tubulin-specific antibody and the expression of microtubules in 3t3 cells after attachment to a...

Experimental Cell Research 10.3 (1976) 331-340

TUBULIN-SPECIFIC ANTIBODY AND THE EXPRESSION OF

MICROTUBULES IN 3T3 CELLS AFTER

ATTACHMENT TO A SUBSTRATUM

Further Evidence for the Polar Growth of CytopEasmic Microtubules in vivo

MARY OSBORN and K. WEBER

Max Planck Znstitutfiir biophysikalische Chemie, GGttingen, West Germany

SUMMARY

Mouse 3T3 cells were allowed to attach to and spread on glass. The expression of cytoplasmic microtubules during the respreading process was monitored by immunofluorescence microscopy using monospecific antibody against tubulin. During radial attachment of the cells a ring of flattened cytoplasm is seen around the nucleus. Cytoplasmic microtubules then enter this spreading ring from the perinuclear region and elongate toward the plasma membrane. At later times microtubules appear perpendicular to the plasma membrane and seem to be in intimate contact with it giving the impression that they “stretch” the cytoplasm. When the cells assume their typical fibroblastic shape numerous microtubules arc seen. They traverse the cytoplasm. Some come close to the plasma membrane and some bend to conform to the shape of the cell. Changes in microtubular organization correlate well with changes in cell shape. These results together with our previous observations on the assembly of cytoplasmic microtubules upon recovery from colcemid treatment suggest that microtubules may grow as polar structures from a microtubular organizing center towards the plasma membrane. The hypothesis that cytoplasmic microtubules might confer polarity on the cell is discussed.

Electron microscopic studies of cells pro- vide evidence for a cytocenter around which other cellular elements are roughly radially arranged. The cytocenter is a cytoplasmic structure housing the centrioles and associated structures, and is usually found near the nuclear membrane. In thin sec- tions one can often find numerous microtubules radiating from this focal point of organization (see e.g., [ 11). We have shown previously that indirect immunofluorescence microscopy with antibody against tubulin allows the direct visualization of cytoplasmic microtubules in a variety of tis- sue culture cells [2. 31. By using a mono-

specific antibody against tubulin. we have provided evidence for a microtubular organising structure in mouse 3T3 cells during interphase. This structure probably corresponds to a cilium close to the cytocenter [4]. When 3T3 cells are treated with mitotic drugs, or are exposed to low temperature. cytoplasmic microtubules depolymerize. During recovery from either colcemid or colchicine treatment, regrowth of cytoplasmic microtubules appeared to occur as a unidirectional process from the microtubular organising structure (cytocenter) towards the plasma membrane [4].

Treatment with trypsin-EDTA solution

332 Osborn and Weber

is commonly used to propagate cells in tis- vested by centrifugation and allowed to reattach to

sue culture, since it causes cells to round up glass coverslips in fresh medium. All operations were carried out at 37°C except for the centrifugation which

and detach from the substratum. After re- was performed at room temperature. At various times

attachment, cells spread out and resump- after seeding, coverslips were removed and processed for immunofluorescence microscoov. as described be-

tion of their typical morphology involves a low. reassembly and reorganisation of their sub- cellular filamentous systems, i.e. micro-

Indirect immunojh~orescence . tubules, microfilaments and 100 A filaments m’croscopy (see e.g. [.5, 61). Here we report studies on

The coverslips were processed for indirect immunofluorescence microscopy, as described in detail previ-

the display of cytoplasmic microtubules ously [2, lo]. Briefly the-procedure involves fixation of

during attachment and respreading. Mouse the cells in 3.7 % formaldehyde in phosphate-buffered saline (PBS) for 8-10 min. The coverslips were then

3T3 cells were studied during respreading treated with prechilled methanol and acetone at -20°C

on glass coverslips by immunofluorescence for 5 and 1 min, respectively. After air-drying, rabbit antitubulin antibody was spread on the cells. After

microscopy using tubulin-specific anti- 45 min at 37°C the coverslips were carefully washed

body. With the onset of “radial attach- with PBS and then treated for 30 min at 37°C with fluorescein-labelled goat anti-rabbit y-globulins. After

ment” , microtubules spread from the peri- careful washes in PBS the coverslips were mounted in

nuclear region into the ring of flattened Elvanol and viewed in a Zeiss fluorescence microscope using epi-illumination and oil immersion objec-

cytoplasm. These microtubules seem to tives at 40 and 63 power. elongate towards the plasma membrane and at certain stages appear perpendicular to it. During later stages of cell spreading, microtubules seem to be in intimate contact with the plasma membrane, and seem to “stretch’ the cytoplasm. Changes in the patterns of cytoplasmic microtubules seem to correlate well with changes in cell shape.

Others have shown that in vitro poly- merization of microtubules occurs pre- ferentially as a unidirectional process [7-91. Thus, assembly of microtubules may be a polar process both in vitro and in vivo. The in vivo results presented here and previously [4] suggest that the cell might use cytoplasmic microtubules to determine direction within the cytoplasm.

MATERIALS AND METHODS

Cell cultures 3T3 cells were from Dr Claudio Basilica, New York University, and were grown in Dulbecco’s modified Eagle’s medium, with 10% calf serum. Plates of non- confluent cells (8.5 cm 0) were washed once with 2 ml trypsin-EDTA solution (0.05 % trypsin (GlBCo), 5X 10ml M ethylenedinitriloacetic acid in phosphate- buffered saline (PBS) [lo]) and allowed to round up in a further 2 ml of the same solution. Cells were har-

Monospecific tubulin antibody The monospecific rabbit antibody against tubulin has been described previously [3, 111. The antigen was homogeneous 6S tubulin from pig brain. The purified y-globulin fraction of the rabbit serum was subjected to afftnity chromatography on tubulin covalently coupled to Sepharose 4B. The monospecific tubulin antibodv was eluted from the column bv PH 2.7 glycinecHC1 buffer [ 111. The fluorescein labelfed goat anti rabbit Y-elobulin was obtained from Miles Co.

The specificity of the antibody against tubulin has been documented in detail previously:

(I) It decorates specifically the following tubulin- containing structures; cytoplasmic microtubules, mitotic spindles and vinblastin-induced paracrystals r2, 3, 101.

(2) The expression of the intracellular structures which can be decorated by the antibody is sensitive to colchicine, or colcemid, and to low temperature [2, 3941.

(3) An antibody with the same properties has been independently prepared and described by Fuller and co-workers [12, 131.

(4) The antibody can be made monospecific by affinitv chromatograohv on tubulin covalentlv bound to Sepharose 4B [i 1,‘14].

(5) The monospecific antibodv reacts specifically with tubulin in i&mnodiffusion~and immunoelectrd- phoresis [2, 10, 141.

Monospecitic antibody was used in the studies presented in this paper.

Nuclear fluorescence Our monospecific antibody against tubulin does not stain the nuclei of interphase 3T3 cells (see e.g. figs 16 and 17) in contrast to our first antibody prepara-

E.~I Cell Res 103 f 19761

tion. The first antibody preparation was not used as a monospecific antibody and stained the nuclei of 3T3 but not of other cells [2]. It is necessary, however, to draw attention to the fact that Brinkley and co- workers. using a similar independently prepared antibody against tubulin found some nuclear staining in several cell lines [13]. Since the nuclear staining. whenever it occurred, was found to be colcemid resistant [2, 131, its nature and importance is still un- clear and more work is necessary to establish why some antibody preparations give rise to it. The pattern and organization of cytopiasmic microtubules was found to be identical with all antibodies so far used [2,4. 13j.

In the current work the centers of cells at early times after respreading appear bright. This is a photographic problem due to the momholorrv of the cells and should be clearly distinguished fr&tt the nuclear fluorescence discussed above. The cells in figs 2-10 are rounded up, and contain in the center a complex array of microtubules which on focusing through the cell can be seen to be in different focal planes. Thus, when one focuses on microtubules in the periphery of the cell the center of the cell is overexposed, and appears bright in the photographs.

RESULTS

Respreading of blhole cells

Non-confluent mouse 3T3 cells were treated with trypsin-EDTA solution and harvested by centrifugation. The cells were resuspended and ailowed to attach to the surface of glass coverslips in fresh medium. At various times after seeding coverslips were removed and subjected to indirect im- munofhiorescence microscopy using monospecific anti-tubulin antibody. The reasons for considering the fluorescent structures to be microtubules have been summarized above (see Materials and Methods) and described in detail in previous publications [2, 4, 10, II].

Shortly after attachment to the substratum the ceils are small, rounded up and brightly fluorescent (e.g. fig. 1). At this stage although individual microtubules can be distinguished by focusing through the plane of the cell it is impossible to document the display of microtubules. With the onset of “radial attachment” a ring of ‘flattened cytoplasm” spreads around the cells. This

ring is at first devoid of microtubules (fig. 2), but at slightly later times a few microtubules appear to spin off from the complex array of microtubules visible in the interior of the cell and to enter the ring of cytoplasm which is spreading around the cell (figs 3-5 j. During this stage the space adjacent to the plasma membrane is still free of microtubules. ahhough the microtubules entering into this space often bend very strongly and seem to reach for the plasma membrane (figs 3-5). With further spreading of the cytoplasm the microtubules reach the plasma membrane. At this stage cells with numerous microtubules nearly perpendicuiar to the plasma membrane are seen (figs 5- 10): as well as cells in which microtubules run for long distances parallel to the membrane (figs 8, IO). Fig. 5 is of particular interest because it shows an asymmetric cell with an asymmetric distribution of microtubules. When the cells star1 iosing their round shape, and assume an individrrah morphology by sending out cellular processes. numerous microtubules are seen in these processes. Almost all cell extensions show microtubules (see figs 7-14). aithough very occasionally cells with processes free of microtubules can be detected (fig. 15).

In the interior of cells shown in figs l-12 microtubules can be visuaiized by focusing through the cell, but are difficult to photo- graph, because the cells are rounded up and the microtubules are crowded, arranged in a complex array and not in the same focal plane. As the cells spread out microtubules in the perinuclear space and in the periphery can be photographed in the same cell because they lie in approximately the same plane (see figs 13, 14). At this stage a fluorescent structure from which many of the cytoplasmic microtubules appear t.o radiate can be distinguished by ditfferential focusing through the cell (fig. 16, arrow,


see also [4]). In these cells the nucleus is not fluorescent (figs 13-16; and see Materials and Methods).

Some support for the assumption that 3T3 cells progress during the respreading process through the stages shown in figs l-14 is given in table 1. Here the cells have been classified in one of four classes at various times after reattachment. The per- centages found for each class at different times suggests that cells progress from class 1 to class 4, i.e. from a rounded up stage to a stage where microtubules are perpendicular to the membrane, and finally to the later stages shown in figs 9-14. Thus far only growing cells have been used for the respreading studies, and it is not known if the patterns obtained are influenced by the cell cycle.

Fully respread cells When cells assume their typical fibroblastic shape (figs 14, 16, 17), numerous cytoplasmic microtubules are seen. They traverse the cytoplasm. Some stop at the plasma membrane and some bend to conform to the shape of the cells. Single microtubules seem to originate in the perinuclear area and some can be traced for long distances (-20 pm). As in our previous study [4] microtubules are not seen originating at the plasma membrane, or freely in the cytoplasm, indicating a requirement for a nu- cleating center.

Respreading of encrcleated cells When 3T3 cells are enucleated with cyto- chalasin B (CB), allowed to recover, and then treated with trypsin-EDTA solution and allowed to reattach, the process of respreading of the enucleates appears to be very similar to that of the normal cell. Again a ring of flattened cytoplasm devoid of microtubules is observed at early times. The

photographed 18 h afterreplating. As shown’ in table 1 at any given time cells are distributed in different classes with respect to their microtubular pattern. The order in which the figures have been placed (figs 1-14) is derived in part from the time at which the cells were fixed, and in part from data for a particular experiment similar to that presented in table 1. Note in figs 2, 3 Gxnnvs) the ring of flattened cytoplasm apparently empty of microtubules, in fig. 3 the microtubules spinning off into the periphery. In figs 6, 7 the microtubules appear to terminate very close to the cell membrane and are perpendicular to it. In the later figures the microtubules bend to conform to the plasma membrane. Most cell processes appear to contain microtubules (see, however, fig. 15). Figs 1-14, approx. x600.

Exp Cell Rer 103 (1976)

organizing structure is maintained in the majority of enucleated cells. In fully respread enucleates the microtubules radiate from the center of the enucleate toward the plasma membrane.

DISCUSSION

The assembly process of cytoplasmic microtubules during the spreading of 3T3 cells after replating has been followed by immunofluorescence microscopy using monospecific tubulin antibody. As the cells spread out microtubules can be visualized coming from the perinuclear area. Figs 1-14 as well as the data shown in table 1 suggest that during respreading microtubules appear to elongate in a unidirectional manner from the perinuclear region toward the plasma membrane. The same direction of assembly was identified previously for the regrowth of microtubules after recovery of 3T3 cells from the influence of microtubular depolymerizing treatments e.g. treatment with mitotic drugs (colcemid or colchicine) or exposure to low temperature

Figs 1-U. 3T3 cells were treated with trypsin-EDTA solution, and allowed to reattach to glass coverslips. At the times after replating indicated below cells were fixed and subjected to immunofluorescence microscopy using monospecific antibody against tubulin (see Materials and Methods). Fig. 1, 30 min;fig. 6, 60 min;figs 2-5, 7, 8, 90 .figs

min; 10-13, 120 min. Fia. 14 is a fullv resnread cell

Polar growth of microtubukes in373 cells 335


Exp Ceil Res 103 (1976)

Polar growth of microtubules in X3 cells 337

Fig. 15. Microtubular profile in cell fixed 90 min after replating. Note that some cellular processes appear to contain no microtubules (arrows). X 1000.

[4]. Thus, under a variety of different experimental conditions cytoplasmic microtubules was found to grow in a preferred direction in the cell, i.e.from the cytocenter toward the plasma membrane.

We have not been able to document the microtubular array in cells prior to attachment to the substratum. It is also difficult to document, with immunofluorescence microscopy at early times after attachment, both the microtubular array and the cytocenter as origin of assembly. This is in part due to the round form of the cells and the concomitant difficulties of focusing. Thus, our results do not distinguish between assembly of new microtubules from an organising structure or de novo elongation of pre-existing shorter microtubules (or a com-

bination of both processes). However, e tron microscopic studies which have shown few microtubules in rounded UI? cells early times after replating, would favour t first possibility [6] ~

Our results show that during replating there is a stage during which cytoplasmic microtubules appear to be aligned almost perpendicular to the spreading plasma membrane (figs 6-9). At this stage some tubules appear to terminate at, or very close to, the membrane, although distances of less than l-2000 A would not be resolved in the light microscope. The figures s cells in which individual microt~b~l~s appear to be bent. These bends are partic- ularly noticeable in cells at earl after respreading (e.g. figs 3, S),

Elvp Cd Res 103 !I3761


Figs 16, 17. Microtubular profiles of fully respread probably corresponds to a cilium close to the cyto- interphase 3T3 cells (24 h after replating). The cylin- center (for details see [4]). x930. drical structure indicated by an arrow in fig. 16

Polargrowdl of microtubules in3E cd!s 339

Table 1. Percentage of cells showiilg four typical patterns of microtrrbde organization as a function of time after replating

Time after 5% of cells in classes replating (min) 1 2 3 4

30 71 21 8 0 60 24 59 :; 0 90 Ii 46 5

I?0 2 15 45 39 180 0 1 1’ L 86

3T3 cells were allowed to attach to glass covet-slips. 4t various times after replating (30. 60, 90, 120 and 180 mitt), coverslips were removed and processed for immunofluorescence microscopy using tubulin specific antibody. At each time point 150 cells were screened and the cells divided into four classes on the basis of their microtubular organization. These classes are

- defined as follo\vs: Class i, cells round and brightlv fluorescent through- out; individual microtubul& not readily visible &I focusing through the cell (e.g. fig. 1). Class 2, cells round with a complex array of microtubules in center; cytoplasm close to plasma membrane appears empty of microtubules (figs 2, 3). Class 3. cells round but microtubules have reached the plasma membrane and are often perpendicular to it (figs 4-7). Class 4. cell has begun to put out processes and to assume an irregular outline; some microtubules appear not to end at the cell membrane but bend and conform to it (figs 9, IO, 11). Also includes cells which are no longer radtally symmetric (figs 13, 14).

also be seen occasionally in fully respread cells (e.g. fig. 17 and [ 131).

It is generally agreed that microtubules are actively involved in the development and maintenance of cell shape, although it is not really determined whether they cause the shape change, or follow passively the morphological changes in cell shape (see e.g. [lS, 161). Our results show that in 3T3 cells changes in cell shape are well cor- related with changes in microtubular distribution. Almost all cell processes contain microtubules. Mowever. the existence of occasional ceils which have some processes devoid of microtubules (such as that shown in fig. 15) might favour the second pos-

sibility, i.e. that microtubules foilow- changes in cell shape rather than induce them. However, spreading of cells induces organization not only of microtubules but also of the submembranous microfila- mentous bundles. which contain actin, myosin, tropomyosin and cy-actinin [17, 1.8, 191. Shtdies with antibodies to actin and myosin ([19] and our unpublished resultsl show that the microfilament system is also laid down shortly after replating. Clearly it would be desirable to follow in parallel .he organization of both microfilaments and microtubules in the same cell.

The apparently unidirectional growth of microtubules in vivo opens the question of the number and location of growing points for a cytoplasmic microtubule. Since the tubule is a polar structure one prefers rhe assumption of only one growing point [16], however, the possibility of two growing points cannot be dismissed. In vitro poly- merization studies have indicated that unidirectional growth is highly preferred under a variety of conditions. but at high con- centrations of tubulin bidirectional growth can be observed [‘i, 8. 91. Since we do not know if the free tubulin in the ce!l is corn- partmentalized and where such compart- mentalization occurs. it is not possible at present to decide where the growing poin; is localized.

Microtubules are actively involved in rhe development and maintenance of asym- metry [ 15, 161, The very nature of a tubule means the setting up of an axis. Since iso- lated tubules have a helicai structure ,with a defined ‘handedness’ the axis of :he tubule should have a fixed polarity IX. 2 i], Thus, the direction in which a tubule grows in vivo should be preserved in the final polarity of the finished structure [lS]. Visu- alization of the complex pattern of cytoplasmic microtubules in interphase 3T3


cells by immunofluorescence microscopy does not reveal this polarity directly. How- ever, under a variety of experimental conditions cytoplasmic microtubules in 3T3 cells appear to grow from the cytocenter towards the plasma membrane. Thus, the microtubules could indeed act as cytoplasmic direction markers indicating the cytocenter in one direction and the plasma membrane in the other. Furthermore, at least the majority of microtubules mark the cytocenter as the origin of the cytoplasmic polarity given by the microtubules. Thus, it remains to be seen if the 3T3 cell makes molecular use of the polarity system set up by its cytoplasmic microtubules, i.e. does it use microtubules to guide intracellular movement or transport of material in a unidirectional manner?

We thank T. Born and A. Hoech for photographic assistance. The expert technical assistance of H. J. Koitzsch is greatly appreciated.

REFERENCES 1. Porter, K R & Bonneville, M A, Fine structure of

cells and tissues, p. 17. Lea & Febiger, Philadelphia, PA (1973).

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2. Weber, K, Pollack, R E & Bibring, T, Proc natl Received May 6, 1976 acad sci US 72 (1975) 459. Accepted July 16, 1976

tubulin-specific antibody and the expression of microtubules in 3t3 cells after attachment to a...

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