Cell, Vol. 61,

135-145,

April 6, 1990. Copyright 0 1990 by Cell Press

Tubulovesicular Processes Emerge fmm 7hns=Gu#gi s, and I Cisternae, Extend along Mlc Adjacent Tram-Golgi Elements into a FkHkMum Mark S. Cooper: Ann H. Cornell-Bell, Alex Chernjavsky: John W. Dani: and Stephen J Smith’ Howard Hughes Medical Institute and Section of Molecular Neurobiology Yale University School of Medicine New Haven, Connecticut 06510

Summary Morphological dynamics and membrane transport within the living Golgi apparatus of astrocytes labeled with NBD-ceramlde were Imaged using both electronically enhanced fluorescence video and laser confocal microscopy. In time-lapse recordings, continuous tubuloveskular processes are observed to emerge from trans-Golgi elements and extend along microtubules at average rates of 0.4 km/s. In addition, discrete fluorescent particles are observed to emerge from the trans&olgl and subsequently migrate along microtubules at comparable velocities. Frequently, tubuloveslcular processes form stable connections that interlink adjacent trans-Golgl elements into an extensive reticulum. Laser photobleaching-recove ry experiments reveal that tubuloveakular processes can provide direct pathways for the diffusion of membrane lipids between joined frans-Golgi elements. These results suggest that mkrotubule-baeed transport and membrane fusion can operate to Interconnect certain clsternal membranes of adjacent Golgi elements within the cell. Introduction The membranous compartments of the Golgi apparatus are involved in biosynthesis of lipids, glycosylation of proteins, and the sorting of these products into transport and secretory vesicles (Palade, 1975; Goldfisher, 1962; Farquhar, 1965; Dawidowicz, 1987). Often seen as a network of stacked flattened cisternae in the pericentrosomal area, the Golgi apparatus is centrally located to receive and direct a variety of exocytic and endocytic materials that pass en route through the organelle (Farquhar, 1985; Pastan and Willingham, 1985). It is of current interest to determine how the three-dimensional morphology of this organelle is formed within the cell and how this morphology subserves its biosynthetic and transport functions. It is well known that the pericentrosomal organization of the Golgi complex is dependent on intact microtubules. Disruption of microtubule networks with colcemid, nocodazole, or anti-tubulin antibodies results in a fragmentation of the Golgi complex and, frequently, its dispersal throughout the cytoplasm (Hiller and Weber, 1982; Pavelka and Ellinger, 1983; Wehland and Willingham, 1983; l

Present address: Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 943055426.

Rogalski and Singer, 1984). In double-label immunofluorescence micrographs, close lateral associations have been observed between Golgi membranes and microtubules in cells recovering from microtubule disassembly (Rogalski and Singer, 1984). A specific association of the Golgi apparatus with microtubules is also suggested by findings that antibodies to a 110 kd protein, which crossreact with microtubule-associated protein MAP-2, also recognize an antigen associated on the cytoplasmic surface of the Golgi complex in a variety of nonneuronal cells (Allan and Kreis, 1986). In addition, a 58 kd protein has recently been isolated that binds both Golgi membranes and microtubules (Bloom and Brashear, 1989). Recently, it has been reported that Golgi stacks, initially dispersed by a nocodazole-induced microtubule disassembly, will recluster around the centrosome by migrating along microtubules, after the cell’s microtubules are allowed to repolymerize (Ho et al., 1989). These results are compelling evidence that the mechanical forces generated by microtubule-based transport motors are involved in the reassembly of fragmented Golgi elements near the centrosome, a process that occurs naturally in many cells during telophase of mitosis (Robbins and Gonatas, 1964; Moskalewski et al., 1977; Lucocq et al., 1987). In this paper, we report a new role for microtubule-based transport in ttie dynamic organization of the Golgi apparatus, namely in the rapid distension of tubulovesicular processes from existing trans-Golgi elements along microtubules. Using both electronically enhanced Ruorescence video and laser confocal microscopy, we have imaged these dynamic structures in the living Golgi apparatus of rat hippocampal astrocytes labeled with the lipid N-(7-[4-nitrobenzo-2oxa-1,3-diazole])-6-aminocaproyl sphingosine (NED-ceramide), a fluorescent vital stain for the elements of the trans-Golgi (Lipsky and Pagano, 1985b; Pagan0 et al., 1969). After being extended from stationary trans.Golgi elements, tubulovesicular processes are observed to form stable contacts with the membranes of adjacent Golgi elements. In separate experiments, using laser photobleaching-recovery, we have found that stable tubulovesicular processes between trarrs-Gofgi elements can serve as direct pathways for the diffusive exchange of lipids, suggesting that newly formed tubular processes are eventually fused into a continuous network with the cisternal membranes of adjacent trans-Golgi elements. These results suggest that microtubule-based transport can influence membrane transport within the Golgi by pulling tubulovesicular processes into contact with the membranes of adjacent Golgi elements where membrane fusion can take place. In addition to observing the dynamics of tubulovesicular transQolgi processes, we have found that scanning laser microscopy allows the imaging of submicron fluorescent particles emerging from the Golgi apparatus. These particles are often observed to traffic along curvilinear paths between Golgi elements or into the cell periphery, at average rates of 0.3 pm/s. These curvilinear tracks also appear to be microtubules on the basis of the

Cell 136

velocities ticles.

and linear trajectories

of the fluorescent

par-

Results Morphologies of the Golgl Apparatus The fluorescent lipid NBD-ceramide is known to stain elements of the Golgi apparatus in living cells, revealing the macroscopic structure of the organelle. At the level of the light microscope, the broad outlines of Golgi elements stained with NBD-ceramide are identical in appearance to Golgi elements stained with conventional Golgi markers (Lipsky and Pagano, 1985b). Recently, using correlative light and electron microscopy, Pagan0 et al. (1989) have found that NBD-ceramide preferentially localizes within elements of the trans-Golgi. The rrans-Golgi of rat hippocampal astrocytes, stained by NBD-ceramide, displays several morphological characteristics that have been previously described in a variety of cell types using light, immunofluorescence, and electron microscopy (Beams and Kessel, 1968; Novikoff et al., 1971; Rambourg et al., 1981; Lin and Queally, 1982; Louvard et al., 1982; Rambourg and Clermont, 1986; de Camilli et al., 1986). In astrocytes, the rrans-Golgi ranges in appearance from small discrete elements to highly elongated elements that frequently form reticular networks (Figure 1). Thin processes are often seen bridging the intervening space between adjacent elements (Figure 1B). The length of these structures ranges from 0.5-14.0 urn (mean = 3.5 f 2.7 urn, N = 62). These structures are probably membranous since they contain NBD-labeled lipids and appear continuous with other rrans-Golgi elements. We will refer to these extensions as tubulovesicular processes, as is consistent with previous terminology used to describe similar membranous extensions in the endoplasmic reticulum (ER) (Dabora and Sheetz, 1988; Lee and Chen, 1988). From light and electron micrographs of various fixed cell types, tubulovesicular processes have

been frequently described as connecting Golgi stacks, or dictyosomes, into a continuous network that courses through the cytoplasm (Beams and Kessel, 1968; Novikoff et al., 1971; Rambourg et al., 1981; Rambourg and Clermont, 1988). In culture, astrocytes extend in diameter up to 200 pm, and are flattened to less than a micron over most of their surface. The perinuclear region is also highly compressed to l-2 brn in thickness, forcing the majority of the Golgi apparatus to lie in a single focal plane of the microscope. The Golgi apparatus frequently extends over distances of 30-60 pm, making it ideal for microscopic observation. The distribution of Golgi elements may be either pericentrosomal or perinuclear, the latter referring to a distribution of stacks more symmetrically disposed around the nucleus (Figure 1C). As cells are kept in culture for 3-4 weeks, Golgi elements often become highly interlinked into an extensive reticulum. The reticulum frequently is composed of interconnected elements of uniform width (Figures 1A and lC), rather than the thin tubulovesicular processes that interconnect adjacent rrans-Golgi elements in cells cultured for shorter periods of time (Figure 1B). In cells cultured for 3-4 weeks, elongated rrans-Golgi elements are occasionally seen suspended both above and below the nucleus (Figure 1C). We have also observed similar Golgi morphologies in astrocytes stained with rhodamine-conjugated wheat germ agglutinin (data not shown), a marker for the rrans-Golgi (Tartakoff and Vassalli, 1983). These morphologies were also observed in astrocytes infected with the O-45 temperature-sensitive mutant of vesicular stomatitis virus and stained with an antibody to its glycoprotein after the cells were held at 20% (unpublished data with H. F?Moore), a condition that causes the virus to accumulate in the rrans-Golgi network (Rogalski et al., 1984). As in many other cells (Thyberg and Moskalewski, 1985) the macroscopic morphology of the Golgi apparatus in astrocytes is highly dependent on intact microtu-

Figure 1. Morphologies of the Golgi Apparatus in Living FlatHippocampalA&ucytes after Staining with the Fluorescent

Lipid NBD-Ceramide

(A) The Golgi apparatus frequently appears as a reticulum of anastomosing branches in the pericentrosomal area. (6) In less reticulated forms, membranous tubules or tubulovesicular processes are often ob served to interconnect adjacent Golgi elements. (C)The elaboration of such membranous interconnections results in a necklace of Golgi elements that may symmetrically surround the nucleus. This perinuclear reticular network of elements is similar to Camillo Golgi’s (1698) original description of the organelle in Purkinje neurons. (D) Disassembfy of microtubules with 10 uM nocodazole results in a fragmentation of the reticulum. The fragmented Golgi elements generally remain immobilized in the psrinuclear area. Scale bar = 10 urn.

F$gi

Dynamics

and Microtubule-Based

Transport

Table 1. Velocities of Tubulovesicular Golgi Processes, Vesicles, and Translocating Organekes in Astrocytes Velocity (wW Extending tubulovesicular processes Retracting tubulovesicular processes Golgiderived particles Mitochondria Lysosomes

Golgi-Derived

Observations

Number Cells

0.49 f

0.41

35

12

0.42

f

0.49

29

13

0.27

f

0.15

62

12

0.26 0.20

f f

0.12 0.10

45 46

5 5

of

In calculating mean organelle velocities from time-lapse recordings, only organelles moving faster than 0.05 pm/s were used in order to prevent large numbers of stationary organelles from contributing to the mean velocities. By employing a low velocity cut-off limit, the means represent the average velocity within a tail distribution of organelle movement.

Brownian movement can be imaged with very little motional blurring. Although the particles correspond to a limited number of pixel elements, this lack of motional blurring allows the particles to be clearly discerned. In our experiments, however, we found it necessary to restrict time-lapse sampling intervals to periods greater than 4 s in order to obtain extended time-lapse recordings with adequate signal-to-noise and minimal photobleaching. After emerging from Golgi stacks, Go@-derived particles are observed to migrate, in a saltatory fashion, along curvilinear paths between Golgi elements or into the cell periphery (Figure 3B). Particles in the cell periphery are also observed to migrate along curvilinear paths toward the nucleus. The average velocity of these Go&Wived particles was measured to be 0.27 urn/s (kO.15 SD, N =

bules. Microtubule disassembly with nocodazole leads to a fragmentation of elongated and reticulated Golgi elements. In astrocytes, the fragmented Golgi elements remain in the perinuclear or pericentrosomal area (Figure 1D). In other cell types, fragmented Golgi elements are observed either to disperse throughout the cytoplasm (Couchman and flees, 1982; Rogalski and Singer, 1984; Lipsky and Pagano, 1985b) or remain near the nucleus (Lin and Queally, 1982) following microtubule disassembly. lbbulowslcular Processes and Vesicles Emerge from 7hns-Qolgi Elements In time-lapse recordings, tubulovesicular processes are observed to emerge rapidly and distend from stationary Vans-Golgi elements, which appear to be immobilized in the perinuclear area. These tubular processes both extend and retract along curvilinear paths at average velocities of about 0.4 urn/s (see Table 1). Often, the motion of processes appears saltatory, with periods of advance and retreat. In some cases, these tubulovesicular projections extend away from existing rrans-Golgi elements, toward the cell periphery (Figure 2A). In other cases, the episodes of extension and retraction bring these processes into transient contact with the membranes of adjacent frans-Golgi elements. It is also observed that tubulovesicular processes can remain extended and contiguous with these trans-Golgi elements after making apparent contact (Figure 28). These newly formed tubular connections appear identical in length and width to the stable membranous connections (Figure 1B) that often interconnect adjacent Pans-Golgi elements. In addition to continuous tubulovesicular processes emerging from &arts-Golgi elements, we have observed discrete submicron fluorescent particles emerging from the Golgi using the scanning laser confocal microscope (Figure 3). Because the scanning laser beam of the confocal microscope passes rapidly over the sample (8 us per pixel), an effective stroboscopic illumination is produced in which small particles undergoing active transport or

Figure ments

2. Extension

of Tubulovesicular

Processes

from

Golgi

Ele-

(A) A SIT camera recording sequence (0,60, and 160 s) shows the extension of a thin membranous process (arrow) from an immobile Golgi element. The constant appearance of a dark spherkzrl lyeoaome adjacent to the Golgi element as well as other landmarks shows that the same plane of focus was preserved during the recording. Scale bar = 5 pm. (B) A confocal fluorescence micrograph showing a tubulovesicular process interconnecting adjacent Golgi elements. The formation of this tub&vesicular process is shown in (C). Scale bar = 10 urn. (C) A thin membranous process (arrow) is extended at 0.20 r&s. ultimately forming a bridge between two Gofgi elements. There are 10 s intervals between frames. After making contact, the tubulovesicular process remained contiguous wkh the second Golgi element until the end of the recording, 4 min later. Same scale as (B).

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Figure 3. Migration of Particles with Scanning Laser Microscopy

from

the Golgi

Apparatus

Observed

(A) Small fluorescent particles are seen in the vicinity of bans-Golgi elements labeled with NED-ceramide. White boxes outline areas in which particles are observed to emerge from trans-Golgi elements. Time-lapse sequences showing the appearance and migration of Golgi particles within the lower and upper boxes are shown in (C) and (D), respectively. (B) A line drawing illustrating the movements of fluorescently labeled Golgi particles in the perinuclear area. Positions of the particles at 20 s intervals are illustrated by dots. Certain particles appear to emerge from rrans-Golgi elements and subsequently migrate along curvilinear paths toward the cell periphery. The trajectory of a particle moving toward the nucleus is also shown. The particles move in a saltatory fashion, with linear excursions and an average velocity of 0.15 urn/s. Scale bar = 25 urn. (C) An example of a small fluorescent particle migrating in the space between two rrans-Golgi elements (area corresponds to lower box in [A]). In the first frame, the open arrow points to a bulge at the side of a Golgi element. As the bulge disappears in the subsequent frame, a fluorescent particle is observed to appear in close proximity to the Golgi element. The particle subsequently migrates along a linear trajectory and disappears from view as it approaches another Golgi element. (D) A second example of a particle that appears to emerge from a rransGolgi element. An open arrow points to a bulge in a Golgi element not present in previous frames. The subsequent frame shows a new fluorescent particle (solid arrow) not present in the field of view before. The particle begins to move away from the Golgi element in subsequent frames. The extended trajectories of this particle and the particle in (C) are illustrated by the line drawing in (8).

82) a mean similar to the average velocity of extending and retracting tubulovesicular Golgi processes (Table 1). As cells labeled with NBD-ceramide are incubated in the dark at 87% for 24 hr, the number of such fluorescent particles appearing in the cell periphery increases substantially, from 0.6 f 1.0 per cell at 2 hr after labeling, to 66.9 f 60.7 per cell at 24 hr postlabeling (N = 75, both time points). This result suggests that the particles emerging from trans-Golgi elements in time-lapse recordings may be secretory or storage vesicles, rather than fragments of the trans-Golgi induced to form by laser illumination.

Tubulovesicular Golgi Processes Coalign with Microtubules lb determine whether tubulovesicular rrans-Golgi processes coaligned with microtubules, images of the rrans-Golgi in the living cell were first acquired using either a SIT camera or a confocal microscope. Cells were then fixed and stained for cytoskeletal elements. In a flow-through perfusion chamber, successive solutions for cytoskeletal staining of both microtubules and actin were applied to the specimen without moving the field of view. The distribution of microtubules in the perinuclear area of astrocytes is so dense that the trajectory of tubulovesicular Golgi processes or Golgi-derived particles cannot be easily followed along single microtubules. However, above the nucleus, the sparser distribution of microtubules sometimes allows such correlations to be made. In a given culture, about 1% of the cells possess Golgi elements above or below the nucleus. For microscopy, the contrast of rransQolgi elements in these areas is enhanced owing to the reduced density of other membranous organelles, such as mitochondria and the ER, which are lightly stained by NBD-ceramide (Lipsky and Pagano, 1985b) and contribute to background fluorescence. In Figure 4, two elongated Golgi elements above a nucleus are seen to coalign with bundles of microtubules. Frequently, these microtubule bundles also coalign with actin stress fibers. Whereas actin stress fibers may be responsible for orienting microtubule bundles above and below the nucleus, they do not provide a sufficient binding substrate to maintain elongated Golgi elements, as evidenced by the fragmentation of elongated elements in cells after microtubule disassembly (Figure 1D). On occasion, it is possible to locate a thin tubulovesicular process extended over the nucleus, together with a sparse distribution of microtubules. Figure 5 shows two such thin tubulovesicular processes that follow trajectories corresponding to single microtubules rather than actin fibers. Several additional observations suggest that both the distension of tubulovesicular processes as well as the migration of Golgi-derived particles are occurring along microtubules. First, the rates of movement of tubulovesicular processes and particles emerging from rrans-Golgi elements are within a factor of 2 of the velocities of translocating mitochondria and lysosomes within astrocytes (Table 1). In other cells, mitochondria (Freed and Lebowitz, 1970; Vale et al., 1985) and lysosomes (Matteoni and Kreis, 19W; Heuser, 1989) are known to be displaced along microtubules at comparable velocities in both anterograde and retrograde directions. Second, the saltatory movements of Golgi-derived particles and tubulovesicular processes appear to be constrained to move along curvilinear paths. These linear movements, without lateral displacements, are indicative of active transport along an extended cytoskeletal element, as opposed to Brownian motion (Rebhun, 1972). By inference, these elements are most likely microtubules since no extensions of tubulovesicular processes, linear particle movements, or other directed organelle movements are seen in astrocytes fol-

&;gi

Dynamics

and Microtubule-Based

Transport

Figure 4. Alignment Microtubules (A) SIT camera

of Golgi

Elements

with

micrograph

of elongated Golgi nucleus. (B) A dttity enhanced Nomarski micrograph of the same region. Several nucleoli are visible in the nuctear region. (C) A microtubule stain reveals that the Golgi elements in (A) coalign with bundles of microtubules. Elongated Go@ elements are large enough to interact with many microtubules and are not generally seen to be coincident with single microtubules. (0) An actin stain (rhodamin&abeted phalloidin) shows that the bundles of microtubules are also coincident with stress fibers over the nucleus. Scale bar = 10 pm.

elements suspended over the

lowing microtubule disassembly with nocodazole (data not shown). Cortical actomyosin activity appears to play little, if any, role in the rapid movements (0.1-2.0 urn/s) of membranous organelles in well-spread astrocytes. Time-lapse Nomarski recordings of these cells show no surface dynamics in the perinuclear area or lamellipodial activity in the cell periphery over the period of several hours. This lack of cortical movement and cell translocation is consistent with the pattern of stained actin in these cells, which shows that actin filaments are arranged in numerous stable stress fibers (Figure 4). Long-Range Diffusion of Lipids Occurs through the Cisternae of Elongated Wns-Golgi Elements Previous electron microscopy reconstructions of the Golgi suggest that the saccular and nonsaccular regions of

Figure

5. Alignment

of Tubulovesicular

Processes

cisternae within elongated Golgi stacks are actually highly interconnected fenestrated sheets (Rambourg et al., 1981; Rambourg and Clermont, 1988). To test this concept experimentally, we used a repetitive laser scan of the confocal microscope to bleach a line across various trans-Golgi elements and examine diffusive transport within these structures by following photobleaching-recovery in timelapse video. After the laser is repeatedly scanned across a portion of the cell, several types of frans-Golgi elements can be simultaneously examined for fluorescence recovery from a single photobleach line (Figure 8). Small discrete transGolgi elements often lie completely within the bleach zone. Large elongated Warts-Golgi elements are usually bleached only at a single segment of their entire length. The ER and mitochondria, which are lightly stained by the NBD-ceramide (Lipsky and Pagano, 1985a), also become

with Microtubules

(A) Thin tubulovesicular processes of tmns-Golgi elements can be seen in the focal plane Slightly above the nucleus of a cell. Image taken with laser confocal microscope. (B) A fluorescent antibody stain of the microtubules above the nucleus. (C) Same microtubules stain as in (6). A general alignment of single microtubules (arrows) with the thin tubulovesicular Golgi processes in (A) is shown. (D) An actin stain (bodipy-labeled phallacidin) of the same region shows a dense tangle of fibers that do not coalign with the tubulovesicular Golgi processes. Scale bar = 15 urn. The micrographs of (B), (C), and (D) are directly superimposable on (A) for comparison.

Cell 140

Figure 6. Photobleaching-Recovery Determines That Elongated TinsGolgi Elements Are an Interconnected Compartment for Diffusion (A) A group of isolated and interconnected trans-Golgi elements prior to photobleaching. The fluorescent background to the bright tramGolgi elements is due to the ER and mitochondria, which are lightly stained by NBDceramide. (B) Using a repetitive laser scan, a line is bleached across a portion of the field. Several short isolated elements have been entirely bleached (arrowheads). The top end of an elongated element is also completely bleached (arrow). The center of an elongated branch appears unbleached because much of its fluorescence recovery already occurred during an 6 s time lag between the end of the photobleach and the recording of the image frame of (B). (C) Twenty seconds later, fluorescence returns to the bleached zones of the elongated element (arrow). The background fluorescence, corresponding to the ER, has also partially recovered. Isolated trans-Golgi elements, which were not connected to other Golgi elements outside the bleach zone, did not recover their fluorescence (arrowheads). Scale bar = 10 urn.

bleached. Their combined fluorescence is seen as the background to the more brightly stained tnns-Golgi elements. After photobleaching, a rapid recovery of fluorescence is observed in bleached Vans-Golgi elements if they extend outside the bleached zone. As fluorescence increases in the bleached zone, a depletion of fluorescence is observed in adjacent unbleached areas of the transGolgi elements (Figure 7D). This transfer of fluorescence is consistent with the notion that photobleaching-recovery proceeds by a lateral diffusion of lipids. The mean time for recovery to a steady-state fluorescence after photobleach-

ing was 38.2 s (kg.8 SD, N = 24), a rate which is also consistent with the photobleaching-recovery of diffusing fluorescent lipids within cell membranes (Koppel, 1979). In 90 photobleached elements with portions extending outside the bleach zone, 51 cases of fluorescence recovery were observed, as opposed to 2 cases of no recovery. Recovery of fluorescence after photobleaching was recorded as positive if the fluorescence of a bleached Golgi element increased to a level exceeding that of the recovery of the background. Certain elongated trans-Golgi elements, frequently in interconnected reticula, appeared unaffected by the laser photobleach. The apparent resistance to photobleaching (see, for example, Figure 66) is probably accounted for by two experimental factors. First, photobleaching requires several seconds of repetitive laser scans in a single line over the cell. During this time, rapid lipid flow into the bleached areas of elongated and connected Golgi elements tends to restore their fluorescence. Second, when using the confocal microscope, an 8 s instrumental time lag occurs between the completion of bleaching and the acquisition of the first image scan. In such cases where Wans-Golgi elements appeared bleach resistant, complete recovery of fluorescence had probably already occurred by the earliest opportunity to observe. In the above experiments, involving 90 elongated elements, 34 appeared to be resistant to photobleaching. In the remaining three cases, recovery of fluorescence within the frans-Golgi element was impossible to discern in the face of a gradual bleaching of the specimen, which occurs in time-lapse recordings due to the required laser scanning. Isolated rmns-Golgi elements, which are not connected to other Golgi elements outside the bleach zone, do not recover fluorescence after photobleaching, even after 15 min of observation. In photobleaching experiments involving a total of 90 isolated elements in 31 cells, 88 elements showed no recovery of fluorescence. In two elements, the recovery of fluorescence was indeterminate due to the gradual bleaching of the specimen generated in timelapse recording. The background fluorescence in photobleached areas frequently recovers at a comparable rate to the recovery of fluorescence in elongated trans-Golgi elements (Figure 6). This is not surprising, since the recovery of fluorescence in the interconnected network of the ER would also be expected to proceed by the lateral diffusion of unbleached lipids within its membrane. Tubulovesicular Processes Fuse the Cisternae of Adjacent kww+Golgi Elements into a Single Compartment Photobleaching experiments were also performed on Golgi stacks interconnected by tubulovesicular processes to determine whether these processes actually fuse with the cisternal membranes of adjacent trans-Golgi elements. Figure 7 shows the photobleaching-recovery of an element connected by a thin tubular process to an extensive reticulum of other Golgi elements. The rate of recovery in this structure is approximately the same as within the bleached elongated elementsof the rrans-Golgi reticulum. This result indicates that thin tubulovesicular processes

Golgi Dynamics 141

and Microtubule-Based

Transport

Figure 7. Photobleaching-Recovery of a 7iisGolgi Element Occurs via Diffusion through a Thin Tub&vesicular Process (A) A highly interconnected reticulum of tramGolgi elements prior to photobleaching. (B) After photobleaching, a small elongated trarts-Goigi element, connected only by a thin tubuloveaicular process to the reticulum, is entirely bleached from view (arrow). Other elongated trans-Golgi elements within the reticulum have been strongly photobleached. (C) Eight seconds after (B). Recovery by diffusion proceeds rapidly both within the interconnected reticulum and the Golgi element connected by the thin tubulovesicular process. (D) Thirty-seven seconds after(C). Toward the end of recovery, diffusion has mixed bleached and unbleached zones to a more uniform fluorescence. The result suggeate that the rapid lateral diffusion of lipids takes place both within elongated tmns-Golgi elements and through tubulovesicular processes into adjacent Golgi elements. Scale bar = 10 pm.

can provide direct pathways for the exchange of lipids by diffusion between trans-Golgi elements. In photobleaching a total of 14 Pans-Golgi elements bridged by thin tubular processes to unbleached tmnsGolgi elements, 10 elements showed recovery of fluorescence, as compared with 3 which showed no recovery. The recovery of fluorescence in one element was indeterminate due to gradual photobleaching. The above data as well as the pattern of fluorescence recovery in Figures 6 and 7 illustrate that rapid lipid flow can take place within elongated and interconnected tmnsGolgi elements. In addition, the data suggest that membrane fusion commonly occurs between the cisternae of frans-Golgi elements as they come into contact. Discussion Our time-lapse studies suggest that microtubule-based transport plays an important role in interlinking the cisternal membranes of adjacent Pans-Golgi elements into continuous compartments in which long-range diffusion can occur. It is possible that both anterograde- and retrogradedirected microtubule-associated motors, such as kinesin and dynein, are involved in the extension and retraction of tubulovesicular Golgi processes as well as in the transport of particles that appear to move between Golgi elements. Recently, it has been shown that Golgi elements, scattered initially by microtubule disassembly, will migrate along repolymerized microtubules and recluster in the perinuclear area (Ho et al., 1969). It is apparent, however, from our time-lapse recordings of astrocyctes, that Golgi elements are not maintained in the perinuclear area solely because of a continuous retrograde transport along microtubules. Golgi elements remain in fixed locations even while tubulovesicular processes appear to be pulled and distended from their surfaces. Two specific proteins have

recently been isolated that are localized to the cytoplasmic surface of Golgi elements and bind these membranes to microtubules in vitro (Allan and Kreis, 1966; Bloom and Brashear, 1989). In vivo, such an immobilization via specific binding to microtubules could explain why Golgi elements remain stationary, while microtubule-associated transport motors apparently exert forces on their membranes. However, it is not clear why such microtubule binding proteins, primarily found on the surface of Golgi elements (Allan and Kreis, 1986; Bloom and Brashear, 1989), would result in the localization of the Golgi in the pericentrosomal area. The function of tubulovesicular processes and interconnections between Golgi elements is also an enigma. Fragmented Golgi stacks, both in vivo and in vitro, continue to process secretory materials in the absence of microtubules, demonstrating that specific transport functions of the Golgi are maintained in the absence of these structures (Rogalski et al., 1984; Orci et al., 1969). The linking of the homologous cisternal membranes of many separated Golgi elements into a continuous network, however, may serve to accelerate protein and lipid trafficking within the cell. Such large compartments might allow a greater pool of ligands and substrates to access specific receptors or glycosylating enzymes. An alternative role for the production of tubulovesicular processes may lie in the morphogenesis of elongated stacks of cisternae within the Golgi apparatus. A tubulovesicular process interconnecting the cisternae of two adjacent Wans-Golgi elements may serve as the scaffolding for future addition of cisternae into a stacked pattern, upon the original tubular bridge. Certainly, the appearance of interconnecting Golgi elements of the same thickness in Figures 1C and 7 suggests that elongated elements might be constructed upon bridging tubulovesicular processes, such as those seen in Figures 1B and 28. Indeed, many saccular portions of Golgi cisternae appear as fenestrated

Cell 142

sheets or networks of fused tubules (Beams and Kessel, 1968; Novikoff et al., 1971; Rambourg et al., 1981; Rambourg and Clermont, 1986). The description of tubulovesicular processes emanating from the trans and cis faces of Golgi stacks in a variety of cells (Rambourg et al., 1981; Lindsey and Ellisman, 1985; Rambourg and Clermont, 1986; Tanaka et al., 1986; Noda and Ogawa, 1988) is consistent with the notion that elongated stacks might be constructed from overlapping tubulovesicular processes extended from existing stacks of cisternae. The fusion of cisternal membranes between adjacent &ens-Golgi elements via tubulovesicular processes raises several issues dealing with protein and lipid transport through the Golgi apparatus. Are Golgi stacks composed of completely separate and distinct cisternae at all times? If this were true, fusion between adjacent stacks via tubulovesicular processes would have to be restricted to homologous membranes (i.e., cis to cis, trans to trans). To preserve such separate compartments, a specific class of membrane fusion proteins or receptor-based fusion machinery, capable of recognizing homologous cisternal membranes,. would be required to operate within the Golgi. This function of promoting membrane fusion between homologous Golgi compartments might be a variational role for the membrane fusion proteins and membrane-targeting machinery (Balch et al., 1984; Malhotra et al., 1988; Beckers et al., 1989) that are believed to mediate the progressive movement of transport vesicles through the stacked heterologous cisternae of the Golgi apparatus (Farquhar, 1978; Farquhar and Palade, 1981; Dunphy and Rothman, 1985). At present, there is conflicting evidence concerning the separateness of the various Golgi cisternae as well as their isolation from the ER. On the basis of both scanning and transmission micrographs, Tanaka et al. (1986) concluded that luminal connections exist between various cisternae of Golgi stacks. In one case, they reported a Golgi stack composed of a continuous, helically wound cisternal element, In addition, they described tubulovesicular connections between the Golgi and the ER. Similar connections, between the cis elements of the Golgi and the ER in neurons have been reported by Lindsey and Ellisman (1985) on the basis of three-dimensional electron micrograph reconstructions. Although connections may exist between the cis-Golgi and the ER, our present photobleaching experiments, which show that NBD-ceramide is highly mobile, suggest that the frans-Golgi is isolated from diffusional contact with the ER. Otherwise, one could expect a rapid mixing of lipids between these compartments and a disappearance of the intense fluorescence from trans-Golgi elements. The recent finding that NBD-ceramide localizes in the trans-Golgi (Pagan0 et al., 1989) is also strong evidence that the trans-Golgi is a compartment that is isolated from diffusional contact with the cis and medial cisternae of the Golgi stack. The distension of tubulovesicular processes along microtubules and the fusion of contacting membranes into a reticulum is not unique to the Golgi apparatus. Similar behaviors are observed in the formation of the ER in vivo

(Terasaki et al., 1986; Lee and Chen, 1988) as well as in membranous extracts believed to be from the organelle in vitro (Dabora and Sheet& 1988; Vale and Hotani, 1988). In addition, lysosomes have been observed to fuse into a tubular reticulum in macrophages treated with phorbol esters or solutions that result in cytoplasmic alkalinization (Swanson et al., 1987; Heuser, 1989). Similar to morphogenesis of the ER and reticular Golgi elements, the formation of these tubular lysosomal networks also requires intact microtubules (Swanson et al., 1987). With the propensity of homologous organelle membranes to fuse into reticula as they are brought into apposition by microtubule-based transport, a major cytological question remains as to why heterologous membranous organelles do not fuse with each other. This question is especially intriguing when considering that the fusion proteins believed to be on the surface of transport vesicles (Malhotra et al., 1988, 1989; Wilson et al., 1989; Beckers et al., 1989) shuttling between different types of organelles (e.g., ER to Golgi, Golgi to lysosomes, or heterologous Golgi compartments) might at some point be located on the cytoplasmic surface of the organelles themselves. Experimental Procedures Cell Culture

and Vital Staining

Astrocytes were isolated from the CA1 region of neonatal rat hip pocampus using the procedure of Finkbeiner and Stevens (1966). Cells were plated at a density of 1000 cells per cm2 on 22 x 22 mm #I coverslips in minimal essential media without phenol red (GIBCO, Grand Island, NY). Three days later, 5 x lo+ M arabinosylcytosine was added to the culture media to suppress additional astrocyte proliferation, allowing cells to spread and flatten. Cells were used for microscopy after 2-4 weeks in culture. These astrocyte cultures stained positively (>96% of cells) with an antibody to glial fibrillary acidic protein. A stock solution of 4 mM NBD-ceramide (Molecular Probes, Eugene, OR), made by dissolving the lipid in DMSO (Aldrich, Milwaukee, WI), was stored as 10 ul aliquots at 4OC in the dark. Aliquots of the fluorescent lipid were diluted to 40 pM (1% DMSO) in a complete medium containing Earl& minimal essential medium, Fen/Strep (50 U/ml), and fetal bovine serum (all media from GIBCO, Grand Island, NY). Astrocyte cultures were labeled with this solution for 10 min. The cells were then washed twice with complete medium and incubated at 3PC for 60 min. After being loaded into the membranes of many types of cells, NBD-ceramide is transferred to the Golgi apparatus, where it is converted to and accumulates as NED-labeled sphingomyelin and glucocerebroside (Lipsky and Pagano, 1965a; Pagano, 1966). After staining, coverslips with labeled cells were mounted for microscopy in a flow-through perfusion chamber that has been described elsewhere (Forscher et al., 1967). In certain experiments, microtubules were disassembled by incubating cells in 10 uM nocodazole (Sigma, St. Louis, MO) at 4°C for 20 min, then warming the cells to 3pc in the continuous presence of the drug.

Confocal,

Fluorescence,

and Nomamkl

MIcroscopy

A Bio-Rad MRC-500 scanning confocal laser microscope (Cambridge, MA) was used to obtain time-lapse recordings of Golgi dynamics in cells labeled with NBD-ceramide. Cells were illuminated with 466 nm light from the instrument’s argon laser through a Zeiss IM-35 inverted microscope using a 63x11.25 numerical aperture (NA) Neofluar objective. The confocal aperture in the laser scanning assemblage was kept completely open to maximize the collection of epifluorescent light from the sample. Light passing through the aperture was filtered by a 515 nm long-pass filter before being detected by the instrument’s photomultiplier tube. The photocurrent coming from the photomultiplier was passed through a 20 kHz low-pass (four pole) RC filter (Krohn-Hite Corp. model 3200, Avon, MA) to reduce pixel noise through temporal

Ft;gi

Dynamics

and Microtubule-Based

Transport

integration before the signal was stored in the frame buffer of the microscop& Nimbus host computer. Time-lapse recordings, made from single sweeps of the instrument’s 4 s slow scan, were stored on a high resolution Panasonic optical memory disk recorder model TC?2026F (Secaucus, NJ). Time-lapse fluorescence and Nomarski video recordings were also made using BIT (silicon intensified target) and Newvicon type video cameras (Hamamatsu Corp., Middlesex, NJ) on aZeiss IM 35 inverted microscope with mercury arc and tungsten illumination, using Zeiss 63xn.25 NA Neofluar and 63x/1.4 NA Planapo objectives and shuttered 450-495 nm excitation and 520 nm long-pass emission optics. To improve the signal-to-noise of time-lapse video signals, an Imaging Technology (Woodburn, MA) Series 151 image processor, controlled by a host IBM-AT computer, was used for boxcar (eight video frames) or rolling frame averaging (0.5 s time constant). Video images were recorded on a Panasonic optical memory disk recorder (model TQ2026F) (Secaucus, NJ). To obtain high contrast single images of Golgi structures stained with the rapidly bleaching NBD fluorophore, a boxcar average of 64 video frames was used. An Imaging Technology PFG plus640 personal frame grabber controlled by an AT compatible personal computer with Image-Pro software from Media Cybernetics (Bethesda, MD), was used to acquire single images from the optical memory disk recorders for digital image processing. Slide-stretch contrast enhancement, two cycles of an unsharp mask nonconvolution filter (Pratt, 1976), and image scaling functions were used to maximize the signal-to-noise of images displayed on a high resolution (1260 x 1064 pixel array) Hitachi monitor (model HM4115-D-&O, Woodbury, NY). Photographs of images were taken from the monitor using a 35 mm camera with a macro lens and Kodak T-Max ASAfilm.

Immunofluoreecence

and Cytochemical

In separate experiments, rhodamine-conjugated wheat germ agglutinin (Sigma, St. Louis, MO) was used as a cytochemical marker of the frans-Golgi (Tartakoff and Vassaili, 1963) to confirm the morphologies of structures stained with NBD-ceramide. After fixation, cells w8re permeabilized with 1% Briton X-100 in calcium-free Ringer’s solution for 1 min. The cells were then stained with 50 pg/ml rhodamine-conjugated wheat germ agglutinin for 15 min.

Photobieeching

Experiments

Using software of the Bio-Rad laser wnfocal microscope, lines were photobleached across Golgi elements by commanding the scanning assemblage of the microscope to sweep the 466 nm laser beam, with no neutral density filter attenuation, 200 times over a prescribed linear path, for a total of 4 s. To assess quantitatively the rate of fluorescencerecovery in bleached Golgi elements, intensity versus time plots were generated by averaging intensity values of a 5 x 5 pixel square in the center of bleached Golgi elements using Image-Pro software. The mean time for bleached Golgi elements to recovery to a steady-state fluorescence after photobleaching was determined from these plots.

Acknowledgments We gratefully acknowledge the financial support of the National Institutes of Health (grant NSl667l-09) to S. J. S. and of the Howard Hughes Medical Institute. We also thank H. I? Moore for providing us with the O-45 mutant of vesicular stomatitis virus and a primary antibody to its coat. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adwtisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact.

Stelnlng

Within 20 s after time-lapse filming, cells were fixed for 5 min with 2% formaldehyde (Polysciences, Warrington, PA) and 0.05% glutaraldehyde in a calcium-free modified Ringer’s solution containing 106 mM NaCI, 5 mM KCI, 5 mM MgClz, 3 mM EGTA. 3 mM HEPES (pH 7.2). Cells were then extracted with 1% Briton X-100 (Polysciences, Warrington, PA) in calcium-free Ringer’s solution for 2 min to permeabilize cell membranes and extract efficiently membranous organelles from the perinuclear area. The following procedure was used for staining microtubules: 5% normal goat serum (Vector Stains, Burlingame, CA) was applied as a block for 5 min. A primary antibody to the b-subunit of tubulin (Ameraham International, UK), diluted from a stock concentration to 50 pg/ml, was then applied for 20 min. After three washes with calcium-free Ringer’s solution, a biotinylated second antibody (anti-mouse raised in horse) (Vector Stains, Burlingame, CA) in calcium-free Ringer’s solution with 1% normal goat serum was applied for 15 min. Following three washes with calcium-free Ringer’s solution, FITC-labeled avidin (50 kg/ml) was applied for 15 min. Actin was stained with 6 IUlml rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR), for 5 min. Simultaneousdouble-labeling with fluorescein and rhodamine was not possible using the laser confocal microscope since the argon laser emits primarily at 466 and 512 nm. Rhodamine is only weakly excited with 512 nm light. To obtain images of microtubules and actin within the same cell, the FITC-labeled avidin microtubule stain was first recorded with the wnfocal microscope. The specimen was then intensely bleached by repetitive laser scans. Actin was subsequently stained with 6 IU/ml bodipy-labeled phallacidin (Molecular Probes, Eugene, OR), which has spectral properties similar to fluorescein. Both fluorophores are excited well by the 466 nm line of the microscope’s argon laser. Nomarski time-lapse video recordings were used to obtain the velocities of migrating mitochondria and lysosomes in the hippocampal astrocytes. To confirm the identity of specific organelles within the cells, several vital stains were used. Lysosomes were stained with 0.01% acridine orange (Sigma, St. Louis, MO) for 2 min and viewed with rhodamine epifluorescence optics, 510-560 nm excitation and 590 long-pass emission (Matteoni and Kreis. 1967). Mitochondria and the ER were labeled, respectively, with 10 fig/ml rhodamine 123 and 10 pg/ml DiOCg) (both from Molecular Probes, Eugene, OR) for 2 min and viewed with fluorescein epifluorescence optics, 450-490 nm excitation and 515-565 nm emission (Johnson et al., 1960; Terasaki et al., 1964).

Received

November

30, 1969; revised

January

30, 1990.

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