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Investigation of tensile regulation of
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presented by

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c:\circ\datedm.pm3—p. 1

INVESTIGATION OF TENSILE REGULATION OF
AXONAL GROWTH

By

J ing Zheng

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology

1992

ABSTRACT

INVESTIGATION OF TENSILE REGULATION OF
AXONAL GROWTH

{?7-#?38

By

J ing Zheng

This dissertation represents an investigation of the role of tension in
neurite initiation and elongation. Neurites of cultured chick sensory neu-
rons were attached by their growth cones to glass needles and were sub-
jected to increasing tensions. This experimental process resembles “towed
growth” normally occurring in situ. The growth sensitivity of neurites
to tension was surprisingly high: an increase in tensiOn of 1 ,udyn in-
creased the elongation rate an average of 1.5 pm/ hr. Neurite elongation
rate increased in proportion to tension magnitude greater than a ten-
sion threshold, which varied among neurons with most between 50-150
udyn. The linear relationship between growth rate and tension provides
a simple control mechanism for axons to accommodate tissue expansion
in growing animal.

The size and pattern of surface addition during the experimental “towed
growth” regime was also examined using microspheres to label the sur-
face of neurites. New membrane is added interstitially throughout the
neurite, but different regions of neurite vary widely in the amount of new

membrane added.
We confirmed that neurites can be initiated de novo by application

of tensions above some thresholds. Initiated neurites developed growth
cones capable of normal motility and axonal elongation. As assessed by
electron microscopy and immunofluorescence, tension-induced neurites
containing an array of MTs indistinguishable from control neurites. Like
growth cone-mediated neurites, MTs of tension-induced neurites are ex-
tensively acetylated, axially oriented and present in large numbers. This
normal microtubule array suggests that tension stimulates a rapid re-
organization of cell body microtubules including microtubule bundling
and/ or de novo assembly of microtubules. To decide the role of reorga-
nization and assembly, we investigated tension-induced neurite initiation
in the presence 4nM vinblastine, which poisons further assembly of mi-
crotubules without depolymerizing extant polymers. We found that only
10% of poisoned neurons would form neurites in contrast to 53% in un—
poisoned group. Almost 25% of poisoned neurons that failed to form
neurites succumbed in a type of the “stretch-and-break” that was struc-
tural failure never observed in unpoisoned neurons. An abnormally low
density of poorly oriented microtubules was also found in vinblastine-
poisoned towed neurites. These data suggest that tension application
rapidly stimulates significant levels of new MT assembly concomitant
with neurite initiation, the reorganization of existing MTs plays little or

no role.

To my parents, Yu Zheng and Kemin Liao, my hunsband
zhonggang Zeng, for their everlasting love, understanding and
dedication. Also to my sweetest daughter Amelie, whose smile

always lightens my heart.

ii

ACKNOWLEDGMENTS

I wish to express my appreciation to Dr. Steve Heidemann, not only
for his guidance and support throuthout my graduate studies, but also
for patiently correcting my “J inglish” for past five years, and encouraging
me not “slackening” for “pulling” until reaching the goal.

I would also like to thank Dr. Buxbaum for his criticle insight and
suggestions, and Dr. Birgit Zipser, for her expert advice and help in my
research program. I also thank ‘my other committee members: Dr. Ron
Meyer and Dr. William Spielman, for their thoughtful contribution to
my education and this dissertation.

A very special thanks is also extended to Dr. Stu Pankratz for his
expert advice, Tim Dennerll, Phil Lamoureux, Chingju Lin, Viv Santi-
ago for technical assistance, encourange and friendship. Many thanks
to the members and friends of physiology department. In particular to
acknoledge, Jerry Lepar, Bob Cole, J ianxiong Song, Viv Steel.

iii

Contents

Introduction 1
1 Tensile Regulation Of Axonal Elongation And Initiation 6
1.1 Introduction .......................... 6
1.2 Material and methods .................... 8
1.2.1 Materials ....................... 8
1.2.2 Cell Culture ...................... 8

1.2.3 Neurite Elongation And Direct Axial Force And
Length Measurement ................. 9
1.2.4 Neurite Initiation ................... 10
1.2.5 Indirect Immunofluorescence ............ 10
1.2.6 Transmission Electron Microscopy ......... 11
1.2.7 Neurite Membrane Addition And Subtraction . . 11
1.3 Results ............................. 12
1.3.1 Control Of Neurite Elongation By Tension ..... 12
1.3.2 Surface Addition In Towed growth ......... 17
1.3.3 Neurite Initiation ................... 26
1.4 Discussion ........................... 35

2 Investigation Of Microtubule Assembly And Organiza-

tion Accompanying Tension Induced Neurite Initiation 40
2.1 Introduction .......................... 40
2.2 Material and methods .................... 42
2.2.1 Materials ....................... 42
2.2.2 Cell Culture ...................... 42
2.2.3 Effects Of Vinblastine ................ 42
2.2.4 Immunofluorescence Microscopy .......... 43

iv

2.3

2.2.5 Immunoelectron Microscopy ............. 44
Results ............................. 45
2.3.1 Acetylated Microtubules In Tension-Induced Neurites 45
2.3.2 Effects Of Kinetic Stabilization Of MTs On Tension-

Induced Neurite Initiation .............. 56
2.3.3 Neurites Formed In The Presence of 4nM Vinblas-
tine Lack Microtubules ................ 65
2.4 Discussion ........................... 71
Reference 75

List of Tables

1 Comparison of analyzed threshold with experimentally ap-
plied tension ......................... 19

2 Effects Of vinblastine on de novo initiation of neurites . . 64

vi

List of Figures

10

N eurite lengthening in response to increasing .......
Growth rates of neurites (elongation rate corrected for elas-
tic stretching) as a function of applied tension for three
typical neurites ........................
Frequency distribution of the tension sensitivity of neurite
elongation for 14 experimental neurites ..........
Frequency distribution of the tension thresholds (tension
value of zero growth intercept from plots as in Figure 2)
for experimentally stimulated neurite elongation from 14
experimental neurites ....................
Surface movements of three neurites subjected to tension-
induced neurite elongation .................
Phase images of neurite retraction, with polyethyleneimine-
treated microspheres marking the neurite surface .....
Surface movements of three neurites allowed to retract in
response to neurite slackening ...............
De novo initiation of neurites from chick sensory neurons
by applied tension. MTs content of the induced neurites
was examined by immunofluorescence and transmission
electron microscopy .....................
De novo initiation of neurites. from chick sensory neurons
by tension experimentally applied to the cell body

The elongation of a neurite initiated by experimentally ap—
plied tension in response to a step function of increasing

tension ............................

vii

13

15

23

25

27

29

11

12

13
14

15

16

17

18

19

2O

21

De novo initiation of neurites from chick sensory neurons
by applied tension. Immunofluorescence image of neuron
stained for acetylated a-tubule ...............
Both growth cone-mediate and tension-induced neurites
contain similar array of acetylated MTs ..........
Acetylation of neuronal cell body microtubules ......
Transition region between the cell body and tension-induced
neurite stained for acetylated a-tubulin ..........
Density of acetylated microtubules in tension-initiated and
normal neurites .......................
Acetylated microtubules in the growth cones of tension-
initiated and normal neurites ................
Comparison of apparent microtubules acetylation in the fi-
broblasts by immunofluorescence and immunoelectron mi-
croscopy ...........................
Microtubules in fibroblasts and neurons following 4 hours
of incubation in medium containing 4 nM Vinblastine sul-
fate. Cells were immunofluorescently stained for ,B-tubulin
Two examples of stretch-and-break failure observed in cell
poisoned with 4 nM Vinblastine ..............
Electron micrograph of a Vinblastine poisoned neuron fixed
while undergoing stretch-and-break failure of the sort shown
in Figure 19 .........................
Phase images and quantitative confocal light microscopic
images of poisoned and unpoisoned neurons with tension-

induced neurites stained for fi-tubulin ...........

viii

46

48
51

53

54

57

59

62

66

67

List of Abbreviations

MT: Microtubule

MTs: Microtubules

MTSB: microtubule-stabilized buffer: 100 mM Pipes, 2 mM
EGTA, 4% (w/v), polyethylene glycol (WM ca. 8000),
0.025% sodium azide, pH 6.8

PBS: Phosphate Buffered Saline

mAb: Monoclonal Antibody

Buffer I: Hanks’ buffered salt solution containing
5 mM Pipes, 2 mM MgC12, and 2 mM EGTA,
pH 6.

TBS I: 10 mM tris, 140 mM NaCl, pH 7.6.

TBS II: 20 mM tris, 140 mM NaCl, pH 8.2.

ix

Introduction

The fundamental tasks of the neuron are to receive, integrate conduct,
and transmit signals. To perform these functions, neurons establish their
connections by extending neuritic processes (axonal or dendritic) away
from the rather fixed position of the cell body. The growth of the axon
is a particularly complex and interesting developmental process. Despite
intensive study, the mechanism that regulates axonal development is still
poorly understood and surrounded by controversy. Axonal development
has been studied largely by observing and examining naturally occurring
neurite elongation [24, 26, 46, 55, 67, 74, 76, 84]. Accumulating evidence
has suggested that mechanical tension is an important intrinsic regula-
tor of axonal development [36]. The evidence that tension is a normal
regulator of axonal development stimulated the development of methods
allowing experimental intervention using tension. This thesis represents
a new approach to the study of axonal development, one in which the
experimenter can control neurite initiation and elongation through appli-
cation of mechanical force.

The growth of axons includes axon/neurite initiation and elongation.
By 1941, Paul Weiss had categorized axonal elongation into three succes—
sive stages that remain entirely relevant today [94]. The first two stages
“pioneering” and “application” are mediated by growth cone activity.
In contrast, the growth cone is quiescent in the final “towing” stage of
growth: once the growth cone has contacted its target, the growth cone
transforms into a nonmotile synaptic terminal. Migrating target cells
pull on their attached axon, causing “towed” axonal growth. This was
the first evidence that tension might regulate axonal elongation.

The role of tension in axonal development was largely ignored until

Dennis Bray [13] showed indirectly by a vectorial analysis of neurites’
geometric pattern on the dish that neurites of culture neurons were under
tension. There is now widespread agreement that growth cone-mediated
elongation also reflected tension stimulated growth via a pulling growth
cones [15, 16, 49, 51, 56, 75, 92]. The best evidence came from Lamoureux
et al [49]. They reported direct measurements of neurite tension as a
function of growth-cone advance. They showed that growth cone advance
and neurite tension are linearly related and accompanied by apparent
neurite growth. No increase in force occurs in neurites whose growth
cone fails to advance. So all three stages of axonal elongation may be
mediated by mechanical tension, whether the tension is provided by the
advance of the growth cone or through the movement of the target tissue
[21]. The question is, “How does the mechanical tension regulate the
process?”

One of the important underlying questions concerns the cytoskeleton.
The cytoskeleton is the major internal structure of axons [30, 56, 58]; it
consists principally of microtubules (MT), neurofilaments (intermediate
filaments in neurons), and actin filaments. Actin filaments form a corti-
cal network below the axon plasma membrane. In growing axons, actin
filaments extand into the motile leading edge termed the growth cone
[20, 48, 51, 96]. Microtubules and neurofilaments are present in parallel
array within the shaft of the axon [20, 51, 96]. It is generally appreci-
ated that neuronal elongation is critically dependent upon the assembly
of microtubules [66]. This raises the question of how mechanical tension
regulates the chemical reactions of cytoskeletal assembly.

One possible answer is the thermodynamic model postulated by
Buxbaum and Heidemann [21]. That is, mechanical force is a source

of (thermodynamic) free energy that can affect the monomer/polymer

assembly equilibrium of any assembling polymer [21, 22, 37]. Compres-
sion of MTs raises their free energy relative to no force load; more com-
pression shifts the tubulin/microtubule equilibrium toward disassembly,
less compression favors assembly [21, 22, 37]. The model assumes that
traction force (tension) exerted by the growth cone on the underlying
substratum shifts tensile support away from the compressed MTs onto
the substratum. The shift of compression away from the MTs to the
substratum with the advance of growth cone lowers the critical concen-
tration of tubulin required for assembly. Consequently, tubulin that a
moment previously was in equilibrium with the high free energy, com—
pressed polymer now adds into the low free-energy, compression-relieved
polymer. This MT assembly continues until equilibrium compression on
the MTs is again reached. If Buxbaum and Heidemann scheme’s is right
, we would expect that there should be a threshold tension for axonal
growth at which mOnomer/ polymer reaches equilibrium, we would also
expect a reproducible relationship between neurite tension and neurite
growth.

Neurite initiation appears to be different from neurite elongation. Ini-
tiation involves a localized protrusion of plasma membraneand formation
an axial array of MTs that is previously absent [61, 62, 86, 87, 94]. The
mechanism that regulates neurite initiation is poorly understood. For
example, the factors that determine the side on a cell body from which a
growth cone will emerge remain unidentified. That is, why does an axon
grow out on one side of the cell body but not at another?

Interestingly, Bray found that, using a “towing motor,” long neurites
could be initiated simply by pulling the margin of previously rounded
neuronal cell bodies. The neurites produced by microelectrode towing

had a normal appearance with abundant, longitudinally aligned MTs and

neurofilaments [16]. This data strongly suggests that mechanical tension
also can regulate neurite initiation. The process of MTs events accompa-
nying neurites initiation is of particular morphogenetic and cell biological
interesting because: First, the centrosome, which has been postulated to
play a major role in determination of cell morphology by inducing an
asymmetry in the distribution of MT [18, 65, 60] has not typically been
found at the base of a neurite [81, 86]. How MTs organize into an axial
array after application of tension is an important and unanswered ques-
tion. Second, axons differ from the cell body by the absence of protein
synthetic machinery [53] and the presence of a dense array of high ordered
cytoskeletal elements [14, 20, 52, 62, 69, 70, 96]. Whether MTs assemble
principally in the cell body or/ and at the growth cone and how MTs or
tubulin are transported in the axon has been controversial for a decade
and is still unclear [7, 6, 52, 54, 57, 68, 76]. An investigation of MTs
assembly and/ or reorganization during neurite initiation, especially at
the very early stages, should be of fundamental importance to answering
these questions.

Another important question is that of membrane addition during neu-
rite elongation. Based on normal, growth cone-mediated growth, elonga-
tion rates of 40-100 um/ hr [11, 12, 59], as much as 3 pm? of surface may
be added per minute [90]. New membrane is added at the growth tip in
growth cone-mediated growth. Labeling studies with fluorescent lectin or
carmine particles [11, 31], pulse-chase experiments with ferritin-labeled
lectins and with phospholipid precursors [71, 72], and autoradiography
of radiolabeled membrane proteins [88, 34] support this concept. In con-
trast, the assumption in towed growth is that mass addition is interstitial
[38], a possibility that is supported by indirect experimental results of

Campenot [23]. Where is new membrane added when the growth cone is

 

not actively involved in elongation?

This dissertation represents an attempt to address the questions men-
tioned in this introduction. In chapter 1, an experimental process sim-
ilar to “towing” growth in situ was used to investigate the relationship
between mechanical tension and neurites growth rates, as well as mem-
brane addition pattern accompanying neurite towed growth. This method
is also used to quantitatively investigate the relationship between ten-
sion and neurite elongation or initiation predicted by the thermodynamic
models of Buxbaum and Heidemann [21, 22].

Chapter 2 focuses on MTs events accompanying the neurite initiation
induced by experimentally applied tension, especially at the very early
stages, about which little is known [29, 55]. To assume that the neurite
initiation by tension resembles growth cone-mediated initiation, I com-
pared the density, orientations, and acetylation pattern of microtubules
in tension-induced and “normal” growth cone mediated neurites. With
the aid of MT “kinetic stabilizer”: Vinblastine [40, 41], the relative roles
of reorganization and assembly of MTs in neurite initiation was also as-

sessed.

1 Tensile Regulation Of Axonal Elongation And Ini-

tiat ion
1 .1 Introduction

Evidence is accumulating that mechanical tension is an important reg-
ulator of axonal development [36]. Tension applied to neurites of chick
sensory neurons and to PC12 cells stimulates their elongation [16, 28].
This mirrors instances of tensile regulation of axonal elongation in viva.
Growth cones pull on neurites, stimulating their elongation [49]. In later
stages of axonal development, migrating target cells pull on their at-
tached axons, causing “towed” axonal growth [94]. Balice-Gordon and
Lichtman [5] reported that a similar mechanical coupling between target
cells and neurons is responsible for the growth of motor nerve terminals
on mouse muscle fibers, thus integrating the size of the synapse with the
size of the muscle fiber. Experimentally applied tension can also initiate
new neurites from chick sensory cells [16]. Conversely, sudden declines in
neurite tension cause chick sensory neurites to develop tension actively
and to retract [28], providing a potential mechanism for axonal retraction
in vivo [77]. Growth and retraction of neurites stimulated by supplying
or withdrawing NGF to/ from different region of rat sympathetic neu-
ron also appeared to be regulated by neurite tension [23]. Both axonal
growth and retraction must involve assembly/disassembly of cytoskele-
tal elements. A complementary force interaction between compressed
axonal microtubules supporting tensile axonal actin may regulate micro-
tubule assembly in the axon by a thermodynamic mechanism, integrating
microtubule assembly with growth cone advance [21, 27].

We report here further investigations of the relationship between ax-

onal growth and mechanical tension. We wish to test the preliminary

6

finding of Dennerll et al. [28] that axonal elongation rate is linearly cor-
related with tension magnitudes. The procedure used for this experiment
is similar to towed growth; for example, the active role of the growth
cone is eliminated. New membrane is added at the growing tip in growth
cone-mediated growth [11, 31, 71]. Where is new membrane added when
the growth cone is not actively involved in elongation? Finally, we wish
to extend the work of Bray [16] on neurite initiation by measuring the
force required to induce neurite initiation to determine if initiation differs

mechanically from elongation of extant neurites.

1.2 Material and methods

1.2.1 Materials

Polystyrene microspheres and Polybed 812 embedding resin were ob-
tained from Polysciences, Inc. Phosphate-buffered saline (PBS) and fetal
calf serum were purchased from Gibco. L—15 medium, Collagen IV, poly-
L—lysine, trypsin, and polyethyleneimine were all purchased from Sigman
Chemical Co. Laminin was obtained from Collaborative Research. A
mouse monoclonal antibody against a-tubulin was the kind gift of Dr.
David Asai of Purdue University.[1] Fluorescei-labeled goat anti-mouse

IgG was purchased from Kirkegaard and Perry Lab. Inc.

1.2.2 Cell Culture

Chick sensory neurons were isolated from lumbosacral dorsal root ganglia
of 10-12 day old chick embryos, as described by Sinclair et al. [83]. These
dorsal root ganglia were rinsed in supplemented L-15 medium (L-15 sup—
plemented with 0.6% glucose, 2 mM L-glutamine, 100 U / ml penicillin, and
136 ug/ ml steptomycin), and treated with 0.25% trypsin for 30 minutes
at 37°C. The trypsin was then removed, the ganglia were rinsed 3 times
with the supplemented L-15 medium containing 10% fetal calf serum.
The ganglia were triturated with pipette into a single cell dispersion,
and cultured in the supplemented L-15 medium containing 10% fetal calf
serum and 100 ng / ml 78 nerve growth factor isolated from mouse salivary
glands [93]. Cells were grown an untreated tissue culture dishes at a den-
sity low enough to render non-neuronal contamination inconsequential.

Neurons were cultured for 16-24 hr prior to experimentation.

1.2.3 Neurite Elongation And Direct Axial Force And Length Measurement

The axial tensions of neurites were measured with force-calibrated glass
needles, and their length was determined as previously described [28].
The bending moduli of the experimental needles were between 5.1 and
15.4 pdyn/pm. A calibrated needle was attached to a neuron’s growth
cone, and the neurite was pulled in 30-60 minutes “steps” of constant
force; that is, a tension magnitude was chosen, beginning at 50-100 pdyn,
and this tension was held constant for 30-60 minutes by moving the mi-
cromanipulator to maintain the appropriate deflection of the calibrated
needle. Subsequently, the same technique was used to apply 30-60 min-
utes periods of higher tension to the neurite, each level typically 25-50
,udyn higher than the previous value. The experiments were recorded on
videotape at 24X time lapse and were subsequently analyzed for neurite
length and tension as described previously [28].

In addition to an expected towed growth response [16], chick sen-
sory neurons have a passive viscoelastic solid response to applied tension
[28]. In order to determine accurately (inelastic) the growth parameters
from neurite length measurements, initial analysis subtracted the elastic
stretching component of neurite length using a previously described the
mechanical model for passive viscoelastic behavior [28]. Individual neu—
rite values for the stiffness of the mechanical elements were obtained as
described by Dennerll et al. [28]. The elastic response of a neurite to
tension over time was calculated, and this value was subtracted from to-
tal neurite length to give a value for growth. These analyses showed that
the rather small stretching increases for each tension step were complete
after the first 10-15 minutes. Consequently, in later experiments growth

rates could be calculated from length data omitting the first 15 minutes

___'_

following tension increases.

1 .2.4 Neurite Initiation

Neurons without extant neurites were attached to calibrated experimental
needles at the cell margins. The response of the cell to the application of
various tension levels was recorded on videotape and analyzed for tension
and neurite length as above. Some experimentally initiated neurites were
examined by immunofluorescence or by transmission electron microscopy

for the presence of microtubules.

1.2.5 Indirect Immunofluorescence

Following initiation, neurites were subjected to tension to allow contin-
ued elongation to various lengths. The distal ends of such neurites were
then micromanipulated from the needle back onto the culture substrate.
A diamond-tripped “objective” was used to mark the experimental neu-
ron by circling the dish beneath. Immunofluorescent staining of micro-
tubules was carried out by a method similar to that of Thompson et
al. [89]: Medium was carefully removed, and the culture was permeabi-
lized in 0.5% Triton X-100 in the MTSB (microtubule-stabilized buffer:
100 mM Pipes, 2 mM EGTA, 4% (w/v), polyethylene glycol (MW ca.
8000), 0.025% sodium azide, pH 6.8), then fixed in 3.7% formaldehyde
solution in MSTB, all at 37°C, followed by extraction in methanol at
-20°C. The neurites were then incubated with the primary antibody to
a-tubulin (kindly provided by Dr. David Asai), rinsed then incubated
with fluorescein-labeled secondary antibody goat anti-mouse IgG. The
prepared samples were viewed with a Nikon inverted microscope equipped

for fluorescence equipment.

10

1.2.6 Transmission Electron Microscopy

Ultrastructural observations of experimentally initiated neurites were
made using standard methods previously described [2, 42]: as soon as the
distal end of neurite was micromanipulated from the glass needle back
onto the culture substrate, concentrated glutaradehyde was added. The
neuron was fixed for 30 minutes with 2% glutaradehyde in supplemented
L-15 medium, postfixed for 5 minutes with 1% 0804 in 0.1M Cacody-
late, dehydrated in ethanol series, and embedded in Polybed 812. Thin
sections cut parallel to the substrate were stained with uranyl acetate
and lead citrate, and observed with a Phillips 300 transmission electron

microscope.

1.2.7 Neurite Membrane Addition And Subtraction

Polystyrene microspheres with diameter of 1.6 pm were treated with 5%
polyethylenimine, then washed 5 times in distilled water and 1 time in
L-15 medium, and stored in L-15 medium at 0.8% (w/v). Ten microliters
of microspheres suspension were added to the cell cultures containing
3-4 ml culture medium. After incubating for 30-60 minutes at 37°C to
allow the microspheres to attach to neurons, excess microspheres were
removed by rinsing the culture with fresh L-15 medium. Calibrated nee-
dles were attached, as above, to the growth cones of neurites judged to be
appropriately labeled with microspheres, and the neurites were pulled or
slackened. The experimental process was videotaped and the distances
between cell body and microspheres were measured by the same proce-

dure used to measure neurite length.

11

1 .3 Results

1.3.1 Control Of Neurite Elongation By Tension

PC12 neurites subjected to experimentally applied tensions of less than
100 pdyn elongated as simple viscoelastic solid showing no long term
extension. However, neurites showed plastic, long term elongation, in-
terpreted as growth, when subjected to tension >100 pdyn. The long
term elongation rate above this threshold was proportional to the mag-
nitude of the initially applied tension [28]. These experiments, however,
suffered from a technical limitation, applied force declined as the neurite
lengthened such that both neurite length and tension were changing at
all time. We wish to confirm the findings of Dennerll et al. [28] using a
better-controlled method on authentic neurons.

Fourteen chick sensory neurites were attached to the calibrated glass
needles at their growth cones and subjected to increase steps of tension.
Each step was a period of constant force of 30-60 minutes duration, and
steps differed from each other by 25-50 pdyn. Figure 1 summarizes one
such neurite-lengthening experiment. As for PC12, chick sensory neu-
rites lengthened continuously above a threshold tension, here 43 pdyn.
Also, neurite lengthening increased with increasing tension level. How-
ever, not all of this length increase was growth. Some portion of this
neurite lengthening was elastic stretching because chick sensory neurites,
like PC12 neurites, have a viscoelastic solid component to their extension
behavior [28]. The lower, broad line of Figure 1 plots the elastic response
of the neurite predicted by a mechanical (spring and dashpot) model for
the viscoelastic behavior of chick sensory neurons [28]. As shown by the
dotted line, this component of the behavior equilibriums a given tension

by a given change in length (i.e. as an elastic solid) over a period of 10-15

12

 

Figure 1. Neurite lengthening in response to increasing steps of ten-
sion. Chick sensory neurons were pulled from their distal ends by a
calibrated glass needle. Bar graph, The deflection of the needle was
stabilized by micromanipulation to exert various levels of force (left
ordinate) on the neurite, which were maintained for 25-60 minutes
before increasing the force to a new level. Upper, dotted line, Overall
neurite length (right ordinate) from cell body to needle; lower, broad
line, passive viscoelastic response of neurite predicted from neurite
mechanical constants and the mechanical model for neurite response
as described by Dennerll et al. [28].

 

 

 

400 ..

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ocifirw)
O 50 100 150 200 250 300 350 400
Time(mins)

 

l3

minutes. Yet the neurite continues to elongate after this time, apparently
at constant rate, and we interpret this neurite elongation that follows the
elastic equilibration period to be towed growth [16, 28]. Like Bray [16], we
found that this lengthening occurred with an apparent increase in volume
of the neurite, that is, in the absence of neurite thinning expected of
stretching except at the very highest elongation rates, and in the absence
of cell body shrinkage (see Figures 8 and 9 for visual confirmation in a
different context). We found, as shown in Figure 2, that this growth rate
(i.e. corrected for elastic stretching) is proportional to tension magnitude
above a tension threshold. That is, plots of growth rate versus tension
for all 14 neurites showed a simple linear relation between tension above
a threshold and growth rate. Correlation coefficients were between 0.83-
0.99 and were greater than 0.9 for 10 / 14 neurites. As seen in Figure 2, this
relationship hold even at quite high, non-physiological rates of elongation,
up to 400 pm/ hr. The application of uniformly increasing steps of force
with accompanying neurite lengthening gave rise to the possibility that
growth rate was proportional to neurite length, rather than tension. To
check this, decrement tension steps were applied among the incremental
steps in a few experiments. In all case, as shown in Figure 2C, growth
rate declined with decreasing tension. This indicates that growth rate
was a function of tension, not of neurite length.

The relationship between growth rate and tension, the slopes of the
lines in Figure 2 reflects the sensitivity of neurite growth rate to tension,
that is, the growth rate per pdyn of tension. Figure 3 is a frequency distri-
bution of this sensitivity for the 14 neurites. N eurites’ tension sensitivity
varied, most neurites elongated between 0.22-2.0 ,um/hr/pdyn. However,
some neurites were more sensitive, one elongated 5.5 pm/hr/ndyn. In-

deed, the sensitivity of sensory neurites to tension was surprisingly high;

14

 

Figure 2. Growth rates of neurites(elongation rate corrected for elastic
stretching) as a function of applied tension for three typical neurites.
The slope of the lines is the sensitivity of neurite elongation to tension
(see Figure 3). The zero growth intercept is the tension threshold for
growth (see Figure 4). The experiments shown in (A) and (B) consist
of incremental steps of tension only, as in Figure 1. The experiment
shown in (C) includes 2 decrement steps of tension( triangles) among
the incremental steps. Growth rate declined during decrement steps
as expected.

 

 

 

 

 

 

 

 

 

 

 

 

 

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15

Figure 3. Frequency distribution of the tension sensitivity of neurite
elongation for 14 experimental neurites. Tension sensitivity is given
as the growth rate per unit tension.

 

 

No. of Neurites

 

 

 

 

 

 

0.5 1 1.5 i 2.5 3 )5
Growth Rate (urn/hr)/Tenslon (udyna)

16

that is, diflerences in force at our method’s detection limit (approx. 5
,udyn) would cause a moderate difference in growth rate in most neu-
rites. We found no correlation between a neurite’s initial length and its
sensitivity to tension.

Dennerll et al. [28] found that PC12 cells had a surprisingly repro-
ducible tension threshold for growth of 100 pdyn. Figure 4 summarizes
the tension threshold (the tension intercept at zero growth rate as in Fig-
ure 2) for all 14 neurites. Thresholds for chick sensory neurites are seen to
vary between 35-560 pdyn with the majority (9 / 14) between 50-159 pdyn.
This is similar to but more variable than the 100 pdyn threshold for the
clonal PC 12 cells. An important confirmation of the tension threshold for
growth was that 8 of 14 neurites that were subjected to initial tensions
lower than their threshold either retracted or showed no growth, shown
in Table 1. For example, a neurite whose zero growth intercept was later
found to be 140 pdyn retracted at 7 pm/ hr when subjected to 115 pdyn of
force. Two of these eight neurites also showed retraction when subjected
to force slightly above their threshold; for example, one neurite with an
intercept of 120 udyn retracted at 138 pdyn. The remaining 6 neurites
were not subjected to force less than their threshold. Also, we found no

correlation between the neurite’s initial length and its threshold tension.

1.3.2 Surface Addition In Towed growth

It was of interest to determine where membrane addition occurs in exper-
iments similar to the above, which resemble towed growth, particularly
in view of work by Campenot [23], who found evidence for “interca-
lated” growth and retraction, that is, mass addition and subtraction oc-
curring in the middle of neurites. Microspheres treated with polyethylen-

imine sticked aggressively to the neuronal membrane and appear to be

17

Figure 4. Frequency distribution of the tension thresholds (tension
value of zero growth intercept from plots as in Figure 2) for experi-
mentally stimulated neurite elongation from 14 experimental neurites.

 

 

 

 

 

 

 

 

 

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Threshold (udyno)

18

 

 

No. of Trials Analyzed Tension Elongation
Threshold Applied Rates ( pm / hr)
1 3.46 2.84 -7.88
2 98.04 83.72 -6.45
3 52.39 34.96 -72.28
4 139.43 115.37 -7.68
5 105.73 46.60 -23.58
6 105.73 74.60 -7.50
7 120.00 138.00 -7.50
8 109.69 111.24 -18.00

 

 

 

 

 

 

Table 1: Comparison of analyzed threshold with experimentally applied tension

19

markers for membrane regions per se, similar to the carmine particles
used by Bray [11]. Labeled chick sensory neurites were elongated by
pulling from the growth cone with glass needles, as above, and the video-
tape was analyzed for the changed distances of microspheres from the
cell body. Figure 5 shows the results for 3 of the 7 neurites analyzed
in this way. As shown in Figure 5a, microspheres remained essentially
stationary on neurons that were not being pulled. In this experiment,
there was some small drift in the position of the micromanipulator, al-
lowing for a small amount of neurite retraction observable over the long
term. Like Bray [11], we found that microspheres were not moved by
neuronal motility processes. However, microspheres did oscillate slightly
when observed at time-lapse speed (24x), as described by Bray [11] for
carmine particles. In pulled neurites, as shown in Figures 5, b and c, the
distance between microspheres and between most microspheres and the
cell body increased. However, different regions of neurites clearly varied
in the extent of change. For example, in Figure 5b, a substantial change
in the distance of the microspheres symbolized by squares (arrowhead)
contrasts with the nearly stable position of the microspheres nearest the
cell body. There was also some tendency for the greatest separation to
occur in the distal half of neurites as shown in Figure 5, b and c.

A similar analysis for marking membrane subtraction during neurite
retraction is shown in Figure 6 and 7. We previously found that slacken-
ing neurites initiated active contraction and tension generation [28]. Six
neurites tethered at their growth cone were slackened in a series of short
steps to cause retraction, as showed in Figure 6. An effort was made to
hold the neurite close to zero tension so that it was under neither tension
nor compression. As shown in Figure 6, this gave rise to small, local sinu-

soids, but the entire neurite was not permitted to slacken into a sinusoid,

20

Figure 5. Surface movements of three neurites subjected to tension-
induced neurite elongation. Neurite surfaces were labeled with
polyethyleneimine-treated vinyl microspheres. The position of these
microspheres relative to the cell body was recorded as the neurite was
lengthened by pulling with a glass needle. The open circles represent
the position of the growth cones. All other symbols represent partic-
ular microspheres, with their time course of position change given as
a connecting solid line. (a). Control neurite tethered onto a glass
needle but not pulled. Some drift in the position of the microma-
nipulator allowed some neurite retraction during the course of the
experiment. (b). A neurite that showed particularly variable surface
movement. As seen here, most of the surface movement occurred be-
tween the second microspheres behind the growth cone (arrowhead)
and the microspheres behind it. As seen at t=0, these microspheres
were originally located in the distal third of the neurite. (c). A neurite
that showed more evenly distributed surface movements as elongation
proceeded.

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Figure 6. Phase images of neurite retraction, with polyethyleneimine-
treated microspheres marking the neurite surface, as photographed
from the videotape of the experiment. (a). Neurite decorated with
microspheres immediately prior to the initial slacking. (b). Five min-
utes later, a small sinusoid typically seen during these experiments
can be seen distal to a microsphere group. (c). Ten minutes later,
the neurite has again been slackened, and small sinusoids can be seen.
(d). Ten minutes later, the sinusoids seen in (c) have been resorbed.
The neurite retracted 50 pm during the 25 minutes of this sequence.
Bar, 20 pm for a-d.

23

 

Figure 7. Surface movements of three neurites allowed to retract in
response to neurite slackening. Surface movements were marked with
polyethyleneimine-treated microspheres as for Figure 6. Symbols are
as in Figure 5. (a). Control neurite tethered'to a glass needle but
no slackened. (b) and (0). Position of microspheres and growth cones
of neurites retracting in response to slackening. In some experiments
microspheres converged due to neurite shortening such that they could
no longer be resolved as separate. This is reflected in the termination
of several lines at various times in Figure 7b.

 

 

 

 

 

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as occurs upon axotomy [2]. The videotape of such experiments was
analyzed for the decrease in the distance of microspheres from the cell
body, data shown in Figure 7. Figure 7a shows the data from a control
neurite that was not slackened and did not retract, microspheres maintain
their position about each other and to the cell body. In slacked neurites,
the distances between microspheres decreased all along the neurite but,
As for elongation, to a highly variable extent. For example, the neurite
shown in Figure 7b lost surface primarily from the most distal regions.
Analysis of retraction was complicated by the fact that, as the neurite
retracted, some microspheres came too close together to resolve. This
is apparent in Figure 7b, in which some lines representing microspheres
position terminate at various time points. In contrast, the neurite shown

in Figure 7c lost surface proportionately throughout its length.

1.3.3 Neurite Initiation

Bray [16] showed that neurites could be initiated de novo by experi-
mentally applied tension. We wished to confirm and extent this results
on neurons without extant neurites. Before experimental intervention,
some experimental neurons were completely devoid of extensions from
the rounded cell margins while others (Figure 8a) had motile lamellipo-
dial extensions extending from the cell margins as described by Collins
[25]. Calibrated glass needles were applied tangent to the margin of cells
in random locations. No differences were noted among various regions
of the margin for the capacity to initiate neurites, with the single excep-
tion of margins near asymmetrically located nuclei; that is, cell margins
overlying a thin layer of cytoplasm between the plasma membrane and
the nuclear membrane could not be induced to form and elongate. Ap-

plied tensions of 60-1000 udyn caused a cell process to form and elongate

26

Figure 8. De nova initiation of neurites from chick sensory neurons
by experimentally applied tension. (a). Neuron prior to a glass nee-
dle attachment. (b). Same cell as in previous frame after 96 minutes
of experimentally applied tension. In this cell, neurite initiation oc-
curred both at the site of needle attachment" to the cell margin and
at the opposite cell margin. The arrowhead points to filopodia on
the distal end of the neurite attached to the needle. Note also the
growth cone-like appearance of the distal end opposite the site of nee-
dle attachment. (c). Eight minutes later, the distal end of the neurite
formerly attached to the glass needle has been manipulated onto the
culture dish. The growth cone-like appearance of this end is apparent.
(d). The same cell as in frame (c) fixed and processed for immunofluo-
rescence with a monoclonal antibody against a-tubulin (see Materials
and Methods). (e). Transmission electron micrograph of a neurite ini-
tiated de nova by experimentally applied tension (not the same cell
as shown in frame a-d). Microtubules are readily apparent. Scale bar,
a-d, 20 pm. e, 0.5 pm.

 

Figure 9. De nova initiation of neurites from chick sensory neurons by
tension experimentally applied to the cell body. These micrographs
were taken from the videotape of the experiment. (a). Prior to the
beginning of tension application. A neuron with no outgrowth is at-
tached to a glass needle. (b). A neurite formed on the margin of'
the cell opposite the pulling needle. This micrograph was taken 22
minutes after tension was first applied to the cell margin. Note the
growth cone-like appearance of the distal end and the attachment
point of this end relative to the cell below. (0). Fifteen minutes later,
the neurite elongated by towing of the cell body, i.e., without a change
in the position of distal end of the neurite. This micrograph reflects
the maximum extent of towed growth for this neurite; i.e., no addi-
tional movements of the micromanipulator were imposed after this
time. (d). Thirty-three minutes later, additional neurite elongation
was caused during this period by the advance of the growth cone, as
seen by the change in the position of the neurite growth cone relative
to the cell below. Bars, 10 pm in a-d.

29

H%HEKI¢R-$ra-.g—

E'aQ‘ET$‘-

 

(Figure 8b) in 80% of the cells to which tension was applied. In the
remaining 20% of cells, applied tensions only caused the cell body to
elongate but failed to cause process formation, even tensions > 1000 udyn.
As shown in Figures 8 and 9, the elongation of initiated neurites occurred
without obvious thinning of the lengthening neurite, or shrinking of the
cell body.

In total, we initiated neurites to varying lengths from 41 neurons and
obtained same force measurements from 35 of these. We generally found
no lag time for neurite formation after force application, 33 of 41 neu-
rons responded immediately with changes of some type. In some of these
cases, however, the cell elongated before an obvious process formed. In
other cases, an obvious processes formed immediately. Process formation
generally occurred at the cell margin attached to the glass needle. In 7
cases, the process formed at the cell margin opposite the needle (Figures
9a-c). That is, micromanipulation pulled the cell body, and the process
remained attached to the dish at its distal end, suggesting that tension
per se, not the needle or its surface treatment, stimulated the initiation
process. As for elongation of extent neurites, there was evidence that
neurite initiation required tension above some threshold; that is, in 10
neurons we found that initial applied forces were insufficient to initiate
neurites, but that subsequent higher tensions were immediately success-
ful. For example, one neuron failed to respond to tensions of 73 and
98 ndyn applied for 5 minutes each, -but rapidly produced a neurite at
123 pdyn. However, we have no information about the thresholds of mast
cells because initiation is a singular occurrence (one cannot “go back” and
try a lower tension if the initial tension is successful), not a continuous
variable (one also cannot extrapolate a graph back to a zero intercept).

The forces at which neurites were successfully initiated are as follows:

31

The majority (21/35) required less than 200 udyn; in five cases forces
< 100 ndyn initiated neurite formation. Four neurons initiated neurites
in response to forces between 200-300 pdyn for process formation, seven
neurons formed processes between 300-600 pdyn and three neurons ini-
tiated neurites in response to forces greater than 800 pdyn. We do not
understand the basis of this wide variability in tension requires to initiate
neurites. Cell body size, for example, was not a factor. The only sys-
tematic difference was that more force was generally required for those
neurites “towed” by the cell body, as in Figure 9, than for those towed
by their distal end, as in Figure 8. Although the variability is poorly
understood, sensory neurons are a heterogeneous group of cells, so the
differences are not entirely surprising.

In many cases neurite initiation appeared to consist of two phases.
Initially, a “bump” or “nubbin” formed, and this was easily drawn out
from the cell margin for a period of some 10-20 minutes. During this
period, too high a force frequently caused the process to break or caused
a loss of attachment to the needle. Subsequent elongation from the nub-
bin was somewhat slower and frequently involved a higher force, though
still less than 200 ,udyn generally. Unlike elongation from extant neu-
rites, elongation of neurites initiated de navo was not linearly related to
force. Rather, neurites appeared to elongate at a particular rate despite
increase in applied tension (Figure 10). Nevertheless, neurites initiated
de novo developed microtubules and growth cones, two important char-
acteristics of “normal” neurites. Motile filopodia were noted on seven
neurites while still attached to the needle (Figure 8b). As Shown in Fig-
ure 80, if the distal ends were manipulated off the needle back onto the
dish, these ends strongly resembled growth cones. As shown in Figure 9,

growth cones also formed on all 7 neurons that were towed by the cell

32

Figure 10. The elongation of a neurite initiated by experimentally
applied tension in response to a step function of increasing tension.
The solid line shows the tension magnitudes applied to a neuron as
30-40 minutes periods of constant force. The solid circles show the
net length of the neurite. In contrast to self-initiated neurites (Figure
1), experimentally initiated neurites did not show a dependence of
elongation rate with tension magnitude.

 

 

 

 

 

 

 

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250-- «250

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33

body rather than the neurite. This indicates that the presence of the
needle was not responsible for growth cone formation. Most importantly,
the growth cones formed by tension engaged in motile activity such as
filopodial extension/ retraction and could advance along the dish, caus-
ing neurite elongation, as shown clearly in Figure 9c and d. We did not
attempt to assess the existence of growth cones on the distal ends of neu-
rites that did not show filopodia while still attached to the needle because
of competing experimental demands on initiated neurites, some for elec-
tron microscopy, some for cytomechanical analysis of long duration, and
so on. In addition to functional growth cones, experimentally initiated
neurites were found to contain microtubules. We examined ten cells for
neurite microtubule content by either immunofluorescence or transmis-
sion electron microscopy. All eight of the initiated neurites longer than 10
nm were found to contain microtubules as shown in Figure 8d and e. As
shown in Figure 8d, the tubulin-immunofluorescence image in initiated
neurites showed a bundle of microtubules in which individual polymers
could not be resolved. This is typical of neurons [3, 35, 57]; and of cells
experimentally induced to express 7', a major microtubule-associated pro-
tein of axons [44]. Of two neurites less than 10 pm long, one was found
to contain microtubules by immunofluorescence and one was found. to be

devoid of microtubules by electron microscopy.

34

1 .4 Discussion

In towed growth, an axon elongates after attachment to its target as a
result of tension exerted on the axon by target movements [94]. The ex-
perimental technique used here, attaching a glass needle to the distal end
of a neurite and pulling, is an in vitra analog of this growth phase. Recent
reports suggest an important role for towed growth in the extension of
retinal ganglion cell dendrites of fish [39] and cats [63], in neuronal path-
way formation within the visual system of Drasophila [85], and in size
matching of pre- and postsynaptic elements of the neuromuscular junc-
tion of rats [5]. Despite this and considerable older evidence concerning
towed growth, studies of tension induced axonal growth have been very
limited; our work can be regarded as an extension of that of Bray [16],
who fixed the elongation rate of neurites by towing with a specially de-
signed towing motor. This allowed investigation of towed growth at slow
rates, but provided no information about forces. We, in contrast, fixed
the force and observed the elongation. This allowed an analysis of the
relationship between force and growth rate. We found that chick sen-
sory neurons are surprisingly sensitive to tension. Some neurites have a
growth rate (pm/ hr) tension (pdyn) ratio of 5:1 (Figure 3). An aspect of
this sensitivity is that the elongation rates we observed were often higher
than the most rapid growth cone-mediated elongation rates reported for
these neurons in culture (approximately 100 um/ hr; [16]). However, we
found no difference in the growth/ tension relationship at elongation rates
above and below this benchmark (e.g., Figure 2B).

We found that the growth rate of chick sensory neurites is proportional
to tension above a threshold. This result confirms our earlier interpre-

tation of tension stimulated growth in PC12 cells and, from a cytome-

35

chanical perspective, confirms a fundamental similarity between axonal
growth and Bingham fluid behavior, that is, a linear relation between
rate of length change and force above a threshold. The linear relation-
ship between tension and growth rate is attractive heuristically in that
it accounts both for axons accommodating small forces without growth
and for large- scale tissue expansion without progressive changes in the
steady-state tension on axons. If growth amount was proportional to ten-
sion, for example, very long axons would be under much greater tension
than short axons and would require increasingly higher tensions as the or-
ganism grew. We observe that tensions below a threshold produced a zero
growth rate. Thus, axons can accommodate tissue growth precisely if the
level of tension on the axon increases with the rate of tissue expansion, an
intuitively reasonable situation. Tension regulation of towed growth may
then resemble typical physiological control mechanisms characterized by
“set points.” In this case there is an axonal tension set point: Axons bear
some tension as shown for cultured neurons [13, 28]. Tissue growth exerts
additional tension on associated axons stimulating their elongation. This
axonal elongation dissipates the tension because the axon is adding mass
to accommodate the increase in length rather than stretching elastically
(imagine a spring that added gyres when pulled). As tissue expansion
slows to a halt, tension on the axon declines with the addition of mass
until it declines below threshold where it remains unless the tissue again
expands. Thus, moderate steady state values for tensions on axons are
maintained.

New membrane is added at the growth tip in growth cone-mediated
axonal growth [11, 31, 71]. Microtubules are also added at the distal end
in growth cone-mediated elongation [7, 57]. In contrast, the assumption

in towed growth is that mass addition is interstitial [38], a possibility

36

that is supported by experimental results of Campenot [23]. We labeled
the surface of towed neurites with polycationic microspheres, similar to
Bray’s [11] use of carmine particles, to determine whether new surface
was added interstitially and to observe the pattern, if any, to the ad-
dition. The polyethyleneimine-treated microspheres did not translocate
significantly relative to the cell body in the absence of pulling. Most
importantly, the sum of the increased distances between microspheres
during towed growth equaled the increased length of the neurite. These
observations provide strong evidence that the movement of microspheres
relative to the cell body during towed growth represents regions of new
surface addition. As expected, we found that new surface is added inter-
stitially along the entire length of the neurite (Figure 5). However, new
surface addition did not occur uniformly throughout the neurite. There
was some tendency for the new surface to be added preferentially at the
distal half of neurites, as shown in Figure 5b and c. Also, as shown in
Figure 5b, new surface tended to be added in particular regions of a given
neurite, while other regions added little or no new surface; that is, towed
growth was similar to growth cone-mediated elongation in that some re-
gions of the neurite were favored for membrane insertion relative to other
regions. New membrane arises from the fusion of membranous vesicles
with the axolemma following the synthesis of the vesicles in the cell soma
and rapid transport down the axon (for review, see [73]). The nature
of the signal that specifies sites for vesicle off-loading from the transport
system and/ or for vesicle fusion are completely unknown. However, our
results complement previous findings on growth cone-mediated growth by
providing evidence for regionalization of axolemmal growth during towed
growth. Also, the differing sites of addition in growth cone-mediated and

towed elongation imply that this regionalization is sensitive to the mode

37

of growth.

Initiation of axons / dendrites appear to be a process distinct from elon-
gation. Motile, “amoeboid” activity is initially characteristic of most, if
not all, of the cell margin of cultured neurons [25, 29, 95]. Motility is then
restricted to particular regions, and ultrastructural observation of such
incipient outgrowths, both in culture and in situ, show cytoskeletal ele-
ments concentrated at these sites [61, 62, 86, 87]. However, a cause and
effect relationship between spatial restriction of motility and the con-
centration of cytoskeletal elements remains elusive. Our evidence that
mechanical force can both initiate neurites and regulate microtubule as-
sembly suggests the possibility that tension may be a link between motile
activity and the cytoskeletal assembly in process outgrowth. Mechanical
tension as a regulator of axonal microtubule assembly has been discussed
previously [21, 66]. The apparently normal density of microtubules within
neurites initiated and elongated by experimentally applied tension (Fig-
ure 8e) provides strong additional evidence that mechanical force can reg-
ulate axonal microtubule assembly both spatially and temporally. More
unexpected was the finding that tension stimulated the formation of func-
tional growth cones at the ends of tension-induced neurites. In neurons
with an appropriately differentiated state, tension appears to be sufficient
to regulate spatially and initiate the formation of two crucial structures
characteristic of sprouting neurons: an axonal microtubule array and a
growth cone. The forces required to initiate neurites, less than 200 udyn
in most case, were in the same range as those for elongation of extant
neurites. It is somewhat surprising that initiation required no greater
tension than did elongation; the initiation and spatial organization of
cytoskeletal assembly might be expected to require an additional energy

input. However, the force magnitudes required for initiation and elonga-

38

tion were entirely consistent with the tension (generally above 200 ,udyn,
up to 500 pdyn) these neurons exert on themselves by their pulling growth
cone [49], and by active neurite retraction [28]. We speculate that the
restriction of motility observed in “normal” neurite initiation in culture
reflects the first mitile region to exert more tension than the threshold
for neurite initiation. Possibly, this effective pulling growth cone then
inhibits motility elsewhere by mechanical effects on the cytoskeleton as
described by Kolega [47] for inhibition of fish epidermal cell motility by
experimentally applied tension. The tension exerted by the newly formed
growth cone may induce neurites by concentrating cytoskeletal elements
or stimulate cytoskeletal assembly in the region [21].

The available evidence on mechanical stimulation of axonal elongation
raised the possibility that tension serves as general regulator, acting as a
kind of “second messenger” [66]. It will be of interest to test whether the
effects of various extrinsic regulators of axonal elongation such as NGF
[23], Ca++ levels [33, 45, 50, 64], and properties of the growth substrate

[56]) act through this “second messenger” to stimulate elongation.

39

2 Investigation Of Microtubule Assembly And Or-
ganization Accompanying Tension Induced Neu-

rite Initiation

2. 1 Introduction

Accumulating evidence suggests that mechanical tension is an important
intrinsic regulator of normal axonal development, including the micro-
tubule (MT) assembly and organization accompanying neurite elongation
[16, 28, 97]. For example, growth cones, which are primary source of ten-
sion, pull on neurites, stimulating their elongation [49]. In later stage of
axonal development, migrating target cells pull on their attached axons,
causing “towed” axonal growth [94]. Growth of the neuromuscular junc-
tion during development also appears to be the result of mechanical force
inputs [5].

In a previous study, we confirmed that neurons without neurites re-
sponded to tension application by forming of functional neurites within
minutes [16, 97]. That is, the tension-induced cell processes developed
two crucial characteristics: a functional growth cone and axial MTs array
of normal density [97]. Since functional neurites could be initiated within
minutes by tension, MT events accompanying neurite initiation must be
occurring in the same short period.

Here, we focuse on two aspects of the MT organization and assembly
in tension-induced neurites. First, is the new assembly of MTs required
for neurite formation or are existing MTs from cell body reorganized or
translocate into a cell process? Second, this is the only experimental sys-
tem, to our knowledge, in which the initiation of neurites can be controlled

by the investigator over the time scale of minutes. As such, it may prove

40

an attractive system for the investigation of the very early cytoskeletal
events of neurite initiation, about which relatively little is known [29, 55].
This would require that neurite initiation by tension resembled growth
cone-mediated initiation. Consequently we compared the density, orien-
tations, and acetylation patterns of microtubules in tension-induced and

“norma ” growth cone-mediated neurites.

41

2.2 Material and methods

2.2.1 Materials

Triton X-100 and Vinblastine sulfate were purchased from Sigma Co.
Mouse monoclonal anti-,B-tubulin, 5nM colloidal gold-labeled goat anti-
mouse IgG were purchased from Amersham Inc. A mouse monoclonal
antibody (mAb) specific for acetylated-a-tubulin (1-6.2 1) was the kind
gift of Dr. David Asai of Purdue University. Fluorescein-labeled goat

anti-mouse IgG was obtained from Kirkegaard and Perry Lab. Inc.

2.2.2 Cell Culture

Embryonic chick sensory neurons were cultured as previously described

[97].

2.2.3 Effects Of Vinblastine

In all experiments, Vinblastine sulfate was used at a concentration of
4 nM. To assess the effect of this drug concentration on growth cone-
mediated elongation, we identified regions of poisoned and unpoisoned
cultures and circled them with a diamond “objective” prior to the begin-
ning of the experiments. These regions were then photographed at one
hour interval for two hours. We measured the distance moved by each
growth cone and divided by the time over which the movement occurred
to obtain advance rates.

Some cell cultures containing both neurons and fibroblasts were al-
lowed to incubate for 4 hours in 4 nM Vinblastine, and then prepared for
tubulin immunofluorescence microscopy as described below.

To assess the effect of Vinblastine on tension-induced neurite initiation

and elongation, we divided culture dishes into two groups: unpoisoned

42

group and poisoned group. In the poisoned group, 1 uM Vinblastine in
PBS was added into culture dishes to achieve a final concentration 4 nM.
After incubating for 1 hour, neurons without extant neurites were at—
tached to calibrated glass needles [27] at the cell margins. The response
of cells to the application of tension was recorded on videotape and an-
alyzed for tension and neurite length as previously described by Zheng
et al. [97]. Some experimentally initiated neurites were examined by
immunofluorescence or by immunoelectron microscopy. The unpoisoned

groups were treated similarly except that Vinblastine was omitted.

2.2.4 Immunofluorescence Microscopy

A diamond-tipped microscope “objective” was used to mark the experi-
mental neuron by circling the dish beneath. Immunofluorescence stain-
ing of microtubules was carried out as previously described [97]. The
neurites were incubated with the one of two primary anti-bodies: (i). A
mouse monoclonal antibody that strongly recognizes acetylated-a-tubulin
at 1210,000 dilution (Keating and Asai, unpublished). (ii). A monoclonal
mouse fl-tubulin antibody used at 1:50 dilution. After incubation with
primary mAb, neurites were rinsed, incubated with fluorescein-labeled
secondary antibody. The prepared samples were viewed with a Nikon
inverted microscope equipped for fluorescence observation.

Some cells were observed through an Odyssey confocal microscope
(Noran Instruments). This instrument allows measurements of fluores-
cence intensity along a line chosen by the investigator. We used this to
visualize and compare fluorescence intensity in the cell body and neurites

of poisoned neurons (see Figure 21).

43

2.2.5 Immunoelectron Microscopy

The same two primary antibodies used for immunofluorescence localiza-
tion were also employed for antigen localization at the ultrastructural
level, using the method of J oshi et al. [43] and Geuens et al. [32]. Briefly,
neurites were fixed and permeabilized simultaneously for 1 minute with
0.5% glutaraldehyde and 1.0% triton X-100 in buffer I (Hanks’ buffered
salt solution containing 5 mM Pipes, 2 mM MgC12, and 2 mM EGTA,
pH 6). Neurites were subsequently taken through the following treat-
ments: 0.5% glutaraldehyde in buffer I for 10 minutes; 0.5% triton X-100
in buffer I for 30 minutes; 0.5mg/ ml NaBH4 in buffer I for 15 minutes;
TBS I (10 mM tris, 140 mM NaCl, pH 7.6 ) for 5 minutes. Cells were
incubated at room temperature overnight with primary antibody against
anti-acetylated-a-tubulin at 1: 10,000 dilution. Then cells were rinsed
with TBS I and incubated for 6 hours with 1:2-1:4 dilation of secondary
antibody labeled with 5 nm gold-conjugated second antibody. Cells were
washed again with TBS II (20 mM Tris, 140 mM NaCl, pH 8.2) and
postfixed with 1% glutaradehyde in buffer I for 10 minutes, 2% 0504 in
0.1 M Cacodylate for 20 minutes, stained with 0.5% uranyl acetate in 1%
phosphotungstic acid for 30 minutes, dehydrated with an ethanol series,
embedded in Polybed 812, and ultrathin sections were examined using

an electron microscope.

44

2.3 Results

2.3.1 Acetylated Microtubules In Tension-Induced Neurites

The MT array of normal cultured neurites contains a post-translationally
acetylated form of a-tubulin [9, 10, 78]. In rat neurons, these acetylated
MTs are abundant in the neurite shaft but largely absent from the growth
cone assessed by immunofluorescent staining [3, 78]. We wished to com-
pare the pattern of acetylation of MTs in neurites initiated by growth
cone advance and in neurites initiated by the application of tension, that
is, by towed with a glass needle, in chick sensory neurons.

Neurites are initiated from neurons without extant neurites (Figure
11a) by attaching a force-calibrated glass needle to the cell margin and
pulling [16, 97]. Typically, a pulling force of about 200 ,udyn causes a
process to form and elongated as shown in Figure 11. Elongation oc-
curs without obvious thinning of the lengthening neurite, or shrinking of
the cell body. Motile filopodia develop on the tips of the induced neu-
rites (Figures. 11b and llc). Tension-induced neurites that had been
towed for periods between 10-60 minutes were micromanipulated to de-
tach them from the needle and affix them to the dish surface. After this,
the cultures were prepared for immunofluorescence with an antibody to
acetylated a-tubulin (see Materials and Methods). We were successful
in this delicate task with six cells and found that all the induced neu-
rites stained brightly for acetylated a-tubulin (Figure 11e). Even at the
earliest time ( 10 minutes), tension-induced neurites were observed to
stain with anti-acetylated a-tubulin antibody in a pattern similar to that
of normal, growth cone-mediated neurites on the same dish (Figure 12).
Both normal and tension-induced neurites fluoresced brightly along the

neurite shaft but not at the growth cone. Robson and Burgoyne [78]

45

Figure 11. De nova initiation of neurites from chick sensory neurons
by applied tension. (a). A neuron prior to a glass needle attachment.
(b). Same cell as in previous frame after 20 minutes of experimentally
applied tension. (c). The distal end of the neurite formerly attached
to the glass needle has been manipulated onto the culture dish. The
growth cone-like appearance of this end is apparent. (d). Phase image
of the same cell as in (c) fixed and processed for immunofluorescence.
(e). Immunofluorescence image of the neuron as in (d) stained with a

monoclonal antibody against acetylated a-tubulin(see Materials and
Methods). Bar, 10 pm.

46

 

Figure 12. Both growth cone-mediate and tension-induced neurites
contain similar array of acetylated MTs. (a). Phase image of an
unmanipulated , cultured neuron that initiated neurites via growth
cone activity. (b). Immunofluorescence image of same cell as in panel
(a) stained with 1-6.2 1, the monoclonal antibody against acetylated
a-tubulin (1:10,000 dilution). (c). Phase image of a towed neuron
whose neurite was experimentally initiated as in Figure 11 and fixed
following 57 minutes of tension. (d). Immunofluorescence image of the
same neuron as panel (c), stained with a monoclonal antibody against
acetylated a-tubulin. Notice the lack of staining in the growth cones
region of both cells. Bar, 10 pm.

48

 

reported a similar result in cultured rat neurons.

The density and pattern of acetylated MTs in these neurons was also
examined using the immunoelectron microscopic method of J oshi et al
[43] and Geuens et al. [32]. As shown in Figure 13, acetylated MTs
of different lengths with a similar pattern of randomly orientations were
found in the cell bodies of all neurons. This is to say that neurons without
neurites and neurons with tension-induced neurites contained a MT array
similar to that in the cell bodies of neurons with normal neurites. None of
the 11 cell bodies we observed had regional concentrations of MTs near
the cell margins. With respect to individual MTs, some were densely
and uniformly decorated with gold particles, while on other MTs the
gold particles occurred in distinct clusters or domains. This differential
labeling of MTs may represent distinct stability classes of MT polymer
[3].

In the transition region between cell bodies and tension-induced neu-
rite shafts, there is generally a higher density of MTs in the proximal
neurite than in the neighboring cell body region (Figure 14). The MTs
in this initial segment of the neurite do not begin at a well defined point,
or an organizing center like a centrosome. Rather, the MTs appeared
to radiate from many different directions, similar to the “initial segment
funnel” found in cells undergoing neurite initiation in sitv. [55] and in
PC12 cells [86].

In the neurite shaft itself, we found a low density of acetylated MTs
even in very short, incipient processes (“nubbin”) formed very shortly
(3.5 minutes) after tension application to the cell margin (Figure 15a).
By ten minutes, tension-induced neurites of approximate one cell body
length contained a normal array of acetylated MTs (Figure 15b), confirm-

ing the light microscopic results. The antibody for acetylated a—tubulin

50

Figure 13. Acetylation of neuronal cell body microtubules. All panels
are transmission electron micrographs of cell bodies immunostained
with the monoclonal antibody against acetylated‘a-tubulin (1-6. 2 1
at 1:10, 000 dilution), and an appropriate second antibody conjugated
to 5 nm colloidal gold particles(see Material and Methods). In all
panels the image is of a region midway between the nucleus and the
plasma membrane. Insets of all three panels show individual MTs
at higher magnification from each group respectively. (A). A neuron
lacking neurites. (B). A neuron with a tension-induced neurite. (C).
A neuron with normal growth cones mediated neurites. Bars, 0.5 pm.

51

 

 

Figure 14. Transition region between the cell body and tension-
induced neurite stained for acetylated a-tubulin as described. Bar,
0.5 pm.

  
  

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53

Figure 15. Density of acetylated microtubules in tension-initiated
and normal neurites. All panels were stained for acetylated tubulin
as described in Material and Methods (a). A low density of acetylated
MTs appears in “nubbin” induced by experimental force applied for
3.5 minutes. (b). Acetylated MTs in neurite shaft 10 minutes after
application of tension. (0). Acetylated MTs in neurite shaft elongated
by growth cone-mediated processes for 24 hours prior to fixation. Bar,
0.5 pm.

54

stained the MTs of both normal and tension-induced neurites intensely
and, in most profiles, along their entire length (Figures 15b and 150).
On occasion, short segments of more sparsely gold-labeled MTs were ob-
served. We examined the MTs array of tension-induced neurites that
were pulled for different periods, 10, 15, 50, 100, and 230 minutes, and
were of correspondingly different lengths. We found no detectable dif-
ferences in apparent length distribution or orientation of acetylated MTs
despite large differences in the extent of neurite outgrowth. In contrast to
the light microscopic results, growth cones of both normal and tension-
induced neurites were observed to contain a low density of acetylated
MTs at the ultrastructural level (Figure 16).

A similar difference in the ability to observe acetylated MTs by light
and electron optics was also noted in fibroblasts, which contaminate our
primary neuronal cultures (Figure 17). Immunofluorescence observations
of fibroblasts frequently indicated a lack of acetylated microtubules, par-
ticularly when fibroblasts were in the vicinity of neurons (Figures 17a
and 17b). When fibroblasts were isolated on the dish, however, or at the
electron microscopic level, they were found to have low levels of tubulin

acetylation (Figure 170).

2.3.2 Effects Of Kinetic Stabilization Of MTs On Tension-Induced Neurite
Initiation

The data above indicate that tension stimulates a rapid reorganization

of cell body MTs to accommodate neurite formation and/ or a tension-

induced assembly of axonal MTs that are then very rapidly acetylated.

To assess the relative roles of these two processes, we exploited the recent

discovery that nanomolar concentrations of vinblastine stabilize MTs by

inhibiting both the “on” and “off” reactions of tubulin exchange [40, 41].

56

Figure 16. Acetylated microtubules in the growth cones of tension-
initiated and normal neurites. All panels were stained for acetylated
a-tubulin by immunogold method described in Materials and Meth-
ods. (A). Electron micrograph from the distal'end of a neurite elon-
gated by a growth cone. (B). Electron micrograph of the distal end of
a neurite elongated by experimentally applied tension for 10 minutes.
At the time this neurite was micromanipulated from the glass needle
for fixation, the distal end showed filopodia, similar to Figure 11b and
11c. Bar, 0.5 pm.

57

 

Figure 17. Comparison of apparent microtubule acetylation in fibrob-
lasts by immunofluorescence and immunoelectron microscopy. (a).
Phase image of dish region containing a neuronwith growth cone-
mediated neurite and contaminating fibroblasts. (b). Immunofluores-
cence image, stained for acetylated a-tubulin, of the same region of
the dish as in panel oz. (c). Immunoelectron microscopic image, again
stained for acetylated oz-tubulin, of a different fibroblast showing the
low staining level seen at this higher resolution. Bar, 1 pm.

59

 

This “kinetic stabilization” of microtubules prevents both polymer disas—
sembly and new assembly [41]. Thus, events depend upon re-organization
of existing MTs should be unaffected by the addition of the vinca alkaloid,
while MT assembly-dependent events should be inhibited.

Jordan et al. [41] found the treatment of Hela cells with 4 nM vinblas-
tine for 18-20 hours arrested cells in mitotic metaphase without serious
spindle disorganization or detectable loss of MT polymer. We wished to
confirm that vinblastine at these low concentrations did not cause ap-
preciable MTs disassembly in our cell types. during the typical durations
of our pulling experiments. We examined anti-B-tubulin immunofluo-
rescence in both chick sensory neurons and the chick fibroblasts that
typically contaminate the neuronal cultures. Cells were incubated for 4
hours in the presence of 4 nM vinblastine, then fixed and processed for im-
munofluorescence microscopy with anti-fl-tubulin antibody. Fibroblasts
were of interest because reports of relatively rapid MT-turnover [79, 80]
suggested that they would be more sensitive to disassembly than neuritic
MTs, which are rich in depolymerization-resistant microtubules [4, 8, 42].
Figure 18 shows that both vinblastine-poisoned fibroblasts and neurons
contain a normal MT network for their cell types. In particular, the fi-
broblasts show the extensive, spidery network of MTs that extend to the
cell margins, not a reduced, primarily peri-nuclear network indicative of
only stable MTs [80].

Normal, growth cone-mediated growth was rapidly inhibited by in-
cubation with 4 nM vinblastine. In a simple assay for growth, marked
regions of culture dishes were photographed at 20x magnification at the
beginning of the experiments and two hour after drug or sham addi-
tion (control). The change in length of neurites during this two hours

period was compared and expressed as an hourly elongation rate. In

61

W18. Microtubules in fibroblasts and neurons followmg 4 hours

of Mon in medium containing 4 nM vinblastine sulfate. Cells
mum‘csccutly stained for fl-tubulin as described in Mar

» ma
terials and Methods. Bar, 10 pm.

 

observations of 6 neurites from 3 unpoisoned neurons, the average growth
rate was 6.6 pm/ hr per neurite. In contrast, 12 neurites from 7 poisoned
neurons were found to remain essentially stationary over 2 hours period
with a growth rate of -0.5 ,um/ hr per neurite.

Tension-induced neurite initiation and growth was seriously compro-
mised by the presence of 4 nM vinblastine as shown in Table 2. Of the
11 unpoisoned neurons studied, more than 50% initiated and elongated
lengthy neurites. (In previous experiments of this kind, a higher per-
centage, 70-80% of all neurons, were induced to form neurites [97]). In
contrast, only 4 of 38 poisoned neurons initiated neurites that subse-
quently elongated for two cell body lengths or more. The “successful”
neurites were normal in appearance and in one case formed filopodia at its
distal end. However, these poisoned neurites did not behave like unpoi-
soned neurites in their response to the manipulations intended to attach
the “towed” neurite onto the dish surface, that is, remove the needle for
immunofluorescent and ultrastructural observations. First, the poisoned
neurites had a tendency to stretch and break along their length during
the manipulation. This occurs rarely in normal neurites but 2 of 4 poi-
soned neurites broke in this way. Also, 2 of 4 poisoned neurites rapidly
retracted to less than half length when their distal end was freed from the
needle. One neurite retracted 38 ,um within 20 seconds, while the other
retracted 40 pm in 100 seconds. In contrast, unpoisoned tension-induced
neurites typically elongate a bit during needle removal because of the
tugging required for detachment. In no case did we observe more than a
2% decrease in the length of unpoisoned neurites after detachment from
the needle.

Among the 38 poisoned neurons, we classified 34 as having failed to

initiate neurites. One type of failure was particular interesting. In about

63

 

Control Group

Vinblastine group

 

 

 

 

 

 

 

(11) < 38 >
Failure* 27% 29%
Nubbin Only 18% 36.8%
Stretch and Break 0% 23.7%
While Towing

Success 54.6% 10.5%

 

Table 2: Effects Of vinblastine on de novo initiation of neurites

* (I) Failure. No process formation. For unknown reasons, a neurite could not be initiated

de novo by experimentally applied tension.

(ll) Nubbin Only. A initial nubbin drawn out from the cell margin by experimentally

applied tension failed to elongate.

(III) Streak and break while towing. Elongation only last for a short period. The elon-

gated length from a formed nubbin was shorter than 1-2 cell body length.

(1V) Success. A neurite could elongate as long as a properly tension applied to the

neurite, that is, cell process is larger than 2 cell body length.

64

 

25% of the poisoned neurons tested, a process formed that elongated
for 1-2 cell body lengths, but suddenly ceased elongating and began to
stretch out and break. The break could occur all along their length,
in the middle of neurite shaft or at the distal end (Figure 19). This
stretching process was not the result of any increase in applied tension, it
generally occurred during periods of steady tension. In some instances,
neurite tension was reduced at the first sign of stretching, nevertheless the
neurite continued to thin out and break. Such stretch-and-break failure
has never been observed in unpoisoned cells in this or previous studies
[97]. Once initiation is well underway in unpoisoned cells, the neurite

continues to elongate for long periods, several hours at least.

2.3.3 Neurites Formed In The Presence of 4nM Vinblastine Lack Micro-

tubules

We were able to successfully prepare vinblastine-poisoned neurons for
analysis of their MTs content. Figure 20 shows transmission electron
micrographs of one neuron in which neurite formation failed in the man-
ner described in the previous paragraph. We were able to fix this cell
before the neurite broke. Figure 20a shows that the cell body of this
neuron contains an apparently normal array of MTs. Yet, as shown in
Figure 20b, the neurite shaft is nearly devoid of MTs, containing only
unusually short fragments that often not axially oriented. Apparently,
the somatic MTs did not invade the transition region between soma and
neurite. Also, the application of tension to the cell margin did not serve
to orient the existing MTs.

Figure 21 shows phase and quantitative immunofluorescence images of
neurons stained for fl-tubulin in poisoned and unpoisoned cells subjected

to tension application. Figure 21a are images of tension-initiated neurite

65

Figure 19. Two examples of stretch-and-break failure observed in cell
poilohed with 4 nM vinblastine, but never observed in unpoisoned
cells. See results for description. Bar, 10 um.

 

 

 

Figure 21. Phase images and quantitative confocal light microscopic
images of poisoned and unpoisoned neurons with tension-induced neu-
rites stained for fi-tubulin as described in Materials and Methods.
(A). Tubulin immunofluorescence in an unpoisoned neuron. The graph
in the upper left corner is a measure of light intensity along the bright
line shown across the image of the cell. Inset is a phase image of the
cell. (B). Tubulin immunofluorescence in a poisoned neuron that ini-
tiated a “successful neurite,” i.e. longer than two cell body lengths,
but which broke during attempting to free the glass needle from the
neurite’s distal end. Once again, the graph in the upper left is a mea-
sure of the light intensity along the bright line shown across the image
of the cell using the same instrument setting as in panel (A). Inset is
a phase image of the cell. Inset bar, 10 pm, main panel bar, 20 ,um.

68

 

 

 

in the absence of vinblastine. Both neurite and cell body are brightly
fluorescent. Figure 21b are images of a vinblastine-poisoned neuron that
was “successfully” initiated in that the neurite was originally more than 2
cell body lengths. However, as described above, this neurite broke during
manipulations to release the needle. Little or no tubulin immunofluores-
cence is visible in this “successful” neurite, while the cell body is brightly

fluorescent.

70

 

2.4 Discussion

In previous studies, we confirmed that functional neurites can be initiated
and elongated by experimentally applied tension [16, 97]. Here, we report
that the organization of MTs accompanying tension—induced neurite ini-
tiation is very similar to growth cone-mediated axonogenesis reported by
Lefcort and Bentley [55] in an identified neuron of the grasshopper limb,
and by Stevens et al. [86] in PC 12 cells. At the earliest times after the
applying tension to margin of chick sensory neurons, relatively short MTs
are located in the “nubbin” that form (Figure 15a). These microtubules
do not begin at a well-defined point organizing center. Rather, they ra-
diate in all direction within the funnel-shaped transition region between
the cell body and the incipient neurite (Figure 14), similar to the “initial
segment funnel” reported by Stevens et al. [86], and consistent with the
accumulation of tubulin seen by Lefcort and Bentley [55] as the growth
cone emerges. By the time the towed neurite is one cell body length,
as early as 10 minutes after tension application, the microtubules have
taken on the dense axial arrangement typical of these neurites. Clearly,
applied tension either induced significant new MT polymerization and / or
cause significant reorganization of existing MTs.

The majority of a—tubulin in axonal neurites of cultured neurons is
acetylated [4, 9, 10]. We found that the pattern of acetylated tubulin
in tension-induced neurites is very similar to that reported for growth
cone-mediated neurites. The axial array of neuritic MTs is densely and
uniformly labeled with anti-acetylated az-tubulin antibody at very early
times after neurite initiation, 10-20 minutes (Figures lle and 15b). At
the light microscopic level, fluorescence is observed throughout the cell

body and the neurite shaft except the growth cone region, as also reported

71

by others [3, 78]. However, at the electron microscopic level, we found
a low density of MTs that were acetylated in both tension-induced and
“normal” growth cones (Figure 16). The data suggests two possible ex-
planations for the apparent absence of anti-acetylated oz-tubulin staining
in growth cones at visible light resolutions. One is that the low density
of MTs within the growth cone does not produce enough fluorescence
intensity to see. The other possibility is that the level of acetylation of
growth cone MTs is sufficiently low that, like fibroblasts (Figure 17), it
produces too weak an immunofluorescence signal.

Although the function of tubulin acetylation is unknown, it has been
suggested that it can be as a marker for MT age. That is, microtubule
persistence, over the time scale of tens of minutes. For example, in African
green monkey kidney cells, the timing of reappearance of a steady-state
level of acetylated MTs lagged 30 minutes behind MT repolymerization
after drug induced disassembly [19]. In neurite initiation experiments, in
contrast, acetylated tubulin was apparent in cells fixed at earliest time
after tension application, 3.5 minutes (Figure 15a). As noted above, the
acetylation pattern was indistinguishable from normal neurites by 10-20
minutes (Figures He and 15b). The substrate for acetylation is known to
be the MT polymer [10]. The degree of labeling of tension—induced neurite
MTs appears to represent an extensive level of acetylation judging by (1)
the much lower levels of antibody staining of fibroblasts on the same dish
and (2) the similarity of labeling of both neuritic and somatic MTs. The
neuronal soma were found to be heavily acetylated in this and previous
studies [3, 4, 78].

To determine the relative roles of MT reorganization and de nova as-
sembly in tension-induced neurite initiation, we compared the effect of

“towing” in the presence or absence of MT assembly. We exploited the

72

recent discovery that very low concentrations of vinblastine act as a “ki-
netic stabilizer,” suppressing both “on” and “off” reactions at MT ends.
The overall effect is that additional MT assembly is poisoned with little
or no loss of extant polymer [40, 41, 91]. We confirmed that 4 nM vin-
blastine causes no observable MT depolymerization in either fibroblasts
or neurons of chick dorsal root ganglia (Figure 18). Further, the vinca
alkaloid at this concentration inhibited growth cone-mediated elongation.
Vinblastine poisoning appeared to arrest growth cone advance, poisoned
neurites did not significantly advance or retract during a two hour period
of observation.

The response of neurons to tension application in the presence of 4
nM vinblastine argues strongly in favor of tubulin assembly underlying
tension induced neurite initiation. First, neurite initiation by pulling was
seriously inhibited by the presence of the poison. Those few neurites / cell
processes that did form were found to be abnormal. Cell processes both
longer and shorter than two cell body lengths formed in the presence of
vinca alkaloid were abnormally prone to breaking under tension. The
appearance of the failures, and the instances in which stretching and
breakage continued despite reduced tension loads are consistent with lack
of an internal structural support from MTs [22, 27]. Although such failure
might also occur from lack of new membrane, which is normally supplied
by microtubule-based transport [73, 82]. Those few poisoned neurites we
were able to examine were found to be nearly devoid of MTs.

Bath electron microscopic and immunofluorescence images indicated
that apparently normal MT arrays in the cell body of poisoned neurons
failed to invade the neurite or even form an initial segment funnel. That
is, we find no clear evidence for reorganization of existing MTs. We are

unable to draw any conclusions about the few MT fragments that are ap-

73

parently a feature of poisoned neurites (Figure 20). These may be exam-
ples of tension-induced tubulin assembly severely limited by the poison.
Alternatively, the fragments may reflect very limited MT reorganization
of cell body MTs. If this latter is the case, it is difficult to understand
why the MTs are so short and poorly aligned unless some fragmentation
process occurs in poisoned cells that is normally suppressed or quickly
ameliorated.

We conclude that application of tension to the margin of chick sen-
sory neurons rapidly stimulates significant levels of new MT assembly
concomitant with neurite initiation. Our data also suggests that tubulin
acetylation in these neurons occurs very rapidly after assembly. Baas et
al. [4] also concluded that tubulin acetylation occurs soon after assembly
and our results show extensive acetylation can occur within a very few
minutes. The ability to experimentally control neurite initiation, elonga-
tion and the accompanying MT assembly by tension application suggests
an attractive method for the study of the questions and controversies that

currently swirl through the field of axonal MT transport and assembly.

74

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[2] Baas, P.W., L.A. White, and SR. Heidemann. 1987. Microtubule polarity reversal
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