EM visualization of nucleocytoplasmic transport processes

oxY2-0354’Yn $0 00 + 0 50 f

1990 Pergamon Pre5r

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EM VISUALIZATION OF NUCLEOCYTOPLASMIC TRANSPORT PROCESSES C. M. FELDHERR

ofAnatomyand

Depurtmrnt

Cell Biology. College

and D. AKIN

ofMedicine,University

of Florida, Gainesville, FL 32610, U.S.A.

nuclear envelope is strategically located between the nucleoplasm and cytoplasm, and, as such, can play a major role in controlling cellular activity by regulating the exchange of macromolecules between these two compartments. The nuclear pore complexes, which are located within circular areas formed by fusion of the inner and outer membranes of the envelope, represent the primary, if not the exclusive, exchange sites. Individual pores are able to function in both protein import and RNA efflux from the nucleus. Translocation of macromolecules occurs by either passive diffusion or facilitated transport through central channels within the pores. The functional size of the diffusion channel is approximately 9 to over 12 nm in diameter depending on the cell type. The width of the transport channel varies as a function of the number and effectiveness of the specific nuclear targeting signals contained within the permeant molecule. The maximum diameter of the channel can be over 26 nm. Nucleocytoplasmic exchanges can be regulated either by (1) differences in the properties of the transported molecule (molecular size and signal content) or (2) changes in the properties of the pore complexes, which can effect both diffusion and transport. Abstract-The

CONTENTS I. Introduction ....................................... A. Scope of the review .............................. B. Permeability of the nuclear envelope ............... C. Morphology of the nuclear envelope ............... 11. Identification and characterization of the exchange sites. III. Possible factors regulating transport .................. A.Signalcomposition ............................... B. Variations in pore function ....................... IV. Functional organization of the pores. ................. Acknowledgements. ................................. References .........................................

1. INTRODUCTION

The exchange of macromolecules between the nucleus and cytoplasm, the primary sites of transcription and translation, respectively, is of fundamental importance in maintaining overall cellular activity. In interphase cells, these exchanges must occur across the nuclear envelope. Recent studies suggest that the envelope is not simply a passive barrier, but plays an active role in regulating interactions between the two major cell compartments.

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73 73 74 75 76 79 79 x0 82 84 x4

A prerequisite for understanding the mechanism(s) of macromolecular exchange is the identification and characterization of the translocation sites. Electron microscopic visualization of the exchange process, utilizing both endogenous and exogenous tracers, has been especially useful in this regard, and has resulted in the formulation of a functional model of the nuclear pores. This review will focus primarily on these studies; however, to provide a broader perspective by which to evaluate the electron microscopic data we will first summarize the permeability properties and the unique ultrastructural features of the nuclear envelope.

Due largely to technical limitations and a lack ol information concerning the properties of specific nuclear proteins, most studies of the permeability of the nuclear envelope performed prior to 1975 utilized synthetic polymers or exogenous. nonnuclear proteins as tracers. These data have been reviewed in detail by Feldherr (1972). Bonner ( 197X), Paine and Horowitz (10X0), and Peters (19X6) and will only be summarized here. The general consensus derived from these earlier investigations is that molecules with molecular masses up to approximately 70 kDa can penetrate the nuclear envelope. The rate of nuclear incorporation of exogenous substances was found to be inversely related to molecular size and it was concluded that uptake occurs by passive diffusion, Kinetic analyses of dextran uptake performed on .~‘cwo/~~~soocytes by Paine et (I/. (1975) :tnd on cultured hcpatocytes by Peters (19X4) indicated that the diffusion channels in these cell types have diameters of approximately 9 and IO I I nm. respectively. Consistent with these findings it has also been determined that ions and small organic molecules readily enter the nucleoplasm. The development of polyacrylamide gel electrophoresis and other experimental techniques made it feasible to investigate the intracellular distribution of specific nuclear proteins. Bonner (1975), Feldherr (1975) and DeRobertis 01 trl. (197X) used gel electrophoresis procedures to analyye the nuclear uptake of metabolically labeled proteins in X~~opu.s oocytes. These cells arc especially suited for studies of this type since nuclei can be rapidly isolated with minimal loss of material. It was found that larger polypeptides (94 kDa and above) are incorporated into the nucleus at greater rates than could be accounted for by passive difl’usion. DeRobertis rt (I/. (197X) suggested that these polypeptides might contain specific signals that promote nuclear uptake. Evidence that certain proteins arc targeted to the nucle~~s and that targeting involves transport across the envelope and not simply intranuclear binding (which would prevent back diffusion and increase accumulation rates) was initially obtained by Feldherr c’/ trl. (1983) and Dingwall @IN/. ( 1982).

In the first of these studies it was found that RN I a 14X kDa karyophilic protein found in K011tr oocytes. enters the nucleus approximately 20 tlmc\ more rapidly than could bc xcounted fi~r I,\ passive ditfusion and 4ubscquent ititr;inuclc;ir binding. Dingwall (11 L/I. ( IYX?) not only dcmonstrated that nucleoplasmin (a pentameric. I22 kDa .Y~JrloJ~~soocyte nuclear protein) is transported across the envelope. but also determined that the nuclear targeting signals are located uithin IO klh .tail’ regions of the monomeric subunits. In later studies. utilizing recombinant I)NA technology. the specific amino acid sequences rcquired for nuclear targeting have been idcntilied in a number of karyophilic proteins. The nature (11’ these signals and their properties have reccntl> been reviewed by Silver and Hall (19X%). The sequences that have been charactcrired are rcI;Itively short. ranging from 5 I4 amino acid\ III length, and are highly basic. The SV40 large T-antigen and nucleoplasmin have been the most frequently used proteins for nuclear permeability studies. including clecrron microscopic localization of the exchange sites. Re4pectiLely. the sequcnccs that initiate transport 01‘ these molecules across the nuclear envelope art‘ ;I\ follows: Pro-Lys-Lys-Lys-Arg-Lys-Val (Kalderon (‘/ t/l.. 1984:~). and Arg-Plo-Ala-Ala-Thr-[~~~-l_) ,Ala-Gly-Gin-Ala-Lys-Lys-Lys (Dingwall (‘f t/l.. I9XX). The latter is ;I minimal sequence and rcquires three additional Ilanking amino acids to hc functional. The signals appear to act autotlomously and when insert& into unmasked \ltc\ the coding sequence of cytoplasmlc within proteins. they are able to target the hyhrld polypeptides to the n~~cleus (Rohcrts 01 (I/.. 10X7). Non-n~dear proteins can also be tran\portcd across the envelope it’ they are conjugated \\ith synthetic peptides containing nuclear targeting signals (Goldfarb c’t III.. 1986: Lanford o/ (//.. I9Xh; Chelsky ct trl.. 1989). RNA and RNP particles arc. in general. too large to difruse across the envelope. suggesting that these substances are transported to the cytoplasm. In support of this view there is a considerable hod! of evidence demonstrating that mRNA ~I~LIY I\ an energy-requiring process. dependent on [he hydrolysis of ATP. The data pertaining to ihe

Nucleocytoplasmic

envelope nucleoside triphosphatase and associated binding proteins that are involved in transport have recently been reviewed by Schroder et al. (1987) and Agutter (1988). The poly(A) tail of mRNA appears to play a role in transport, but it cannot be considered a universal targeting signal since not all messages are polyadenylated. The efflux of tRNA from Xenopus oocyte nuclei has been investigated by Zasloff (1983) who found that the process is saturable, temperature dependent and sensitive to a G to U substitution at position 57. In summary, the permeability characteristics of the nuclear envelope are different than those of any other intracellular membrane system, not only are macromolecules able to diffuse across the envelope, but mechanisms are also available for the transport of both karyophilic proteins and RNA. Consistent with these unique functional properties, the envelope also exhibits highly specialized morphological features, which will be considered next. C. Morphology

of the Nuclear

Enrelope

The nuclear envelope consists of two parallel membranes separated by a cisternal (perinuclear) space approximately 40 nm thick. The chemical composition of the membranes is very similar to that of the rough endoplasmic reticulum (Richardson and Agutter, 1980) which is consistent with the morphological observation that the outer nuclear membrane (facing the cytoplasm) is sometimes continuous with the rough endoplasmic reticulum (Watson, 1955). The nuclear surface of the inner membrane of the envelope is closely associated with a fibrous structure, approximately 10-l 5 nm thick in vertebrate cells, that is referred to as the nuclear lamina. The specific polypeptides (lamins) that make up the lamina vary in different cells and also during development (Krohne and Benavente, 1986; Gerace and Burke, 1988); however, all have molecular masses in the range of 50-80 kDa and are biochemically similar to intermediate filament proteins. There is evidence that the lamina functions (1) in nuclear envelope breakdown and reassembly during cell division, (2) as an attachment site for interphase chromatin. which could serve to order the chromosomes for subsequent cell

Transport

75

division and (3) as an attachment site for the pore complexes. The most distinctive features of the envelope are the nuclear pore complexes. At numerous points along the surface of the envelope, the inner and outer membranes are fused to form circular pores approximately 70-80 nm in diameter. These structures can occupy from 5 to over 30% of the nuclear surface, depending on the cell type (Feldherr, 1972). Although the pores are not spanned by membranes, they each contain a cylindrical proteinaceous structure, the pore complex, that appears to be attached to the lamina (Aaronson and Blobel, 1974). The ultrastructure of the pore complex has been the subject of numerous investigations, and several comprehensive reviews are available (e.g. Stevens and Andre, 1969; Franke, 1974; Maul, 1977; Scheer et al., 1988). The most recent models of the pore complex, proposed by Unwin and Milligan (1982) and Akey (1989) are based on analysis of Xenopus oocyte nuclear envelopes. The model proposed by Akey is shown in Fig. 1. The major components are (1) ring structures located at both the nuclear and cytoplasmic surfaces of the pores, (2) eight spokes that extend from the pore margin toward the central axis, and (3) a central structure, the transporter assembly, that occupies the spaces outlined by the spokes, and could function in regulating the exchange pathway through the pores. In addition, peripheral granules were observed associated with the ring structures at the cytoplasmic surfaces; however, these granules were not always present and could represent transient rather than fixed pore components. The composition and function of these peripheral structures is not known. Although the above model has many of the elements proposed earlier by other investigators (see Franke and Scheer, 1970a,b; Maul, 1977, for details) including eight-fold radial symmetry. a number of differences also exist, especially with regard to the nature of the peripheral granules and the dimensions of the subunits. Several explanations have been proposed to account for these differences (Feldherr, 1972; Maul, 1977). They could be due to variations in fixation and processing for electron microscopy; more interestingly, they could represent real differences in pore struc-

c‘. M. Feldherr

and II. Akln

Fig. I. A three-dimensional model of the nuclear port complex proposed by Akcy. The complex is located wthln a ctrcuI;it space formed by fusion of the inner (INM) and outer (ONM) nuclear membrane. and is composed of cytoplasmlc (CR) and nuclear (NR) rings. spokes (S). vertical supports (VS) and a central transporter asscmblq (T). Reproduced v.ith permisson of The Rockefeller- University Press. C’ourte\y of Dr C. W. Akq

ture among the different cell types that have been examined, or reflect changes in the functional state of the pores. as suggested by Kessel (1969). Based on the morphology of the nuclear envelope, three possible routes exist for macromolecular exchanges: (1) directly across the outer and inner membranes, (2) through the rough endoplasmic reticulum to the perinuclear space, and (3) through the pore complexes. The studies that have been performed to distinguish between these possibilities will be considered next.

II. IDENTIFICATION AND CHARACTERIZATION OF THE EXCHANGE SITES Since the nuclear pores were first described by Callan and Tomlin (1950) they have been considered the most likely pathways for nuclearcytoplasmic exchange. Early evidence supporting by Anderson and the view was obtained Beams (1956). who detected dense aggregates of basophilic granules adjacent to and extending through the centers of the pores of Rhodnnizrs nurse cell nuclei. Similar findings were reported by Kessel (1966) who examined tunicate oocytes and by Franke and Scheer (1970b) in amphibian oocytes

(see Fig. 2). The latter investigators also dctcrmined that the central channel available thl translocation was at least I@ I5 nm in diameter. Since the material that was seen crossing the pores was basophlhc and in close proximity to the nucleoli, it was assumed to be ribonucleoprotein in the process of leaving the nucleus. Stevens and Swift (1966) and, more recently.

Nucleocytoplasmic

Skoglund et al. (1986) investigated the formation and subsequent translocation of RNP particles that are produced in association with specific Balbiani ring genes in Chironomus. Within the nucleus, the hnRNA complexes with proteins to eventually form spherical 50 nm particles. As the particles leave the nucleus, they elongate and pass through the central regions of the pores as rod-like structures 135 nm long and 25530 nm in diameter. The mRNA release codes for salivary polypeptides. The investigations described above indicate that rRNA and mRNA can leave the nucleus through central channels that can be over 25 nm in width. Unfortunately, observations such as these are limited to a few cell types in which nucleocytoplasmic exchanges are occurring at an unusually high rate or in which the transported material has identifiable morphological features. A method of visualizing exchanges that can be applied to a variety of different cell types has been used extensively by Feldherr and his co-workers. This procedure involves the microinjection of coated colloidal gold particles into cells, and has the following advantages: First, the properties of the tracer particles can be altered by varying the coating (stabilizing) agent. Stabilization is accomplished by the adsorption of molecules to the surface of the gold, thereby preventing aggregations of colloid at increased salt concentrations, and simultaneously modifying the properties of the particles. Second, by employing different procedures for preparing the colloid, particles ranging in diameter from 2 to over 30 nm can be obtained. This includes the size range of material that normally passes between the nucleus and the cytoplasm. Third, due to the density of the particles, they can be precisely localized in relation to specific cell structures by electron microscopy. Fourth, the tracers can be injected into either the nucleus or the cytoplasm permitting analysis of both nuclear uptake and efflux. General techniques for preparing and stabilizing colloidal gold have been discussed by Horisberger (1979) DeRoe et al. (1987) and Geoghegan (1988). The specific application of these procedures to nuclear transport studies has been considered in detail by Feldherr (1986) and Feldherr and Dworetzky (1989).

Transport

77

As with any technique, the approach is not without its limitations. Not all macromolecules of interest can stabilize gold at physiological pH and ionic strength. In addition, adsorption to the surface of the gold particles can modify the properties of the coating molecules. These factors must be considered when designing and interpreting experiments that utilize colloidal tracers. The gold procedure was initially used to investigate the nuclear uptake of polyvinylpyrrolidone (PVP)-coated gold particles following injection into the cytoplasm of amoebae (Feldherr, 1962, 1965). Electron microscopic analysis showed that particles as large as 12.5-14.5 nm entered the nucleoplasm by passing through the centers of the pores. (These, and all subsequent particle dimensions that will be reported include the thickness of the coat material, which was found to be approximately 3 nm.) Since PVP does not contain a targeting signal, it can be assumed that exchange occurred by passive diffusion. Thus, the diameter of the diffusion channel in these cells could be up to 3.5 nm greater than in oocytes or hepatocytes (see above). According to Paine et ul. (1975), a difference of this magnitude could have a significant effect on nucleocytoplasmic exchange. The identification of signals that are capable of transporting proteins into the nucleus, especially proteins too large to diffuse through the pores, raised the possibility that there are specific exchange sites that function exclusively in transport. To localize the pathways utilized by targeted polypeptides, Feldherr et al. (1984) Richardson et al. (1988) and Dworetzky et al. (1988) injected nucleoplasmin-coated gold particles into the cytoplasm of Xenopus oocytes and later determined their intracellular distribution by electron microscopy. Particles ranging in diameter from 15-25 nm rapidly entered and eventually concentrated in the nuclei; in addition, gold accumulated at the surfaces of the pores and was frequently observed extending through the centers of these structures (Fig. 3). In control experiments, particles coated with PVP, BSA or nucleoplasmin digested with trypsin, which removes the targeting domains, did not associate with the pores and were largely excluded from the nucleus. Gold was never observed within the perinuclear space, as would be

Fig. 3. A X~wopu.v oocyte lixed I hr after a cytoplasmic injection 01 nucleoplasmln-coated gold particles. The tracer- GIII be seen just adjacent to and wthin the nuclear ports. Part&s up to 25 nm 111diameter arc present in the IILIC‘ICLI\I h 1 c‘. cytoplasm. Scale marker represents 200 nm. Reproduced with pcrmissit-rn of The Rockefeller lJnivcrsit\i Prcs\ (‘ourlca\ of Dr S. I

expected if exchange occurred directly across the membrane. Similar results were obtained in an irr rifro system by Newmeyer and Forbes (1988). Thus. transport, like diffusion. occurs through the centers of the pores; however. the diameter of the channels that function in transport are considerably larger than those involved in diffusion (approximately 25 nm compared to 9 nm). Transport channels with diameters of approximately 7.5 nm have recently been identified in HeLa cells (Feldherr and Akin. unpublished), Dworetzky and Feldherr (1988) analyzed the efflux of RNA-coated gold particles that were injected into the nucleoplasm of Xenopus oocytes. The particles were coated with tRNA, 5s RNA, poly(A). poly(1) or poly(dA), and the cells were fixed 15 min. 1 hr or 6 hr after injection. Essentially the same results were obtained regardless of the coating agent used. At all time intervals, particles were observed just adjacent to and within the centers of the pores (Fig. 4). At 15 min and I hr intervals, 97% of the pores appeared to be active in translocation of the tracers; this percentage

decreased at 6 hr. The largest particle\ able 10 penetrate the pores were about 35 nm in diameter. These findings are consistent with carlicr clectron microscopic observations showing that rRNA and mRNA exit the nucleus through the ccntcrs ot the pores (see above). The gold results furthetdemonstrate that the same pathways arc available for other classes of RNA and that essentially all ot the pores are able to transport these molecules. a~ least in oocytes. The latter finding also implies that individual pores can function in both the uptake and efflux of macromolecules. If this were not the cast only about 3% of the pores would be activsc in protein translocation, an unlikely possibility considering the results obtained with nuclcoplasmin -gold (see Fig. 3). As I direct test for bifunctionality. Dworetzky and Feldherr ( 19Xx) performed double-labeling experiments in which I5 25 nm gold particles coated with nucleoplasmin were injected into the cytoplasm and 5 X nm tRNA-coated particles were injected into the nucleus of the same cell. Frequently, pores were seen that had large (protein-coated) particles just adja

Nucleocytoplasmic

Transport

79

Fig. 4. Gold particles coated with 5s RNA were injected into the nucleus of a Xwwpu,s oocyte. After 1hr particles were found associated with almost all of the pore complexes. The presence of gold at the cytoplasmic surface of the pores provides direct evidence that translocation occurs at these sites. N, nucleus; C. cytoplasm. Scale marker represents 200nm. Reproduced with permission of the Rockefeller llniversity Press. Courtesy of Dr S. I. Dworetzky.

cent to their nuclear surface and small (tRNAcoated) particles adjacent to their cytoplasmic surface, demonstrating that individual pores are capable of transporting both classes of macromolecules (Fig. 5).

III. POSSIBLE

FACTORS REGULATING TRANSPORT

A. Signal

Composition

There are several lines of evidence suggesting that both the amino acid composition and number of targeting signals are important factors in the regulation of transport across the nuclear envelope. The effectiveness of the SV40 signal sequence, for example, can be altered by single amino acid substitutions, as demonstrated by Kalderon et ul. (1984b), and Lanford et al. (1988). In the extreme case, activity is almost completely abolished by substituting lysine at the 128 position with

asparagine or threonine. Recently, Lanford et al. (1989) and Chelsky et ~1. (1989) conjugated proteins that are not normally located in the containing synthetic peptides with nucleus targeting sequences that have been identified in several diverse karyophilic proteins. They reported that the different sequences were not equally effective in nuclear targeting. The number of signals per protein molecule has been experimentally altered by recombinant DNA methodology (Roberts et ul., 1987), conjugation with synthetic peptides (Lanford et ~1.. 1986), and proteolytic digestion (Dingwall et al., 1982). In all of these studies it was found that the transport rates increase as the signal number increases. Dworetzky et ul. (1988) utilized colloidal tracers, in conjunction with microinjection, to evaluate the effects of altering the signal composition on nuclear transport in Xenopus oocytes. In this study, both the rates of uptake and the functional size of the transport channel were analyzed. Gold particles that varied from S--30 nm in

x0

C‘. M. I-‘cldherr and I). Akln

Table

Coating 9’40 tBSA BSA BSA BSA

I. EH’ect of Signal

agent

large T-antigen WT. + cT WT, WT, WT,,

Number

on (‘hannel

Average number of signals per molecule

SIK

‘Maximum wc of tran\portcd parllclc (nm)

I 3 5 x II

I2 I2 71 T 25 2s

* These dimensions mcludc the coat material. t In this conjugate the active. utld type signal (WT) \\;I, diluted wth mactive (cT) sigxils to give an a\crage of 1 \~gnal~ per BSA molecule.

Fig. 5. A double injection experiment demonstrating that individual pores can function in both protein uptake and RNA efflux. The larger nucleoplasmin-coated particles that are located on the nuclear side (N) of the pores were Injected into the cytoplasm (C). The small. tRNA-coated particles seen adjacent to the cytoplasmic surfxe of the same porch were injected into the nucleus. Scale marker reprcscnts IOOnm. Reproduced with permlwon of The Rockefeller Unlkersity Press. Courtesy of Dr S. 1. Dworet;lkq.

diameter were coated with one of the following targeted polypeptides: nucleoplasmin, which has live signals per molecule; SV40 large T-antigen, which has a single signal per monomer; BSA conjugated with synthetic peptides containing the SV40 transport sequence. The conjugates contained 5, 8 or I I signals per BSA molecule. Increasing the number of SV40 signals resulted in statistically significant increases in both the rate of transport and the size of the gold particles present in the nucleus. The approximate exclusion size as a function of signal number is shown in Table I. These data imply that the functional diameter of the transport channel is variable and related to signal input. In control experiments, particles coated with BSA alone, or BSA cross-linked with inactive SV40 signals (lysine- I28 substitution) were retained in the cytoplasm. With regard to signal activity, nucleoplasmin proved to be a much more effective coating agent than any of the BSA-signal conjugates. For exam-

ple, I hr after injection the nuclear to cytoplasmic (N/C) ratios obtained for 15 25 nm gold particles coated with nucleoplasmin and BSA conjugates containing I I signals were 0.51 and 0.24. respectively. These results take on added signiticancc when one considers that the conjugate-coated particles contained over twice the number of signals. A direct relationship was found to exist between the rate of transport of a given tracer and its ability to accumulate at the surface of the pores. Thi\ suggests that the effectiveness of a specifc signal sequence might be related to its binding affinity l01 receptors. at the pore surf-ace. R. Vrrriutiom in Pow Fut~ctiorr Theoretically. regulation of nucleocytoplasmic exchanges can be accomplished by altering the properties of tither the permeant molcculc or the pores themselves. As discussed above. numerous investigations have focused on the first of these possibilities. and it is now evident that variations in molecular size and signal content can significantly affect the exchange process. It has also been suggested that the permeability properties of‘ the pores can change as a function of cell activit). however, the supporting data is limited. Jiang and Schindler ( 1988) reported an increase in the uptake of dextran into the nucleus of non-transformed 3T3 libroblasts following treatment with insulin and epidermal growth factor, indicating a change in passive diffusion across the envelope. Slavicek (‘/ c/l. (1989) identified a targeting sequence in adenovirus EIA gene product. located within residue5 I45 185. that initiates transport into the nucki 01‘

Nucleocytoplasmic

oocytes, but not somatic cells. Feldherr (1966, 1968) studied the nuclear uptake of PVPcolloidal gold particles during the cell cycle in amoebae and found that the envelopes were most permeable for the first 2 hr after division. Not only were the relative uptake rates greatest during this interval, but larger particles were incorporated into the nucleoplasm than at other stages of the cell cycle. In contrast to these findings, Swanson and McNeil (1987) failed to detect an increase in the nuclear uptake of fluorescein-labeled dextrans in dividing fibroblasts; however, due to differences in the experimental procedures, a direct comparison of these results with those obtained for amoebae is difficult. We have recently undertaken an investigation of nucleocytoplasmic exchanges during the cell cycle in HeLa cells, and also in dividing and non-dividing 3T3 Ll cells. Passive diffusion and transport were studied by analyzing the nuclear uptake of microinjected BSA- and nucleoplasmin-coated gold particles, respectively. HeLa cells in specific stages of the cell cycle were obtained by either mitotic shake-off procedures or by tracking individual cells in culture starting at the onset of anaphase. The latter technique was term experiments employed in short (15 min4.5 hr), when more precise timing was required. The cells were grown on collagen-coated ACLAR cover slips. ACLAR is a fluoroplastic and is an excellent substrate for both culturing and processing cells for electron microscopy (Kingsley and Cole, 1988). A grid pattern (with approximately 1 mm spacing) was scratched on the surface of the cover slips prior to culturing (Masurovsky et al., 1971). This provided a convenient means of mapping injected cells and, since an impression of the grid was visible on the surface of the blocks after embedding, the same cells could be identified for subsequent sectioning and electron microscopic analysis. Microinjection was performed as described by Graessmann et al. (1980). Cells injected with BSA-gold (5515 nm in diameter) were fixed for electron microscopy after 30 min. Particles were counted in equal and adjacent areas of nucleoplasm and cytoplasm, and relative nuclear uptake at different times in the cell cycle is expressed as (N/C) ratios. It can be seen in

81

Transport

Table 2. BSA-Gold

Nuclear Uptake of During the Cell Cycle

Time after anaphase (hr) 0.25-l l-2 2-3 45 7 I9

N/C ratio 0.115 0.022(s)* 0.004(s) 0.032(s) 0.007(s) 0.005(n)

* The letters in parentheses indicate whether the results are (s) or are not (n) significantly different from the immediately preceding values.

Table 2 that maximum nuclear uptake occurred between 15 min (by which time the nuclear envelope had completely reformed) and 1 hr after the onset of anaphase. This was followed by significant decreases at l-2 and 2-3 hr, an increase at 45 hr, and further decreases at the 7 and 19 hr time periods. The maximum size of the particles that entered the nucleoplasm was approximately 12 nm in diameter. The observed variations in the relative nuclear uptake of BSA-gold do not correlate with changes in nuclear surface area to volume ratios; however, a close correlation does exist between uptake and the rates of pore formation. Although the number of pores increases throughout the HeLa cell cycle, the rates of formation are greatest 61 and 4-5 hr after mitosis (Maul et ul., 1972). This relationship suggests that newly forming pores are more permeable than older, mature pores. The significance of this is not known, but rapid diffusion, especially just after mitosis, could facilitate reconstitution of the nucleus. Particles (15-35 nm in diameter) coated with either nucleoplasmin or BSA conjugated with SV40 targeting signals readily entered and concentrated in the nuclei. Figure 6 shows the distribution of nucleoplasmingold 15 min after a cytoplasmic injection. Gold was observed passing through the pores and particles as large as 25 nm in diameter were present in the nucleoplasm. Unlike BSA-gold. no significant differences in the nuclear uptakes of these tracers were observed during division. Cells were analyzed 15-30 min, 4.5 hr, 8.5 hr, and 17-20 hr after mitosis.

Although no cell cycle-dependent changes in nuclear transport were detected. highly significant differences in nucleoplasmin gold uptake were observed in dividing versus confluent. growth itrrested 3T3 LI cultures. The N/C gold ratio. analyzed 30 min after injection, was approximately 7 timcs greater in dividing cells: furthermore, the diameter of the transport channel was about 4 nm larger than that observed in growth arrested cells (approximately 23 nm versus 19 nm). These results demonstrate that the transport process can vary in ;I given ccl1 type during different functional states. In general. these data. as well as earlier work cited above. suggest that in addition to the properties of the permeant molecule (e.g. size and signal content), nucleocytoplasmic exchanges might be regulated by modulation of the properties of the pores themselves. IV. FUNCTIONAL ORGANIZATION OF THE PORES Based on electron microscopic cndogenous RNA efflux. combined

analysis of with data

obtained using colloidal gold tracers. it can be concluded that nuclcocytoplasmic cxchanpc\ 01‘ both RNA and protein occur through central channels located within the nuclear pores. and that individual pores are bifunctional and can transport both classes ol‘ molecules. The central channel appears to be ;I variable pore component. In (hc absence of a targeting signal it has ;I diamctcr- of’ approximately Y to over I2 nm. depending on the 01‘ cell type. and allows the passive dilfuGon molecules that have the appropriate Dimensions. III the presence of a nuclear targeting sequcncc the functional size of the channel can increase. permitting the transport of materials o\er 75 nm in diameter. Feldherr 1’~ rrl. (1984) proposed that protan transport is a multistep process involving binding to the cytoplasmic surface of the pores. followed hl translocation through central channels. Evidence supporting this hypothesis was subsequently ohtained by Newmeyer and Forbca ( IYXX). and Richardson 1’1rrl. ( 1988). Theae investigators e\tahlished that initial binding requires a nuclear targctThe ing sequence. but is not energy dependent.

Nucleocytoplasmic

translocation process, which is initiated by signal binding, is ATP and temperature dependent. The results reported by Dworetzky et al. (1988), indicate that translocation is a gated, rather than an all-or-none process. Variations in the functional size of the transport channel and the rates of exchange appear to be related to the quantity and effectiveness of the targeting signals. This suggests that multiple signal receptors are present at the pore surface and that the translocation process can be modulated by the number of binding events occurring at a given time. The number of simultaneous events would be dependent, in turn, on both the number of signals and their binding affinity for the receptor. Akey and Goldfarb (1989) visualized the transport of nucleoplasmin-coated gold through the pores of Xrnopus oocytes using cryo-electron microscopy. They suggested that translocation is a multistep process, that culminates in docking to sites located at the centers of the pores, followed by dilation of the central channel. It was also found that the degree of dilation varied in proportion to

Fig. 7. A composite, computer averaged image of nuclear pore complexes that contain nucleoplasmin-coated gold particles in the process of translocation. In this illustration the gold (G) appears white. T, transporter; R, ring structures; outer (OS) and inner (IS) spoke domains; VS. vertical supports. Reproduced with permission of the The Rockefeller University Press. Courtesy of Dr C. W. Akey.

Transport

83

the diameter of the transported gold particles (Fig. 5). The ability to match the size of the channel to the dimensions of the targeted molecule could be an important factor in maintaining pore selectivity during transport by limiting the fortuitous exchange of non-targeted materials. Cellular proteins that bind specifically with nuclear targeting signals, and which might represent transport receptors, have been identified in hepatocytes. Adam rt al. (1989), identified two such proteins with molecular masses of 60 and 70 kDa that bind to the SV40 signal. Yamasaki et ul. (1989) analyzed binding to several different targeting signals and obtained evidence for four putative receptor proteins (140, 100, 70 and 55 kDa). Binding proteins were found to be present in cytoplasand nuclear envelope fractions, mic, nuclear suggesting that they might function by initially complexing with signal-containing polypeptides and subsequently bind to other receptors located at the pore surface. A general lack of data concerning the composition and molecular organization of the pore complex has impeded progress toward understanding the mechanisms of translocation; however. some important information on the area is available. Several laboratories have identified a novel class of glycoproteins localized within the pore complexes that contain O-linked N-acetylglucosamine (Davis and Blobel, 1986; Finlay et al., 1987; Park et al., 1987; Snow ef al., 1987). At least eight such proteins have been reported and they each appear to be present in multiple copies (2-8 per pore). Transport through the pores is inhibited by wheat germ agglutinin that binds to N-acetylglucosamine (Finlay et ul., 1987; Yoneda et al.. 1987; Dabauvalle et al., 1988; Newmeyer and Forbes. 1988). or monoclonal antibodies that recognize the carbohydrate-containing epitope in these glycoproteins (Featherstone et al., 1988). Interestingly, blocking the carbohydrate moieties does not effect either binding of signal-containing proteins or passive diffusion, arguing that the glycoproteins function in the translocation step. Berrios et ul. (1983) and Berrios and Fisher (1986) identified and characterized a myosin-like ATPase that is present in nuclear envelope fractions obtained from several different cell types.

X4

C

M. Fcldhcrr

Recently, Berrios et ul. (1988) localized this protein to the pore complex. Considering the involvement of similar proteins in contractile processes, it is conceivable that the myosin-like ATPase functions in dilation of the central channel during transport. Consistent with this hypothesis, Schindler and .liang (1986) reported that anti-actin and antimyosin antibodies prevent ATP enhancement of dextran uptake into isolated liver nuclei. It would be of interest to determine if molecules containing targeting sequences are similarly affected. Despite the recent progress, our understanding of the composition of the pores is still at a very rudimentary level. This is illustrated by calculations made by Snow r’t al. (1987), who estimated the combined mass of the O-linked pore glycoproteins to be approximately 3.5 x IO” Da. whereas the mass of the pore complex is thought to be 25 100 x 10”Da. Thus. it appears that the great majority of pore proteins have yet to be identified. characterized and localized to specific structural elements.

.-ld,,Oll IlY~,Lyrlrl~!lr.s The author would hkc to thank Dr. Ailene Feldhcrr for her help in preparing the manuscript. The ccl1 cycle studies ~CI-C‘ supported by Grant DCB X71 I??0 from the National Science Foundation

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