Probing the mobility of membrane proteins inside the cell

MISCELLANEA

In comparison with the case for cellsurface proteins embedded in the plasma membrane, little is known about the in situ environment of intracellular membrane proteins. Experimental access to intracellular membrane proteins is blocked by the plasma membrane and often also by organellar membrane(s). Hence the mobility and, by inference, the environment of intracellular membrane proteins remain little studied. However, new techniques offer the ability to penetrate the cell and the opportunity to apply to intracellular membrane proteins approaches that have already been successful in characterizing the environment of cell-surface membrane proteins. The mobility of cell-surface membrane proteins has been determined by three major techniques: fluorescence recovery after photobleaching (FRAP)‘, single particle tracking (SPT)2,3 and optical tweezers3. The molecular specificity of antibodies and their monovalent Fab fragments is a key element in all these approaches. With FRAP, the oldest technique, a diffusion constant is derived from the average behaviour of many molecules. Cell-surface proteins are specifically photolabelled by binding of fluorescently conjugated monovalent antibody fragments (fl-Fabs). Fluorescence can be irreversibly bleached, usually over a circular area of -1 pm by an intense laser flash, and recovery subsequently determined with an attenuated ‘query’ laser beam. Mobility parameters are then derived from the kinetics of fluorescence recovery. Typically, recovery is incomplete in such experiments, and incomplete recovery is equated with an immobile fraction. The occurrence of an immobile fraction has led to the conclusion that the mobility of many membrane proteins is restricted. SPT and optical tweezers are recent techniques and are also dependent on antibody binding for specificity. In these approaches, the antibody is coated onto small (20-40 nm) gold particles or similarly sized fluorescent latex beads. With video-enhanced microscopy (nanovid microscopy), the movement of these particles can be tracked with a resolution of a few nanometres4. In SPT, the diffusion properties of individual membrane proteins are inferred from the movements of individual particles tracked over periods of seconds to a few minuteG. On a spatial scale of tenths of microns, SPT particleprotein complexes exhibit random,

Probing membrane

the mobility of proteins inside the cell

Brian Storrie and Thomas E. Kreis Studies using a variety of microscopy-basedapproacheshave led to a consensusthat most cell-surface proteinsare highly mobile and difFnserapidly within fenced microdomains. Little attention, however, has so far beengiven to the analysis of the mobility of intracellular membraneproteins becauseof their comparative inaccessibility. Recent advances in microinjection, confocal microscopy and the construction of epitope-taggedproteins or of hybrids with an intrinsically fluorescent protein have allowed intracellular membraneproteins to be studied using approaches previously applied to characterize the mobility of cell-surfaceproteins. Confocal fluorescencerecoveryafter photobleaching (c-FRAP) experiments show that intracellular membraneproteins may also be highly mobile.

Brownian movement. On a scale approaching a micron or more, free, Brownian diffusion appears to be confined to microdomains of a few tenths of a square micron in area6. With optical tweezers, the ability of a givenstrength laser field to translocate a trapped particle about the cell surface is scored. At low laser strength, barriers to diffusion are encountered every few to several tenths of a micron7. This again indicates that free diffusion of cell-surface transmembrane proteins is restricted to microdomains similar in size to those seen by SPT. For the typical transmembrane cellsurface protein, the consensus interpretation emerging from FRAP, SPT and optical tweezer experiments is the cytoskeletal fence model. Several investigators have proposed that the plasma membrane is underlain by a meshwork of elements creating fences that block or hinder the movement of transmembrane proteins across microdomain boundaries2j8sg. Within a given fenced microdomain, proteins are free to diffuse randomly. Movement between microdomains is rare and restricted to dynamic breaks in the fence. Treatment of cells with cytochalasins or vinblastine indicates that cytoskeletal proteins such as actin and tubulin are important elements of the fences’. In conclusion, these

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cell-surface studies clearly demonstrate the usefulness of very small scale techniques such as SPT in probing the environment of membrane proteins.

Characterizing of intracellular proteins

the environment membrane

Unlike the situation for cell-surface membrane proteins, access to intracellular membrane proteins is limited by the plasma membrane, and, for organellar proteins, may also be limited by the organellar membrane(s). Fluorescent reporter groups [e.g. rhodamine or caged rhodamine Q (Ref. lo)] conjugated to di- or monovalent antibodies, or antibodycoated 40-nm gold or fluorescent latex particles for intracellular SPT, are large and membrane impermeant. However, ‘battering ram’ or ‘Trojan horse’ approaches have helped to explore the intracellular space. In the ‘battering ram’ approach, scrape loading or microinjection, electroporation is used to introduce specificfl-Fabs or particles coated with antibodies directed against exposed epitopes into the cytosol. Currently, only a handful of antibodies directed against the cytoplasmic domains of intracellular transmembrane proteins exist. Many organellar transmembrane proteins have only a small cytoplasmic

0 1996 Elsevier Science Ltd PII: SOSU-8924(96)40004-6

Brian Storrie is at the Dept of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308, USA; and Thomas Kreis is at the Dept of Cell Biology, SciencesIII, University of Geneva, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.

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domain and possess a much larger lumenal domain. Thus, monoclonal antibodies made against the whole protein are rarely directed against a cytoplasmic epitope. However, several anti-peptide antibodies to the cytoplasmic tails of transmembrane proteins have been generated, and some of these have no apparent effect on the processing and localization of the target protein in viva”. Epitopetagged proteins carrying a sequence recognized by a well-characterized monoclonal antibody and stably expressed in cells provide an alternative approach. For visualization of intracellular transmembrane proteins in vivo, such tags must be attached to the cytoplasmic domain’*. Microinjection with a microprocessor-controlled manipulator is quick, and hundreds of cells may be injected in a reasonable period of time (minutes) without cell damage13. This approach delivers picolitre quantities of solution into the cytoplasm - typically 510% of the cell volume. Scrape loading14 and electroporation15 are technically and instrumentally less demanding mass procedures in which millions of cells can be processed as a batch. However, larger quantities of antibody are required and cell morphology is typically transiently distorted. In the ‘Trojan horse’ approach, the cell itself is ‘tricked’ into expressing the protein of interest as a chimera with an inherently fluorescent polypeptide. This recent development was made possible by the key finding that cloned green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea Victoria fluoresces when expressed in prokaryotic (Eschericbia co/i) or eukaryotic (Caenorhabditis elegans) cells’6. GFP is a small protein of 238 amino acids and has a molecular mass of 27 kDa (Ref. 17). It absorbs blue light (maximally at 395 nm, with a minor peak at 470 nm) and emits green light. The CFP chromophore is derived from the primary amino acid sequence through the cyclization of adjacent serinedehydrotyrosine-glycine residues18. Cyclization of wild-type CFP is a relatively slow process with a first-order rate constant of 4 h (Ref. 19). It is a temperature-sensitive process that occurs at 30°C or lower but not at 37°C (Ref. 20). Consequently, achieving efficient maturation of GFP expressed in mammalian cells requires a down shift in culture temperature. When the cells are returned to 37”C, fluorescence is stable. GFP fluorescence may also be photoactivated2’. Tsien1v,22 and others23 have recently mutated GFP to

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produce a protein that cyclizes more rapidly, fluoresces brighter and has better spectral characteristics. GFP fusions with secreted proteins, after appropriate temperature shift, fluoresce brightly and are transported normally through the secretory pathway’“. GFP fusion proteins are an attractive and promising ‘Trojan horse’ for in situ intracellular protein mobility experiments using either a FRAP or photoactivation approach. Isolation of GFP mutants that fluoresce blue raises the intriguing possibility of co-transfection with blue and green GFP fusion proteins as a means to test for close protein interactions through resonance energy transfer2*. Crossexcitation would be possible only if the two proteins are part of a molecular complex. Undoubtedly the potential of GFPs will be tested fully and exploited in the years to come. Finally, problems may arise from the geometry of intracellular organelles. The plasma membrane of well-spread, cultured mammalian cells is, to a reasonable approximation, planar. In striking contrast, most intracellular organelles must be considered to be a veritable can of worms. To give just two examples, the endoplasmic reticulum (ER) is a complicated, interlocking meshwork of dynamic tubules that extends throughout the entire cytoplasm, and the Golgi complex is a compact, juxtanuclear complex of cisternae and tubules. Such complicated objects are ideally observed with confocal optics; single optical planes may be better viewed in focus with confocal optics than with conventional optics, where significant blurring of the image can occur25. Alternatively, experimental conditions may be created in which out-of-focal-plane contributions may be treated as a constant. This is a reasonable approximation under low-magnitude fluor bleaching conditions such as those of fringe-pattern photobleaching26. Here, photobleach levels as low as -10% can be detected owing to the increase in sensitivity associated with the spatial averaging of the contrast of the fringe pattern. Because of the low photobleach level, the intensity of fluorescence over the course of the experiment is approximately constant and hence the intensity of blur contribution is held constant. One application of fringe-pattern photobleaching to the characterization of a complex intracellular structure has been the study of microtubule dynamics27. The technique could also be applied to intracellular membranes. Particularly interestingly,

it should in principle allow the determination of flux of molecules within or through an organelle. Target intracellular organelles As two key components of the secretory pathway, the ER and Golgi complex are interesting targets for the characterization of in situ membrane protein mobility. The ER is the site of membrane protein and lipid biosynthesis, whereas the Golgi complex is a protein- and glycolipid-processing factory in which proteins are also concentrated and sorted. Newly synthesized proteins enter the Golgi at the cis Golgi network (CGN) and exit it at the trans Golgi network (TGN). One of the major and persistent mechanistic questions regarding these organelles is how resident proteins are discriminated from transient proteins en route to other locations. In the case of ER proteins, retrieval signals have been clearly identified28. Yet, in the absence of these signals, secretion is much slower than might be expected. Possible additional mechanisms include selection of transported proteins by direct or indirect interactions with coat proteins, and segregation of resident from transported proteins in the ER on the basis of physical properties related to general mechanisms of quality control within the ER2v,30. Several different mechanisms have been proposed to explain the retention of Golgi proteins such as glycosyltransferases in the organelle. These include kin-protein aggregation through transmembrane and lumenal domains30,31, localization based on transmembrane domain length32, interaction with intercisternal matrix components33, retrieva13’ and cholesteroldependent sorting interactions34. By domain swapping and site-directed mutagenesis, the Golgi-localization signal of type-11 membrane proteins has been narrowed to positions within and surrounding the transmembrane domain, but no obvious homology exists that might imply a common Golgi-targeting or -retention motif35. The Golgi complex is associated in an unknown manner with microtubules36; in addition, various coat protein complexes (including clathrin with API and COP-I) associate reversibly with the cytoplasmic face of membranes of the Golgi cisternae and intermediate compartment (IC) and regulate traffic through these compartments37. Moreover, recently described putative Golgi matrix proteins may also be important in the maintenance of organelle organization33,38.

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MlSCELlANEA

All these components may interact in some manner, be it direct or indirect, with Colgi transmembrane proteins. In summary, the ER and Golgi complex pose formidable challenges in understanding protein residency versus export and, therefore, are an interesting starting point for probing the intracellular membrane protein environment. Measuring the mobility of intracellular transmembrane proteins by confocal FRAP The first application of FRAP with a confocal microscope (c-FRAP) was to characterize the mobility of a temperature-sensitive mutant of the glycoprotein of vesicular stomatitis virus (ts-045-G)3g. ts-045-G is a wellcharacterized transmembrane protein and has been a powerful tool in dissecting steps along the secretory pathway. At the nonpermissive temperature (39.5”(I), it is blocked in the ER, and, at 15°C and 2O”C, it accumulates in the IC and TCN, respectively. Shifting infected cells to the permissive temperature (31°C) releases these blocks and induces transport of ts-045-C to the cell surface. Transport of ts-045-C from the ER to the Golgi complex depends on coat proteins (COPI and COPII) associated with transport vesicles37, Microinjected antibodies against B-COP block transport of the viral glycoprotein at the interface between the IC and the Golgi complex13. Most importantly, Fab fragments of the monoclonal antibody P5D4 directed against the C-terminal epitope of the cytoplasmic domain of ts-045-G bind with high affinity to the viral glycoprotein in vivo and have no effect on the transport of the glycoprotein through the secretory pathway”. Thus, temperature blocks and microinjected rhodaminelabelled P5D4-Fabs (rh-l?SD4-Fabs) allow visualization of ts-045-G moving through the compartments of the secretory pathway. Combining visualization of movement of ts-045-G with microinjected rh-PSDCFabs and c-FRAP now allows determination and manipulation of the mobility of this model transmembrane protein 39. A typical c-FRAP experiment is illustrated in Figure 1. Rh-PSD4-Fabs were injected into the cytosol of Vero cells on glass coverslips (prescored to provide a reference mark for locating the cells). Since confocal microscopy eliminates fluorescence blur, three-dimensional structures like the Golgi complex can be visualized easily, despite its rather extended volume

(on average, -2-31*m in width, 6-7Pm in length and 2-3Pm in depth). For these studies, a pointscanning confocal microscope was used with an oscillating mirror to move a laser beam across the sample in a linear raster scan25. With appropriate software, this system easily allows line bleaching rather than a point bleach common to conventional FRAP. Monitoring a 30 x 30-Pm field with a 100x, 1.3 numerical aperture objective, bleaching was achieved by increasing the laser dwell time at each of 10 evenly spaced spots along FIGURE 1 a line of I-3Pm length from 720 ns to 30 ms. Line c-FRAP of ts-045-G associated with the Golgi complex. Following placement was carried injection with rh-PSD4-Fabs, cultured primate cells (Vera) with ts-045-C out with a mouse to mark accumulated in the TCN were visualized with the confocal microscope. the start and finish spots An area [box in (a)] was selected for line photobleaching. on a pre-bleach Golgi Arrowheads in (b) (1 s after bleach), (c) (6 s after bleach) and image. The diffraction(d) (16 s after bleach) indicate fluorescence recovery in the bleached area. limited circular bleach at See also Ref. 39. Bar, 2.0 km. each spot was -0.5 pm, as determined in fixed this viral glycoprotein at the cell surcells labelled with a cytosolic fluorface by conventional FRAP, where the escent probe that reacted with free reported diffusion constant is threetubulin in nocodazole-treated cells. As to fourfold lower and -50% of the depicted in Figure 2, the overlapping protein is immobile41,42. The c-FRAP respots give a continuous line bleach. sults for the intracellular glycoprotein The width of the bleach in the X-Y are in agreement with those for other plane in vivo is also -0.5 Pm and the cell-surface proteins measured by bleach is a few microns deep in the SPT. The movement of transferrin and X-Z plane. This gives a readily recoge.2-macroglobulin receptors when moninizable bleach pattern across a comtored by video-enhanced light microsplex tubular organelle such as the copy and ligand-coated nanometreGolgi. Post-bleach images were colsized colloidal gold particles were lected at the short laser dwell time to avoid significant bleaching of the rhoBrownian within confined diffusion domains of -0.25 Pm2 (0.5-0.7 km in damine reporter group. These basic diagonal length) 7,8. Interestingly, the principles can be readily applied with commercial equipment, and, in fact, a only conditions found to give an appreciable immobile fraction for the variation of this procedure - manyintracellular viral glycoprotein were fold repeat laser scanning of the same X-Y line - has been used by those in which ts-045-G was released Cooper and colleagues to bleach NBD-ceramide metabolites accumu-2 pm lated in the Golgi40.

I*

Intracellular membrane proteins can be highly mobile c-FRAP revealed that ts-045-G protein within the TGN or in transit to the Golgi is highly mobile39. No immobile fraction was observed and the diffusion constant of -1 Ob9cm2s-l was consistent with that expected for free, Brownian diffusion of proteins in the Golgi membrane. These results are in striking contrast to those observed for

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k

FIGURE 2 Line bleach procedure. Bleaching of 10 evenly spaced dots with an argon laser along an -2 km linear path produces a continuous line of overlapping, diffraction-limited, -0.5 pm spots.

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MlSCELlANEA

confocal microscopy approaches to the measurement of endogenous protein mobility within intracellular organellar membranes. A harvest now needs to be reaped - through ingenuity and persistence.

References I

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FIGURE 3 Divalent antibodies that bind to coatomer in vivo partially immobilize ts-045-C. Cells infected with ts-045 vesicular stomatitis were incubated at the nonpermissive temperature, microinjected with rh-PSD4-Fabs and antibodies against P-COP [anti&ICE (v), or anti-Al, which does not bind to coatomer in vivo (A)] and then shifted to the permissive temperature. Photobleaching was at t = 0. The smooth lines are drawn as fitted power functions. See also Refs 13 and 39.

from the ER in cells co-injected with rh-PSD4-Fabs and divalent antibodies that bind to P-COP in V~VO~~ (Fig. 3). Thus, c-FRAP clearly is a valid and fairly easy procedure for measuring the mobility of an intracellular transmembrane protein. The diffusion coefficient observed and the lack of an immobile fraction are entirely consistent with measurements of cellAcknowledgements surface transmembrane protein moWe thank Rainer bility by the now accepted procedure Pepperkok of SPT. (University of Geneva) for Future prospects introducing B. S. to A prime goal now will be the microinjection and characterization of the mobilities of thank Ernst resident intracellular transmembrane H. K. Stelzer proteins and comparison with the (EMBL-Heidelberg) dynamic properties of transient organfor helpful ellar proteins. The key obstacle to this endiscussionson using fusion- or epitope-tagged confocal dogenous proteins as target molecules microscopy. B. S. is the achievement of normal protein was supported by a localization and function. It may well Fogarty Senior turn out that lumenal fusions with modified CFPs will be the most International appropriate approach. Coexpression Fellowship from the of CFP conjugates with different specUS NIH while at tral properties will allow resonance EMBL-Heidelberg. energy transfer experiments to deterSupport for mine protein proximity. With CFP experimental work conjugates, there is no need for introfrom the US NSF ducing fluorescently modified anti(DCB-9022817 to body into the cell. Cytoplasmic tagB. 5.) and the Swiss ging with defined short epitopes National Science may, however, be a promising alterFoundation and the native’*. One clear advantage to this Canton de Cenke approach is that alternatives to c-FRAP (T. E. K.) is such as SPT with microinjected partigratefully cles are possible. In conclusion, the has been laid to apply acknowledged. groundwork

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