NEURONAL MOTILITY: THE ULTRASTRUCTURE OF VEILS AND … · 2005. 8. 25. · VEILS AND MICROSPIKES...

J. Cell Sci. 61, 389-411 (1983) 389Printed in Great Britain © The Company of Biologists Limited 1983

NEURONAL MOTILITY: THE ULTRASTRUCTURE OF

VEILS AND MICROSPIKES CORRELATES WITH

THEIR MOTILE ACTIVITIES

K. W. TOSNEY* AND N. K. WESSELLSDepartment of Biological Sciences, Stanford University, Stanford, CA 94305 andMCDB, University of Colorado, Boulder, CO 80309, U.SA.

SUMMARY

We have documented the ultrastructural characteristics that correlate with protrusion, adhesionand retraction of neuronal veils and microspikes, by filming individual neurons of the chick ciliaryganglion and examining the same cells with high-voltage electron microscopy.

We find that new veils invariably contain only a cortical meshwork of filaments and are devoidof microtubules, groups of vesicles and other organelles. At sites of recent veil retraction a corticalmeshwork on the substratum side underlies a filament-free space containing vesicle clusters and acomplexly folded upper membrane. Areas without veil activity are smooth-surfaced and contain athree-dimensional lattice of filaments. We discuss the implications of these observations for themechanisms of surface recruitment and retrieval during motile activity.

We also find that the ultrastructure of moving and attached extensions of the cell surface differsdramatically. Unattached microspikes and actively extending veils have an open, criss-cross arrayof filaments, whereas attached microspikes contain more aligned filaments, which extend as a smallbundle into the growth cone. These results suggest that cell surface protrusion is mediated bymeshworks of loosely packed filaments. More compact bundles of filaments are probably generatedonly at adhesion points.

INTRODUCTION

In order to understand the intracellular basis of motility, we must comprehend howprotrusion and retraction of cellular processes are controlled as well as ways in whichthe basic motile machinery is adapted to fit the specific requirements of particular celltypes. The motile style of the neuron, the subject of this study, is a case in point. Inneurons, the activities of protrusion and retraction are spatially restricted to the'growth cone' at the tip of the neurite. The soma and sides of the neurite have a latentcapacity to produce protrusions, but unlike the temporarily inactive sides of movingfibroblasts, they normally remain quiescent (Bray, Thomas & Shaw, 1978; Wessells& Nuttall, 1978).

Despite this regionalization, there are no compelling reasons for believing thatneurons use essentially different cellular mechanisms for motility. In order to moveforward the growth cone, like the fibroblast, must rapidly recruit and retrieve cellsurface, mobilize the elements that generate force and stabilize the forms of cellularprocesses, and make adhesions to the substratum. This study asks what distributions

•Author for offprint requests at: Biological Sciences Group, University of Connecticut, Storrs,CT, 06268, U.S.A.

390 K. W. Tosney and N. K. Wessells

of cytoplasmic elements characteristically accompany the events of protrusion, retrac-tion and adhesion. Two issues are of particular interest: how are cytoskeletal elementsarranged within processes that are engaged in different activities, and how is cellsurface recruited and retrieved during protrusive activity?

Ultrastructural descriptions of neurons show that microfilaments, arranged asmeshworks or bundles, are invariably associated with cellular processes, while areasthat remain quiescent with regard to motility contain microtubules and membrane-bound organelles, as well as microfilaments (Bunge, 1973; Bray & Bunge, 1973;Luduena& Wessells, 1973; Spooner e£ a/. 1973; Spooner, Yamada & Wessells, 1971;Yamada, Spooner & Wessells, 1970, 1971). Neurons do not have stress fibres, thelarge bundles of microfilaments that terminate at the plasma membrane at adhesionpoints. It has been reported, however, that all neuronal microspikes contain alignedarrangements of microfilaments, similar to but smaller than stress fibres (Kuczmarski& Rosenbaum, 1979; Spooner et al. 1973; Wessells, Spooner & Luduena, 1973).Whether filament bundles are necessary for the protrusion of processes (as suggestedby Goldman & Knipe, 1972; Kuczmarski & Rosenbaum, 1979) or whether meshworkarrays mediate cell protrusion and bundles are generated only at adhesion points (assuggested by Buckley & Porter, 1967, 1975; Buckley & Raju, 1976; Heath & Dunn,1978; and others) remains unclear, and demands a careful analysis of the filamentarrangements that correlate with cellular protrusion and adhesion.

A second issue in neuronal motility is the recruitment of cell surface for neuriteelongation and for cellular protrusion. A large net increase in surface area occurs whenneurites elongate. Based on neurite elongation rates of 40— 100|Um/h (Bray, 1970,1973a; Luduena, 1973), as much as 3|im2 of surface may be added per minute.Although the location and mode of surface addition is not known for fibroblasts, inneurons there is convincing evidence that addition occurs predominantly at thegrowth cone (Bray, 1970) and is mediated by fusion of internal vesicles transportedfrom the soma (Pfenninger & Bunge, 1974; Pfenninger, 1980).

Cell surface protrusion also requires a local increase in surface. Since a microspikeor veil 1 ,um2 can appear in 60 s (Wessells & Nuttall, 1978), calculations show that localrates of increase are 0-5-2-0 jum/min, figures suggestively similar to the calculated netrate of increase. In this respect, Bray (1973a) has suggested that cellular processeselongate because surface is added at their tips. This hypothesis implies that cellularprotrusion is obligatorily coupled to the addition of local vesicles. Alternatively,membrane could be added at diverse points on the growth cone, and recruited fromone part of the growth cone to another for either elongation or protrusion (cf. Trin-kaus, 1980).

The present study was designed to correlate the local motile activity of growthcones from chick ciliary ganglion neurons with the local ultrastructure. We askedwhether regions of the growth cone caught in the act of protrusion differed consis-tently in ultrastructure from areas that were retracting, and whether movingprocesses differed from adhering processes. To detect order in the intracellular fibresystems without laboriously reconstructing cells from thin sections, we viewed wholeneurons with high-voltage electron microscopy (HVEM). This technique can give

dnemicrography and HVEM of neuronal motility 391

detailed, three-dimensional images of portions of the cell that are 1 ^m or less inthickness, with less damage from beam impingement that conventional transmissionelectron microscopy (TEM; Buckley, 1974; Buckley & Porter, 1975). The methodnecessitated culturing, filming and fixing the neurons directly on Formvar-coatedgrids, and viewing them as whole mounts.

When cells are viewed as whole mounts with HVEM or TEM, the filaments thatinterconnect microtubules, intermediate filaments and vescicles are resolved as a fineanastomosing network of microtrabeculae (Buckley, 1974; Buckley & Porter, 1975).Individual trabeculae vary in diameter from about 30—100 A, and probably corres-pond to microfilaments, microtubule-associated proteins, and other filamentous ele-ments (Ellisman & Porter, 1980). Evidence that microtrabeculae are one real imageof the cytoplasm comes from the work of Porter and his colleagues (Byers & Porter,1977; Ellisman & Porter, 1980; Luby & Porter, 1980; Stearns, 1982; Wolosewick &Porter, 1977), which shows that differences in the state of lattice elements as well astransitions in lattice arrangements correlate with cellular physiology and are,therefore, significant for the function of cells. Since microtrabeculae do not appearto have a single chemical identity, we will refer to them by the descriptive term,filaments.

A common aspect of trabeculae is their irregular contours and the appearance offusion where they intersect one another. Heuser & Kischner (1980) have providedevidence that this image is the result of agglutination and condensation of solublecytoplasmic proteins on the filaments during processing. In addition, Ris (1980) hasshown that the rounded contours of trabecular intersections result from waterretention during critical-point drying; when all water is extracted, the lattice appearsas meshworks or bundles of filaments. In these studies, neither fixation nor waterretention artifactually transformed meshwork arrangements of filaments into bund-les.

MATERIALS AND METHODS

Preparation of substrataCleaned gold grids were dipped in a solution of 1 % (v/v) rubber cement in xylene, dried,

attached to films made from 028 % Formvar dissolved in ethelene dichloride, picked up on cleanedflamed coverslips, and lightly carbon-coated to stabilize the film. To render the Formvar properlyadhesive for axon outgrowth, coated grids were incubated overnight in poly-DL-ornithine in 0-15M-borate buffer (pH 8-3), followed by 1-8 h in acetic acid solubilized collagen and 12-36 h in heart-conditioned medium (HCM). The HCM was prepared by culturing confluent monolayers of 8- to9-day-old chick embryo heart ventricle cells in modified medium based on Ham's F-12 (see Helfand,Smith & Wessells, 1976).

Preparation of neuronsCiliary ganglia were dissected from 8- to 9-day-old chick embryos and dissociated into single cells

with trypsin in modified Puck's glucose. Since trypsin may affect the cytoskeletal organization ofcells (cf. Furcht & Wendelschafer-Crabb, 1978), the neurons were allowed to recover from dissocia-tion for 12-72 h in Petri dishes containing 2 ml of HCM at 37 °C in a humidified atmosphere of 5 %CO2 and 95 % air. Neurons survive at least 4 days in such suspension cultures, but do not attachor extend axons, as the substratum is not adhesive enough (see Collins, 1978).


dnemicrographyPrepared grids were introduced into suspension cultures of ciliary ganglion cells, incubated for

5-10 min while neurons attached, and removed to a coverslip-bottomed filming dish containing 2 mlof HCM. A well-attached neuron was selected and filmed at 6-10 frames/min at a final filmmagnification of 80-320X for a median time of 25 min before fixation. Temperature was maintainedat 37 °C with an air-curtain incubator, and pH remained constant over the short term used. Opticswere suboptimal, since the cell lay above a Formvar film, and a variable amount of mediumremained between the grid and the coverslip.

Choice of fixativeIn preliminary trials, we selected one fixation scheme from 30 tried, based on the criteria that it

had preserved regional differences in filamentous arrangements better than other fixatives andminimized surface blistering. We also found that quick and total immersion in primary fixative isessential. If the fluid in the filming chamber is lowered and fixative added, or if 25 % glutaraldehydeis added dropwise to a final concentration of 2%, the cells blister badly. In addition, fixation isslower with these methods, and the cell processes continue to move for some minutes, as noted byBunge (1973). Since it is possible that cells undergoing such slow fixation alter their motilebehaviour or undergo ultrastructural changes, we adopted the immersion methods, even though thefinal 6-12s of motile activity were unrecorded due to the transfer. Since extension and retractiongenerally occur over a longer time course, we felt the unrecorded activity to be a smaller disadvan-tage.

Fixation and preparation for HVEM viewingGrids were transferred from the filming dish to a dish containing 2 ml of 2% glutaraldehyde in

0-1 M-cacodylate buffer at 22°C where they remained for IS min before being rinsed in 0-1 M-cacodylate. The cells were filmed for a dozen frames in this buffer to determine sites of blistering.New bulbous excresences in post-fixation frames were blisters. The cells were then post-fixed for10 min in 1 % osmium tetroxide in 0 ' 1 M Sorensen's phosphate buffer at 0°C. This treatmentminimizes the osmium-induced destruction of actin filaments (Maupin-Szamier & Pollard, 1978).The cells were rinsed in 0-1 M Sorensen's buffer and in distilled water, stained for 1-2 min in 0-5 %uranyl acetate in 0-2M-collidine buffer, rinsed in 0-2M-collidine buffer and in distilled water,dehydrated rapidly in a graded acetone series, and critical point-dried from CO2 . Grids were storedin sealed containers with Drierite until they could be viewed. The films were projected and studiedto determine which cells showed interesting motile activity in the last minutes before fixation, and20 of the 101 cells filmed were selected for detailed analysis with HVEM. This gave 14 sequencesof extending veils, 17 of retracting veils, 16 of quiescent growth cones, 5 of varicosity formation, and4 of complexly fasciculated neurons. Twenty other cells studied with TEM support the HVEMobservations.

RESULTS

Veil protrusion

The protrusion of a small veil in the last 20 s before fixation is shown in prints andtracings from individual movie frames in Fig. lg—h. Stereo HVEM micrographs ofthe same veil (Fig. 2) show that the newly protruded area contains a nearly planarmeshwork of filaments that lies within and unites the cortical regions between theupper and lower membranes. Microtubules and membrane-bound organelles are onlyrarely found and vesicle clusters are invariably absent. All of the 14 veils examined inHVEM that had protruded outward within the last 2 min prior to fixation and hadremained fully extended show these ultrastructural features, whether they extend

Cinemicrography and HVEM ofneuronal motility 393

upwards into the medium or along the substratum. Veils lying close to the substratummay contain bundles of filaments; such bundles always extend from attached micro-spikes.

Areas of veil protrusion display the ultrastructural features described above, irres-pective of the motile activity of adjacent areas of the growth cone. For instance (in Fig.3), tracings from single movie frames show a single growth cone with adjacent regionsthat protrude and retract in the first minute of filming, while a neighbouring area, aswell as the base of the growth cone, remains quiescent. As shown in HVEM (Fig. 4),the newly expanded area (A) contains a cortical meshwork of filaments. Areas of veilretraction and quiescence, described below, also retain their ultrastructural charac-teristics regardless of the activity of adjacent regions, suggesting that each ultrastruc-tural profile portrays locally autonomous motile events. This also appears to be thecase in fibroblasts (Abercrombie, Heaysman & Pegrum, 1971; Trinkaus, Betchaku& Kruilikowski, 1971).

Veil retraction

Areas of complete veil retraction, illustrated in Figs 3, 4 and 5, have several charac-teristic features. (1) Strings of vesicles accumulate beneath the upper plasma mem-brane in a region sparse in filaments. These vesicle strings occasionally lie along orabut upon the ends of filaments. We never saw continuity between these vesicles andeither the plasma membrane or membraneous organelles. (2) The upper plasmamembrane is not attached to filaments, bulges upward, and is thrown into complexfolds and ridges. (3) A nearly planar meshwork of filaments, reminiscent of areas ofveil extension, lies in the cortical region adjacent to the lower plasma membrane, andis continuous with the cortical cytoplasm of the rest of the growth cone. Free ends offilaments may lie interior to the cortical meshwork in the regions where veil retractionareas grade into the more space-filling meshworks of the proximal growth cone.

Areas of incomplete veil retraction are defined as sites where a large veil hasdiminished in size just before fixation. These areas display ultrastructural featurescommon to both fully extended and fully retracted veils (Fig. 6). Small groups ofvesicles are present, but clusters are smaller than in completely retracted veils and donot tend to associate in strings. The plasma membrane is often complexly folded, butappears to retain attachments to the cortical lattice. Microtubules and cellular organ-elles are excluded from the veil.

Quiescent regionsQuiescent regions are defined by the absence of veil activity in the last minutes

before fixation. Three types of quiescent regions were often sufficiently thin to beexamined with HVEM: the branch points of neurites (Fig. 7), the trailing edges ofgrowth cones (Figs 4, 8), and quiescent growth cones (Fig. 9). Fig. 1 illustrates threeways in which neurites branch in vitro (see also Bray, 19736; Wessells & Nuttall,1978; Wessells, Johnston & Nuttall, 1978), and also shows assumption of a roundedform and quiescence in the trailing edge of growth cones. Quiescent growth conesshow no veil activity and, in contradistinction to varicosities (below), remain fully

394 K. W, Tosney and N. K. Wessells

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dnemicrography and HVEM ofneuronal motility

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Fig. 2. Veil protrusion: the veil shown in this stereo pair has protruded in the directionof the large arrow within the last 10 s before fixation (Fig. lg-h) . The newly spread areais about 0-1-0-2 fixn thick, and contains a nearly planar meshwork of filaments that is inintimate contact with both upper and lower plasma membranes. Note the absence ofvesicle clusters. Two attached microspikes send bundles of filaments into the newly spreadarea (small arrows). Bundles are associated only with attached microspikes (see Figs12-14). Compare the low relief of this veil with stereo micrographs of retracted veils andquiescent areas (Figs 5, 8). = X 12000. Stereo tilt angle 8°.

Fig. 1. Cinemicrography of neuronal motility. These prints and corresponding tracingsare selected from movie frames taken at 10 s intervals. Time is given at the lower right foreach frame in count-down fashion (m, min), as that remaining before fixation. Frame lawas taken 5 min after the cell soma attached to the substratum. = X 500.

The first four frames illustrate the non-synchronous restriction oiprotrusive activity togrowth cones during axon initiation. In la, two active growth cones (A and B) extend froma quiescent area of the soma, while the opposite side of the soma exhibits extensive veilactivity. This activity resolves in the formation of three growth cones (C, D and E) byframe lc. The trailing edges of the growth cones become quiescent and round up to formneurites, illustrated by E in lb -c and by C in lc-d (see HVEM Fig. 8).

Branch points along neurites are semi-rounded quiescent areas (see HVEM Fig. 7).Three types of neuronal branching occur during the sequence. (1) Broad growth cones Cand E become quiescent at their midlines (lb-c) and form separate growth cones at theiropposite apices. (2) Separate neurites A and E are pulled together to form a branch point(arrowheads, lc-d) as the soma shifts relative to the substratum (large arrow, lb) inresponse to vigorous protrusive activity on the upper right. This rare form of branchinghas not been reported before. (3) Collateral fibres may develop when adherent regionsregain motility and extend. For instance, a small veil, V, stranded along the neurite in Id,regains protrusive activity, as illustrated in the last four frames. The veil broadens in framelh, 10 s before fixation. The same veil protrusion is shown in stereo HVEM Fig. 2.

While several growth cones remain active, two others (A and B) form phase-brightvaricosities, in lb and lc. Neurite A retains tight adhesions to the substratum and thevaricosity moves proximally toward the soma. Neurite B regains protrusive activity in Id,but does not actively advance before fixation. HVEM Fig. 10 shows a typical variscosity.


Figs 3, 4. Motile history and HVEM of a single growth cone.Fig. 3. Tracings from enlarged movie frames illustrate the complex activity of the single

growth cone shown in HVEM Fig. 4. Time before fixation is 30, 20, and 10 s in 3a, 3b,and 3c, respectively. Three areas of the growth cone have distinctly different motilehistories. At A, a veil protrudes in frame 3b and remains extended through fixation. At B,a veil that is fully extended in frames 3a and 3b is shown in frame 3c to be undergoingretraction, 10 s before fixation. Area C has remained quiescent with regard to veil activityfor the last 10 min before fixation. These areas are indicated by the same letters in Fig. 4.« x 600.

Fig. 4. Adjacent areas on the growth cone shown in this HVEM micrograph differedin motile activity immediately prior to fixation, as illustrated in Fig. 3. Each area displaysthe ultrastructure characteristically associated with its motile history, showing that themotile events are locally autonomous. The extended veil at A contains a cortical meshworkof filaments and lacks vesicle clusters, as is typical of new protrusions. The area of recentveil retraction at B has prominent vesicle strings, a sparse cortical meshwork of filaments,and a folded upper membrane (see Fig. 5). The quiescent area, C (also shown in stereoin Fig. 8), displays a three-dimensional meshwork of filaments that grades into the quies-cent base of the growth cone. The marked differences in depth and relief among the threetypes are best appreciated by comparing stereo images (Figs 2, 5, 8). ~ X 17 000.

Figs 5, 6. Veil retraction.Fig. 5. As shown at = X 20 000 in stereo, this area of complete veil retraction has several

characteristic features. (1) Vesicles accumulate beneath the upper plasma membrane in aregion sparse in filaments. Some strings of vesicles align with or abut upon filaments (smallarrows). (2) The upper plasma membrane is not attached to filaments, bulges upward, andis thrown into complex folds (curved arrows). (3) A planar meshwork of filaments,reminiscent of areas of veil protrusion, lies in the cortical region adjacent to the lowerplasma membrane (large arrow). This veil retracted in the last seconds before fixation (Bin Fig. 3). Fig. 14 shows another retracted veil. Stereo tilt angle 10°.

Fig. 6. Areas of incomplete veil retraction, where a veil observed in cine has becomesmaller but is still detectable before fixation, have ultrastructural features intermediatebetween fully extended and fully retracted veils, as shown here in stereo. Small groups ofvesicles are present (short arrow) and the plasma membrane is crenulated (long arrow).Cortical filaments are detectable when the veil is viewed from other angles, butmicrotubules and other organelles are excluded ~ X 20000. Stereo tilt angle 10°.

Figs 7-9. Quiescent areas.Fig. 7. A branch point of a neurite shown here in stereo has a complex three-

dimensional lattice of filaments with embedded microtubules, mitochondria and otherorganelles. Many individual filaments in this area do not show the rounded contours andanastomosis typical of the microtrabecular image of filaments present in other regions ofthe same cell (see Figs 4, 5). The long axes of many of these filaments parallel the outercontours of the cell. = X 12000. Stereo tilt angle 8°.

Fig. 8. This quiescent edge of the growth cone shown in Fig. 4 has been without veilactivity for at least 10 min. Although the edge of the cell is attached to the substratum ina manner similar to veil attachment, the meshwork of filaments is space-filling and in-cludes membraneous organelles (arrow). = X 30000. Stereo tilt angle, 10°.

Fig. 9. Fully flattened growth cones that were devoid of veil activity before fixation,such as the growth cone illustrated here, do not have large strings of vesicles, filament-freespaces or amorphous areas, and the surface is smooth rather than corrugated. The filamen-tous meshwork is not confined to cortical regions, as it is in areas of new protrusion.Smooth endoplasmic reticulum, mitochondria and other organelles lie embedded withinthe three-dimensional lattice. Microtubules extend from the axon and splay out in theproximal portions of the growth cone, which is generally quiescent during axon elongation(see Fig. lb -d) . The last veil activity on this growth cone occurred more than 10 minbefore fixation. = X 10 000.

K. W. Tosney and N. K. Wessells

Figs 5 and 6. For legend see p. 397.

Cinemicrosraphy and HVEM ofneuronal motility 399

, • . . * , . . . . » t

Figs 7-9. For legend see p. 397.


spread upon the substratum. Inactive growth cones may have produced microspikesthat were not detected in the films.

The lattice elements of all three quiescent regions are densely packed, complexlyinterconnected, are not confined to cortical regions, and are accompanied bymicrotubules and cellular organelles (Figs 4, 7-9; see also Ellisman & Porter, 1980).Elements of the lattice close to the plasma membrane may be cross-linked into anetwork in which the majority of the elements follow the outer contour of the cell (Fig.7). Large clusters of vesicles were uncommon, and neither filament-free nor amor-phous cytoplasmic areas were seen. The surface of quiescent regions provides asmooth profile and a noteable absence of folds or surface corrugations.

These ultrastructural features are present even in areas that are partially spread andattached to the substratum via retraction fibres (Fig. 8). Although branch points mayform in different manners, they share a common ultrastructure; there are no dif-ferences that correlate with their manner of formation.

Varicosities

Occasionally, growth cones cease protrusive activity and form a phase-brightvaricosity, also known as a 'retraction bulb' (Fig. lb—c; see also Shaw & Bray, 1977).Varicosities may loose their adhesions and be retracted. If tight adhesions areretained, the varicosity may remain quiescent, may travel proximally along the neurite(Fig. lc), or the neurite may regain protrusive activity at its tip (Fig. Id) and, in somecases, advance along the substratum.

All the varicosities observed with HVEM contained closely packed clusters ofvesicles (Fig. 10). Some varicosities had surface corrugations while others weresmooth-surfaced.

Blisters

Blisters (also called bubbles or mounds) are glutaraldehyde-induced or -alteredstructures that occur in all types of embryonic cells (Shelton & Mowczko, 1977; Hasty& Hay, 1978; Nuttall & Wessells, 1979). We find blisters to be ultrastructurallysimilar to areas of veil retraction in that the surface membrane is detached from theground substance and vesicles usually cluster within the blistered area (Fig. 11).

Microspikes

Although microspikes are often difficult to detect in printed still frames due to thegranularity of the film, they can be seen when the film is projected, since the eye candetect movement better than slight differences in contrast, and can also reduce back-ground noise by averaging. Since we are unable to show the reader either movies orconvincing still frames of moving microspikes, we adopt the classification of attachedand unattached ('moving') microspikes, which can be distinguished in stereo HVEMmicrographs (Fig. 12).

We find a consistent difference in ultrastructure between adhering and unattachedmicrospikes. Attached microspikes are straight, with relatively smooth outer surfaces


10

\ *

Fig. 10. Varicosities. Varicosities at the tip of a neurite contain closely packed vesicles,as shown in this HVEM micrograph. Fig. lb-d illustrates the formation of varicosities,which appear in cin6 as phase-bright bulbs. ~ X 30000.

Fig. 11. Blistering. On neurons, blisters commonly form during fixation at branch points(curved arrows), at the tips (arrows) or bases of microspikes, and on the soma. Typically,as shown in this HVEM stereo pair, a portion of the surface membrane balloons outwardand vesicles may cluster within a filament-free space (small arrow). These neurites lieupon the spread surface of a glial cell. = X 8000. Stereo tilt angle 10°.

and internal bundles of filaments that extend into the growth cone (Figs 12, 13). Themicrospike is attached to the substratum only at its tip, and the adjacent margins ofthe growth cone are concave in outline, suggesting that the microspike is undertension.

In contrast, unattached ('moving') microspikes have convoluted surfaces and werenever observed to extend bundles of filaments into the interior of the growth cone.The microspikes pictured in stereo HVEM (Fig. 14) were detectable in cine and were

K. W. Tosney and N. K. Wessells

dnemicrography and HVEM ofneuronal motility 403

moving in the direction of the large arrow before fixation. These microspikes displaythe corrugated surface common to moving processes. The filaments within and ad-joining such processes are arranged as an open, criss-cross meshwork (Fig. 12).

Fasciculation

We have examined four instances in which neurites from a single soma meet toform complex fasciculations (e.g. Fig. 15; see Wessells et al. 1980, for a recentdiscussion of neuron—neuron contacts in fasciculation). We find that the ultrastruc-ture of growth cones in fascicles is identical to that of growth cones advancing alonga culture substratum. For instance, areas of veil retraction along fasciculated neuritescontain vesicle accumulations (Fig. 16) similar to those seen after veil collapse insingle growth cones. In addition, in cases where fasciculated neurites have becomeseparated, small adhesions that persist between the two are identical to retractionfibres seen between lone neurites and the culture substratum (Fig. 16). Like themicrospikes they superficially resemble, the retraction fibres are thin, elongated,stretch from adhesion points, and form concave outlines where they meet the bulkof the neuron; unlike microspikes, however, they are by definition produced by theretraction of the cell surface from points of adhesion, rather than by active protrusionfollowed by adhesion. These observations on fasciculated neurites rule out thepossibility that the differences in ultrastructure correlating with correspondingmotile events are merely responses of cultured neurons to a biologically unusualsubstratum.

DISCUSSION

The ultrastructural features that are characteristic of different motile activities ingrowth cones are summarized schematically in Fig. 17. We will discuss how theseobservations relate to differences in function subserved by meshwork or bundle ar-rangements of filaments and to the mechanisms of surface recruitment and retrieval.

Figs 12-14. Microspikes.Fig. 12. This small growth cone displayed extensive microspike activity before fixation.

In this stereo HVEM, it can be seen that the unattached ('moving') microspikes have acorrugated surface and do not extend bundles of filaments into the body of the growth cone(small arrows). In contrast, the attached microspikes have relatively smooth surfaces andinternal bundles of filaments that extend into the growth cone (large arrow). The occur-rence of both ultrastructural types of microspikes on the same growth cone suggests thattheir differences are not a global artifact of fixation. = X 5000. Stereo tilt angle 8°.

Fig. 13. The two microspikes shown are attached to the substratum at their tips. Thesides of the microspikes are smooth and the adjacent surfaces of the growth cone areconcave in outline, suggesting that the microspikes are under tension. Bundles of filamentsextend through the cellular process into the body of the growth cone (arrows). « X 20 000.Stereo tilt angle 10°.

Fig. 14. The microspikes pictured in this HVEM stereo pair were moving in thedirection of the arrow before fixation. Note the convoluted surfaces of the microspikes.The lower portion of the figure shows an area of veil retraction. = X 20000. Stereo tiltangle 10°.


16Figs 15-16. Fasiculation.

Fig. 15. These neurites, which all emmanate from the same soma, meet at the lowerright to form a complex fasciculation. The stereo image shows that the neurites may lieupon the substratum and make close side-to-side associations, or may overlap one another.The growth cone at the top, which had enthusiastic veil activity before fixation, containsvesicles clusters typical of veil retraction areas. Elongated, sinuous mitochondria areenmeshed in a three-dimensional filamentous lattice. « X 5000. Stereo tilt angle 4°.

Fig. 16. Retraction fibres between formerly fasciculated neurites appear as thin, elon-gated extensions with little visible filamentous structure (large arrow). Retraction fibresadherent to the substratum have the same morphology (small arrows). Large vesicles (v)mark the site where a veil retracted 60s before fixation. = X 24000.

Cortical meshworks

Our study shows that cortical meshworks of filaments are characteristic of veils andmicrospikes when they are protruding and moving. This lends support to the conceptthat meshwork arrangements of microfilaments provide both the force and the struc-tural elements for the protrusion of cellular processes (see recent reviews by Pollard,

Cinemicrography and HVEM of neuronal motility 405

Microspikesmovingattached

Veil protrusionarea retraction

Fig. 17. Summary of ultrastructural features correlating with different motile activities.This schematic represents a cross-section through a growth cone with a pattern of motileactivity similar to that shown in Fig. 4. The broken line represents the substratum. Movingmicrospikes have corrugated surfaces and a meshwork arrangement of filaments. Incontrast, attached microspikes are smooth-surfaced and extend bundles of filaments intothe growth cone. Quiescent areas have three-dimensional filamentous lattices with em-bedded microtubules and cellular organelles. In veil retraction areas, a cortical meshworkof filaments on the substratum side underlies a filament-free space containing strings ofvesicles and a complexly folded upper membrane. Areas of veil protrusion contain acortical meshwork of filaments that contacts the upper and lower plasma membranes.

1976; Trinkaus, 1980; Johnston & Wessells, 1980). Individual elements in the mesh-work often extend across the process, from upper to lower plasma membrane. At thebase of the process, the filaments are continuous with the cortical filaments in the restof the growth cone. The cellular process can thus be thought of as an extension of thecortical cytoplasm.

How the cortical lattice is mobilized to form the interior of cellular processes as theyprotrude is not understood. It is probable that the existing lattice is structurallyreorganized at the site of protrusion. Filaments in the cortical region of both micro-spikes and veils appear to extend from membrane to membrane, while in other regionsof the cell, the cortical lattice intermeshes with filaments that lie internally. Transitionfrom a smooth surface to a protrusion is, in a sense, a maximizing of filament-membrane contacts, at the expense of these internal filament—filament interactions.These transitions may require alterations in the interactions between actin andmyosin-like proteins (see Pollard, 1976), or alterations in the gel-sol condition of thecytoplasm (Condeelis & Taylor, 1977).

Another model suggests that the material and the force required for protrusion areprovided by the polymerization of new elements. The analogy is to the explosivepolymerization of actin, which forms the acrosomal process in certain spermatozoa(Tilney, 1977). Observations in support of this hypothesis include studies on thepolarity of actin at the leading edge of cells (Small, Isenberg & Celis, 1978) and


reports of amorphous or finely granular material, which may represent polymerizingfilaments, within newly spread areas of non-neuronal cells (Ausprunk & Bergman,1978; Bragina, Vaseliev & Gelfand, 1976) and within veils in neurons (Wessells &Nuttall, 1978).

We did not find large expanses of amorphous material in newly extending veils.Since the veils that we analysed had all attained a size visible in phase microscopy, itis possible that we missed an earlier, transient polymerization event. In support of thisit should be noted that the veils with amorphous material in the report of Wessells &Nuttall (1978) were generally smaller than the fully extended veils studied here.

We cannot exclude the possibility that amorphous material seen in other studies isan artifact of the section angle (the planar cortical meshwork typical of veils andmicrospikes might appear amorphous in thin section) or of the mode of preparation.For instance, studies in which whole mounts were viewed with the conventionalelectron microscope may have been subject either to damage by beam impingement,or to incomplete critical-point drying, which produces a 'homogeneous' or 'glassy'cytoplasm (see Buckley & Porter, 1975). When, in our early preparatory trials, wenoted amorphous areas in thin cytoplasmic extensions between microspikes identicalto those shown by Wessells & Nuttall (1978), other cells in the same preparation oftenhad large expanses of homogeneous-appearing cytoplasm in non-veil areas.

Bundles of filaments

Our study shows that bundles of filaments are associated only with adhering micro-spikes in growth cones, and do not occur in unattached veils or microspikes. Previousreports that all microspikes on neurons contain aligned filament bundles (Spooner etal. 1971; Wessells et al. 1973) probably resulted from sampling error: since theseneurons were sectioned parallel to the substratum, microspikes visible in individualsections would generally have been attached to the substratum. If the unattachedmicrospikes, which extended much deeper into the Epon block than the bulk of thegrowth cone, were examined at all, they would have been cut obliquely or tangenti-ally, so that criss-cross arrangements of filaments would not be appreciated.

The clear relationship between bundle formation and prior adhesion of processesshown here is consistent with studies of non-neuronal cells that show filament bundlesextending from focal or plaque contacts (Abercrombie et al. 1971; Buckley & Porter,1967; Heath & Dunn, 1978; Heaysman & Pegrum, 1973; Izzard & Lochnar, 1976,1980; Wehland, Osborn & Weber, 1979). Similar small bundles extend from the weakadhesions made by some epithelial cells in vitro (Radice, 1980). Letourneau (1979),using interference microscopy, showed similar attachments, which apparently cor-relate with bundle formation in growth cones. As suggested by many workers, ad-hesion of the cell may induce an isometric contraction that causes filaments to bundletogether (Buckley & Porter, 1967; Albrecht-Buehler & Goldman, 1976; Izzard &Lochnar, 1980; Chen; 1979, 1981a; Bragina et al. 1976; Fleischer & Wohlfarth-Botterman, 1975; Heath & Dunn, 1978; Radice, 1980; Buckley, 1974). Alternatively,if a spreading or moving cell is invariably under tension, the tension would naturallybe greatest at points of greatest resistance, i.e. focal adhesions. In this case, fibres

dnemicrography and HVEM ofneuronal motility 407

could become aligned by tension alone, rather than by an active contraction (Chen,19816).

Finally, we must consider two possible and opposing effects of fixation on themorphology of filaments within microspikes. First, attached microspikes may be putunder tension and artifactually form bundles during fixation and concomitant shrin-kage of the cell. We feel this is unlikely, since interference microscopy of living growthcones shows linear adhesive contacts, which probably correspond to intercellularbundles of filaments extending from attached microspikes (Letourneau, 1979).Second, actin filaments may be broken or shortened during post-fixation in osmiumtetroxide (Maupin-Szamier & Pollard, 1978). For this phenomenon to produce thedichotomy between attached and unattached microspikes that we document here, wewould have to assume that some feature of attachment preserves internal filaments.This is plausible if the filaments are protected by the development of tension or byan association with a regulatory protein, such as tropomysin (reported to preserveactin from glutaraldehyde-induced damage; Lehrer, 1981), that is absent from theprotecting site in unattached microspikes.

Surface recruitment

The results reported here show that sites of local, rapid increase in surface are notassociated with vesicle clusters. Vesicles are preserved by our procedures and, indeed,are characteristic of areas of veil retraction and varicosity formation. These vesiclesare immediate precursors of new surface material only under the unlikely assumptionthat all 17 of the retracted areas and all five of the varicosities we studied were aboutto give rise to a new veil.

We cannot, however, rule out the possibility that internal vesicles contribute direct-ly to the protrusion of processes. Simple calculations show that more than 60 vesicles0-l jUm in diameter would be needed to provide surface area for a new veil of 1 |Um2,which can form in 1 min. If the 60 vesicles were transported with the efficiency ofrapid axon transport, of the order of 300^m/min, they could come from far down theneurite, and would be present so transiently in the vicinity of the veil that they wouldnot be detected by our methods. We can conclude that, if internal stores of vesiclesare required for protrusion, they do not cluster near the site of new extension, but areimported from elsewhere.

Another attractive possibility exists for the rapid mobilization of surface intocellular protrusions. The cell surface required may be retrieved from surface storedin nearby crenations, folds or microspikes. This surface-recycling mechanism hasbeen suggested to operate in fibroblastic cells, where the total surface area remainsrelatively constant, despite demands for the spreading or retraction of surface duringmotility or cell division (Follett & Goldman, 1970; Erickson & Trinkaus, 1976;discussed in detail by Trinkaus, 1980). In these cells, surface irregularities appear tofunction as a reserve of material. We commonly see similar surface corrugations inactive growth cones, whereas quiescent growth cones and many varicosities havesmooth surfaces. The surfaces on retracting veils and microspikes may accommodateprotrusive activity at other areas of the leading edge of the growth cone, or may


provide surface for neurite elongation at its base. Another line of evidence consistentwith this sort of surface recruitment comes from experiments in vitro: when retractionfibres on growth cones are cut, the neuron tends to increase its protrusive activity onthe opposite side (Wessells & Nuttall, 1978), as though increased protrusive activityresulted from retraction elsewhere.

Surface retrieval

The case for vesicles acting as a surface retrieval mechanism during process retrac-tion is guilt by association as well as by degree. Vesicles are invariably present at thesites of veil retraction. In addition, the number of local vesicles is proportional to thedegree of retraction: few vesicles are found in corrugated, incompletely retractedveils, while vesicles are abundant in areas of complete veil retraction. Similar stringsof vesicles are present in the peripheral cytoplasm of those keratinocytes that areactively motile in culture (Macartney & Parkinson, 1981). We know that vesicles areinternalized from the surface of growth cones: when cell surface is labelled withvarious tracers, labelled vesicles appear in a short time (Bunge, 1977; Wessells et al.1974; Wessells, Nuttall, Wrenn & Johnston, 1976). It has also been reported thatmicrospikes may fuse with the plasma membrane during retraction, producing astring of vesicles that lie along micronlament concentrations (Spooner, Luduena &Wessells, 1974). We think it probable that the vesicle strings in retracting veils areinternalized remnants of veils or of the microspikes that are often associated with veils.

An alternative possibility is that the vesicles associated with retracting veils havebeen produced during fixation. Frank blisters and areas of veil retraction share thestriking ultrastructural similarities of vesicle accumulation and absence of contactbetween the surface membrane and the cytoplasmic matrix. The question is, whyshould all withdrawing veils blister? The answer requires a discussion of possiblemeans by which blisters form.

First, glutaraldehyde may stabilize membrane proteins before stabilizing lipids,allowing surface added during the lag time to buckle out from the matrix of the cell.If this is the case, blisters may mark the site of incipient natural membrane addition(cf. Johnston & Wessells, 1980). This argument for the significance of blisters tomotile phenomena is contrary to our observation that the only veils to appear blister-like are those that are retracting.

A second possibility is that fixation induces an adventitious addition of surface fromchance associations of membranous organelles or local reservoirs of membrane in anunstable state. We do not invariably see membranous organelles associated with thebase of veils, before or during retraction, but previously noted considerations on thespeed of vesicle transport do not allow us to rule out this possibility.

A third possibility is that blisters occur wherever connections between the plasmamembrane and cytoskeleton are very labile. The connections may be more thanusually labile during the delicate process of cell surface retreival. This interpretationis supported by our observation of corrugated outlines in processes caught in the actof retraction, which suggests that contacts are altered or diminished between thesurface membrane and the internal filamentous lattice. If this is the case, then sites


of veil retraction (and, on a smaller scale, of microspike retraction as well) wouldconsistently blister in response to fixation with glutaraldehyde.

We thank K. Barald, E. Ferguson, L. Landmesser, E. Roederer, J. Rosenbaum, J. Trinkaus andJ. Wrenn for helpful criticism and F. Thomas for technical assistance. Particular thanks go to G.Wray, without whose enthusiastic assistance this study would not have been possible. Supported byNIH grants HD04708, 5P41 RR00592 and 5 T32GM072604.

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{Received 6 October 1982-Accepted 9 November 1982)

NEURONAL MOTILITY: THE ULTRASTRUCTURE OF VEILS AND … · 2005. 8. 25. · VEILS AND MICROSPIKES...

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