brandizzi F. ARL1plays a role in the binding of the GRIP domain of a peripheralmatrix protein to the Golgi apparatus in plant cells. Plant Mol Biol2006;61:431–449.

22. Latijnhouwers M, Gillespie T, Boevink P, Kriechbaumer V, Hawes C,Carvalho CM. Localization and domain characterization of Arabidopsisgolgin candidates. J Exp Bot 2007;58:4373–4386.

23. Kang BH, Staehelin LA. ER-to-Golgi transport by COPII vesicles inArabidopsis involves a ribosome-excluding scaffold that is transferredwith the vesicles to the Golgi matrix. Protoplasma 2008;234:51–64.

24. Fridmann-Sirkis Y, Siniossoglou S, Pelham HR. TMF is a golginthat binds Rab6 and influences Golgi morphology. BMC Cell Biol2004;5:18.

25. Yamane J, Kubo A, Nakayama K, Yuba-Kubo A, Katsuno T, Tsukita S,Tsukita S. Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic. Exp Cell Res2007;313:3472–3485.

26. Munro S, Nichols BJ. The GRIP domain – a novel Golgi-targeting domain found in several coiled-coil proteins. Curr Biol1999;9:377–380.

27. Kjer-Nielsen L, Teasdale RD, van Vliet C, Gleeson PA. A novel Golgi-localisation domain shared by a class of coiled-coil peripheralmembrane proteins. Curr Biol 1999;9:385–388.

28. McConville MJ, Ilgoutz SC, Teasdale RD, Foth BJ, Matthews A,Mullin KA, Gleeson PA. Targeting of the GRIP domain to the trans-Golgi network is conserved from protists to animals. Eur J Cell Biol2002;81:485–495.

29. Setty SR, Shin ME, Yoshino A, Marks MS, Burd CG. Golgi recruit-ment of GRIP domain proteins by Arf-like GTPase 1 is regulated byArf-like GTPase 3. Curr Biol 2003;13:401–404.

30. Hawes C, Osterrieder A, Sparkes I. Features of the plant Golgiapparatus. In: Mironov A, Pavelka M, editors. The Golgi ApparatusState of the Art 110 Years after Camillo Golgi’s Discovery. Wien,New York: Springer; 2008;p. 611–623.

31. Siniossoglou S, Pelham HR. An effector of Ypt6p binds the SNARETlg1p and mediates selective fusion of vesicles with late Golgimembranes. EMBO 2001;20:5991–5998.

32. Johansen JN, Chow CM, Moore I, Hawes C. AtRAB-H1b and AtRAB-H1c GTPases, homologues of the yeast Ypt6, target reporter proteinsto the Golgi when expressed in Nicotiana tabacum and Arabidopsisthaliana. J Exp Biol 2009;(In press).

33. Panic B, Whyte JR, Munro S. The ARF-like GTPases Arl1p and Arl3pact in a pathway that interacts with vesicle-tethering factors at theGolgi apparatus. Curr Biol 2003;13:405–410.

34. Lu L, Hong W. Interaction of Arl1-GTP with GRIP domains recruitsautoantigens Golgin-97 and Golgin-245/p230 onto the Golgi. Mol BiolCell 2003;14:3767–3781.

35. Barr FA. A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Curr Biol 1999;9:381–384.

36. Conibear E, Stevens TH. Vps52p, Vps53p, and Vps54p form a novelmultisubunit complex required for protein sorting at the yeast lateGolgi. Mol Biol Cell 2000;11:305–323.

37. Conibear E, Cleck JN, Stevens TH. Vps51p mediates the associationof the GARP (Vps52/53/54) complex with the late Golgi t-SNARETlg1p. Mol Biol Cell 2003;14:1610–1623.

38. Siniossoglou S, Pelham HR. Vps51p links the VFT complex to theSNARE Tlg1p. J Biol Chem 2002;277:48318–48324.

39. Liewen H, Meinhold-Heerlein I, Oliveira V, Schwarzenbacher R,Luo G, Wadle A, Jung M, Pfreundschuh M, Stenner-Liewen F. Char-acterization of the human GARP (Golgi associated retrograde protein)complex. Exp Cell Res 2005;306:24–34.

40. Lobstein E, Guyon A, Ferault M, Twell D, Pelletier G, Bonhomme S.The putative Arabidopsis homolog of yeast vps52p is required forpollen tube elongation, localizes to Golgi, and might be involved invesicle trafficking. Plant Physiol 2004;135:1480–1490.

41. Guermonprez H, Smertenko A, Crosnier MT, Durandet M,Vrielynck N, Guerche P, Hussey PJ, Satiat-Jeunemaitre B, Bon-homme S. The POK/AtVPS52 protein localizes to several distinctpost-Golgi compartments in sporophytic and gametophytic cells. JExp Bot 2008;59:3087–3098.

42. Guan H, Kiss-Toth E. Advanced technologies for studies on proteininteractomes. Adv Biochem Eng Biotechnol 2008;110:1–24.

43. Suter B, Kittanakom S, Stagljar I. Two-hybrid technologies in pro-teomics research. Curr Opin Biotechnol 2008;19:316–323.

44. Lleres D, Swift S, Lamond AI. Detecting protein-protein interactionsin vivo with FRET using multiphoton fluorescence lifetime imagingmicroscopy (FLIM). Curr Protoc Cytom 2007;Chapter 12: Unit 12 10.

45. Bhat RA, Lahaye T, Panstruga R. The visible touch:in planta visual-ization of protein-protein interactions by fluorophore-based methods.Plant Methods 2006;2:12.

46. Shyu YJ, Hu CD. Fluorescence complementation: an emerging toolfor biological research. Trends Biotechnol 2008;26:622–630.

47. Wallrabe H, Periasamy A. Imaging protein molecules using FRET andFLIM microscopy. Curr Opin Biotechnol 2005;16:19–27.

48. Dixit R, Cyr R, Gilroy S. Using intrinsically fluorescent proteins forplant cell imaging. Plant J 2006;45:599–615.

49. Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML. Fluorescencelifetime imaging of free and protein-bound NADH. Proc Natl Acad SciU S A 1992;89:1271–1275.

50. Gadella TWJ, Jovin TM, Clegg RM. Fluorescence lifetime imagingmicroscopy (FLIM) - spatial resolution of microstructures on thenanosecond time-scale. Biophys Chem 1993;48:221–239.

51. Chen Y, Mills JD, Periasamy A. Protein localization in living cells andtissues using FRET and FLIM. Differentiation 2003;71:528–541.

52. Majoul I, Jia Y, Duden R. Practical fluorescence resonance energytransfer of molecular nanobioscopy of living cells., 3rd edn. In:Pawley JB, editor. Handbook of Biological Confocal Microscopy. NewYork: Springer; 2006;p. 788–808.

53. Vermeer JE, Van Munster EB, Vischer NO, Gadella TW Jr. Probingplasma membrane microdomains in cowpea protoplasts usinglipidated GFP-fusion proteins and multimode FRET microscopy. JMicrosc 2004;214:190–200.

54. Tonaco IA, Borst JW, de Vries SC, Angenent GC, Immink RG. In vivoimaging of MADS-box transcription factor interactions. J Exp Bot2006;57:33–42.

55. Adjobo-Hermans MJ, Goedhart J, Gadella TW, Jr. Plant G proteinheterotrimers require dual lipidation motifs of Galpha and Ggammaand do not dissociate upon activation. J Cell Sci 2006;119:5087–5097.

56. Immink RG, Gadella TW Jr., Ferrario S, Busscher M, Angenent GC.Analysis of MADS box protein-protein interactions in living plant cells.Proc Natl Acad Sci USA 2002;99:2416–2421.

57. Ribeiro D, Borst JW, Goldbach R, Kormelink R. Tomato spotted wiltvirus nucleocapsid protein interacts with both viral glycoproteins Gnand Gc in planta. Virology 2009;383:121–130.

58. Zelazny E, Borst JW, Muylaert M, Batoko H, Hemminga MA, Chau-mont F. FRET imaging in living maize cells reveals that plasmamembrane aquaporins interact to regulate their subcellular local-ization. Proc Natl Acad Sci U S A 2007;104:12359–12364.

59. Aker J, Borst JW, Karlova R, de Vries S. The Arabidopsis thaliana AAAProtein CDC48A interacts in vivo with the somatic embryogenesis

Traffic 2009; 10: 1034–1046 1045

Osterrieder et al.

receptor-like kinase 1 receptor at the plasma membrane. J Struct Biol2006;156:62–71.

60. Aker J, Hesselink R, Engel R, Karlova R, Borst JW, Visser AJ, deVries SC. In vivo hexamerization and characterization of the Arabidop-sis AAA ATPase CDC48A complex using Forster resonance energytransfer-fluorescence lifetime imaging microscopy and fluorescencecorrelation spectroscopy. Plant Physiol 2007;145:339–350.

61. Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R.Recruitment and interaction dynamics of plant penetration resistancecomponents in a plasma membrane microdomain. Proc Natl Acad SciU S A 2005;102:3135–3140.

62. Brandner K, Sambade A, Boutant E, Didier P, Mely Y, Ritzenthaler C,Heinlein M. Tobacco mosaic virus movement protein interacts withgreen fluorescent protein-tagged microtubule end-binding protein 1.Plant Physiol 2008;147:611–623.

63. Bayle V, Nussaume L, Bhat RA. Combination of novel greenfluorescent protein mutant TSapphire and DsRed variant mOrangeto set up a versatile in planta FRET-FLIM assay. Plant Physiol2008;148:51–60.

64. Larijani B, Hume AN, Tarafder AK, Seabra MC. Multiple factorscontribute to inefficient prenylation of Rab27a in Rab prenylationdiseases. J Biol Chem 2003;278:46798–46804.

65. Miserey-Lenkei S, Waharte F, Boulet A, Cuif MH, Tenza D, ElMarjou A, Raposo G, Salamero J, Heliot L, Goud B, Monier S. Rab6-interacting protein 1 links Rab6 and Rab11 function. Traffic2007;8:1385–1403.

66. Botchway SW, Parker AW, Bisby RH, Crisostomo AG. Real-timecellular uptake of serotonin using fluorescence lifetime imaging withtwo-photon excitation. Microsc Res Tech 2008;71:267–273.

67. Helmchen F, Denk W. Deep tissue two-photon microscopy. NatMethods 2005;2:932–940.

68. Chien CT, Bartel PL, Sternglanz R, Fields S. The two-hybrid system:a method to identify and clone genes for proteins that interact with aprotein of interest. Proc Natl Acad Sci U S A 1991;88:9578–9582.

69. Sinka R, Gillingham AK, Kondylis V, Munro S. Golgi coiled-coilproteins contain multiple binding sites for Rab family G proteins.J Cell Biol 2008;183:607–615.

70. Haseloff J. GFP variants for multispectral imaging of living cells.Methods Cell Biol 1999;58:139–151.

71. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS,Zacharias DA, Tsien RY. A monomeric red fluorescent protein. ProcNatl Acad Sci U S A 2002;99:7877–7882.

72. Held MA, Boulaflous A, Brandizzi F. Advances in fluorescent protein-based imaging for the analysis of plant endomembranes. Plant Physiol2008;147:1469–1481.

73. Peter M, Ameer-Beg SM, Hughes MK, Keppler MD, Prag S,Marsh M, Vojnovic B, Ng T. Multiphoton-FLIM quantification of theEGFP-mRFP1 FRET pair for localization of membrane receptor-kinaseinteractions. Biophys J 2005;88:1224–1237.

74. Tramier M, Gautier I, Piolot T, Ravalet S, Kemnitz K, Coppey J,Durieux C, Mignotte V, Coppey-Moisan M. Picosecond-hetero-FRETmicroscopy to probe protein-protein interactions in live cells. BiophysJ 2002;83:3570–3577.

75. Jakobs S, Subramaniam V, Schonle A, Jovin TM, Hell SW. EFGPand DsRed expressing cultures of Escherichia coli imaged byconfocal, two-photon and fluorescence lifetime microscopy. FEBSLett 2000;479:131–135.

76. Curtis MD, Grossniklaus U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol2003;133:462–469.

77. Suhling K, Siegel J, Phillips D, French PM, Leveque-Fort S, Webb SE,Davis DM. Imaging the environment of green fluorescent protein.Biophys J 2002;83:3589–3595.

78. Burguete AS, Fenn TD, Brunger AT, Pfeffer SR. Rab and Arl GTPasefamily members cooperate in the localization of the golgin GCC185.Cell 2008;132:286–298.

79. Sambrook J, Russell DW. Molecular Cloning: A laboratory Manual.3rd edn. Cold Spring Harbor, New York: Cold Spring Harbor LaboratoryPress; 2001.

80. Haseloff J, Siemering KR, Prasher DC, Hodge S. Removal of a crypticintron and subcellular localization of green fluorescent protein arerequired to mark transgenic Arabidopsis plants brightly. Proc NatlAcad Sci U S A 1997;94:2122–2127.

81. Gietz RD, Schiestl RH, Willems AR, Woods RA. Studies on thetransformation of intact yeast cells by the LiAc/SS-DNA/PEGprocedure. Yeast 1995;11:355–360.

82. Sparkes IA, Runions J, Kearns A, Hawes C. Rapid, transient expres-sion of fluorescent fusion proteins in tobacco plants and generationof stably transformed plants. Nat Protoc 2006;1:2019–2025.

1046 Traffic 2009; 10: 1034–1046