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A physical model of axonal damagedue to oxidative stressAnne E.
Counterman*†, Terrence G. D’Onofrio*‡, Anne Milasincic Andrews§,
and Paul S. Weiss*¶
*Departments of Chemistry and Physics, and §Department of
Veterinary and Biomedical Science and Huck Institutes of the Life
Sciences, Pennsylvania StateUniversity, University Park, PA
16802-6300
Edited by Harry B. Gray, California Institute of Technology,
Pasadena, CA, and approved January 17, 2006 (received for review
May 18, 2005)
Oxidative damage is implicated in the pathogenesis of
neurode-generative disorders, including Alzheimer’s, Parkinson’s,
and Hun-tington’s diseases, and in normal aging. Here, we model
oxidativestress in neurons using photogenerated radicals in a
simplifiedmembrane-encapsulated microtubule system. Using
fluorescenceand differential interference contrast microscopies, we
monitorphotochemically induced microtubule breakdown on the
sup-ported region of membrane in encapsulating synthetic
liposomesas a function of lipid composition and environment.
Degradationof vesicle-encapsulated microtubules is caused by attack
from freeradicals formed upon UV excitation of the lipid-soluble
fluorescentprobe, 6-(9-anthroyloxy)stearic acid. Probe
concentration was typ-ically limited to a regime in which
microtubule degradation wasslow, and microtubule degradation was
monitored by changes inthe observed protrusion of the membrane
surface. The kinetics ofmicrotubule degradation are influenced by
lipid saturation level,fluorescent probe concentration, and the
presence of free-radicalscavengers. This system is sufficient to
reproduce some degener-ative morphologies found in vivo.
membrane � neurodegeneration � Alzheimer’s disease �
Parkinson’sdisease � microtubule
Oxidative modifications to proteins in neurons have
beenimplicated in a number of degenerative disorders and havebecome
a controversial topic in elucidating the progression ofParkinson’s,
Huntington’s, and Alzheimer’s diseases (1–7). Mi-crotubules (MTs)
are key cytoskeletal proteins in nerve cellaxons. These protein
polymers are responsible for many dispar-ate functions of cells,
including structural support and impartingpolarity, and they are
involved in motility and transport ofintracellular cargo (8).
Fundamental studies of MTs in vivo arecomplicated by the presence
of the full complement of biomol-ecules that are required for cell
survival. Thus, a substantialeffort has been devoted to
characterizing MT dynamics in vitro,and these studies have led to
increased understanding of issuessuch as dynamic instability (9)
and the involvement of molecularmotors in producing motility (10).
To mimic the confinement ofa bilayer, MT assemblies (asters) have
previously been examinedin rigid, microfabricated wells (11). The
drawback of such studiesis that interactions between MTs and
components of the elasticlipid membrane are neglected. It is
important to be able to isolateand to quantify specific factors
that impact the structural integ-rity of the cytoskeleton, while
maintaining a simplified cell-likeenvironment.
Toward this aim, we have developed a minimal physical systemwith
which to investigate the effects of oxidative stress on
thecytoskeleton. Previous studies have demonstrated that
incorpo-rating actin and tubulin into liposomes and inducing
theseproteins to polymerize results in a morphological change in
theliposome; a protrusion of the membrane is observed at one orboth
ends of the protein polymer (12–15). The asymmetry
occursfrequently, when the MT wraps around within the
encapsulatingliposome (16). Here, we employ the membrane as a
chemicalreservoir to examine individual factors that lead to
degradationor stabilization of MTs. In our model, the chemical
composition
of the membrane is controlled during liposome assembly and
theelasticity of the bilayer is retained (and is tunable according
tolipid composition). The roles of individual components
(e.g.,specific lipids, MT-associated proteins, or protective
moleculesthat function as radical scavengers) can be elucidated by
recon-stituting the liposomes with varying concentrations of
thesespecies. We demonstrate that the rate of MT degradation
isinfluenced by the lipid saturation level, f luorescent probe
(rad-ical source) concentration, f luorescent probe location, and
pres-ence of free-radical scavengers.
ResultsOverview. An intrinsic property of MTs is their dynamic
instability:the ability to alternate between periods of
polymerization and rapiddepolymerization, or collapse (9). We focus
here on inducingcatastrophic collapse and measuring the rate of
that collapse. Ageneral schematic of our experiment is shown in
Fig. 1. Wepolymerize MTs within lipid vesicles prepared from known
com-positions of synthetic lipids and fluorescent probe(s). Upon
heatingvesicles containing tubulin and GTP to 37°C, tubulin
polymerizesto form MTs. A mechanism that explains the formation of
a singlefilament of MTs by alignment of individual filaments (and
thuscreating a single-membrane protrusion) has previously been
out-lined by Hotani and coworkers (17). Most of the experiments
thatwe describe here incorporate the UV-excitable probe
6-(9-anthroyloxy)stearic acid (6-AS) into the lipid membrane.
UVexcitation of anthracene has been shown to promote
free-radicalformation (18); in our studies, we exploit
anthracene-based fluo-rescent probes to photoinitiate a
free-radical cascade that leads toMT degradation. We track the
extent of MT degradation bymeasuring the perturbation of the
membrane extension. Fig. 2illustrates this process for a single
50-msec exposure of a vesicle-encapsulated MT to UV light.
Comparison of the micrographsrecorded before and after
photoexcitation of the 6-AS probe showsa progression of decreasing
membrane extension, corresponding todegradation of the supporting
MT filament. Note that the place-ment of the radical source in the
membrane enables chemicalcoupling of the photogenerated radical to
the adjacent (encapsu-lated) MT (vide infra).
In an initial experiment, after obtaining a fluorescence
micro-graph of the vesicle with the tubulin extension, we observed
that theextended region of the membrane exhibited pearling and that
the
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS
office.
Abbreviations: 2-AS, 2-(9-anthroyloxy)stearic acid; 6-AS,
6-(9-anthroyloxy)stearic acid; 16-AP, 16-(9-anthroyloxy)palmitic
acid; DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine;DOPS,
1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]; MT, microtubule.
†Present address: Department of Molecular Biophysics and
Biochemistry, Yale University,New Haven, CT 06520.
‡Present address: U.S. Army Edgewood Chemical Biological Center,
Aberdeen ProvingGround, MD 21010.
¶To whom correspondence should be addressed at: 104 Davey
Laboratory, Departments ofChemistry and Physics, Pennsylvania State
University, University Park, PA 16802-6300.E-mail: [email protected].
© 2006 by The National Academy of Sciences of the USA
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MT had depolymerized (19). Pearling was typically observed
onlyat high 6-AS concentrations, and is consistent with the
behavior ofa membrane tether after the release of stress (20, 21).
Importantly,pearling has been noted in axonal dystrophy of aging
primates andin Alzheimer’s disease patients, both of which have
been attributedto axon degradation but were thought to require the
MT-associatedprotein tau, which is not included in our physical
model (22–25). Wehave systematically examined the dynamics of this
system to un-derstand and to quantify factors that affect MT
degradation.
Confirming Free-Radical Involvement. Control experiments
werecarried out to confirm that free-radical formation from 6-AS
wasresponsible for MT degradation. To generate free radicals in
vitro,MTs were polymerized on a slide in the presence of low levels
ofiron, and then hydrogen peroxide was introduced. The mixture
ofiron and hydrogen peroxide is a well known system for
free-radicalgeneration. Within minutes, degradation of the in vitro
MTs was
observed. Next, to confirm that 6-AS was required for
degradationof the membrane-encapsulated MTs, two sets of
experiments wereperformed: UV irradiation of MT-containing
liposomes that didnot contain a fluorescent probe and substitution
of other fluores-cent probes (that were not expected to produce
free radicals) inplace of 6-AS. No MT degradation was observed for
UV irradiationof non-fluorophore-containing liposomes or for
vesicles containing1,1�-didodecyl-3,3,3�,3�-
tetramethylindocarbocyanine (DiI-C12) or1,1�-dioctadecyl-3,3,3�,3�-
tetramethylindodicarbocyanine (DiD)(excited at their respective
peak absorption wavelengths). Thus, weconclude that (i) MT
degradation can be induced by free-radicalattack and (ii) the
membrane-soluble free-radical source 6-AS isresponsible for the
photoinduced degradation that we observe inour experiments on MTs
encapsulated in liposomes.
In Fig. 2, the length of the stretched membrane region is
plottedas a function of time elapsed after 50-msec photoinitiation.
Thedegradation trend that we observed under these conditions
wasapproximately linear over the first minute and appeared to
plateauafter 2 min. Periods of zero degradation (which appear as
steps inthe degradation plot, e.g., at 70–78 sec, 95–101 sec, and
117–128sec) were typical. There did not appear to be a consistent
patternin the frequency or duration of these ‘‘pauses’’ in MT
degradation.The variability we observed during MT catastrophic
collapse mayarise from factors such as lattice discontinuities in
the MT assemblyor variations in MT capping (26).
Below, we demonstrate that this model system can be used
toquantify effects associated with modulating components of
thelipid membrane that influence MT degradation, by altering
thefree-radical source (concentration and location
longitudinallywithin the lipid leaflet), the lipid bilayer (lipid
unsaturation), andthe presence of free-radical scavengers in the
membrane or thevesicle-encapsulated fluid volume. We note that
within a givensample of synthetic lipid vesicles, the lengths and
widths ofmembrane protrusions observed varied widely. To ensure
thereliability of quantitative comparisons for degradation
ratesmeasured from different samples, we examined numerous
ves-icles from each preparation and also compared different
prep-arations having the same composition.
Effect of Varying the Free-Radical Source: Concentration and
Local-ization. To monitor the effect of changing the concentration
offree radicals on MT degradation rate, we performed
severalexperiments on vesicles synthesized by using different
concen-trations of 6-AS (spanning a range of two orders of
magnitude)in a matrix of 5:2
1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC):1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]
(DOPS)(Fig. 3). As expected, increasing the concentration of
6-ASincreased the rate at which MTs were degraded. However, wenote
that the variability among vesicles was high (Fig. 3).
A question that arises when considering the free-radical
sourceis the exact location of the anthracene moieties with respect
to theencapsulated MT. We cannot control the lateral position of
theincorporated fluorophore within the lipid bilayer; in this
case,because 6-AS partitions into fluid-phase lipids, it is found
through-out the bilayer. However, the position of the anthracene
grouplongitudinally with respect to the lipid leaflet (i.e.,
farther away orcloser to the leaflet interior) can be controlled by
using differentfluorescent probes. We synthesized batches of 5:2
DLPC:DOPSvesicles using identical 1,800:1 lipid:probe
concentrations of 2-(9-anthroyloxy)stearic acid (2-AS) (in which
the anthracene groupshould be incorporated closest to the lipid
headgroup and thusclosest to the encapsulated MT in the inner
leaflet), 6-AS, and16-(9-anthroyloxy)palmitic acid (16-AP) (in
which the anthracenegroup should reside near the interior of the
lipid bilayer). Com-parison of the MT-degradation rates for these
batches of vesiclesgives a relative ordering of 2-AS � 6-AS �
16-AP; MT-degradationrates measured for 2-AS-containing vesicles
were �30 times higherthan those measured for the 16-AP-containing
vesicles (Fig. 4).
Fig. 1. Schematic diagram of the experiment. MTs are
encapsulated in lipidvesicles fluorescently labeled with 6-AS, a
UV-excitable probe. Upon photo-excitation (t � 0), a free-radical
cascade is initiated, causing breakdown of theMTs and relaxation of
the membrane (t � �).
Fig. 2. Time progression of a representative experiment. In
micrograph A,MT polymerization inside the vesicle causes
deformation of the membraneinto a long axon-like extension.
Micrograph B shows a 50-msec excitation ofthe 6-AS
membrane-associated probe. Micrographs C–E show the
progressivechanges in length of the MT-supported membrane
extension. The change inlength of the membrane extension is plotted
as a function of time-elapsedpost-photoexcitation.
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Effect of Incorporating Free-Radical Scavengers. To inhibit
free-radical-induced MT degradation, we incorporated
free-radicalscavengers into the vesicle-encapsulated system.
Studies were per-formed by using vitamin C, which is water-soluble,
and vitamins Eand K, which are soluble in the lipid bilayer. Note
that the resultsfor vitamin C cannot be directly compared with
those for vitaminsE and K because of the difference in
localization, solution vs.membrane; thus, the experiments described
here are each refer-
enced to a control (which contained no free-radical
scavengers).Fig. 5 shows that inclusion of these free-radical
scavengers in thevesicle-encapsulated MT system substantially
slowed MT degrada-tion relative to control experiments. A dramatic
effect was observedupon inclusion of vitamin C in the solution; on
average, MTdegradation was observed to be very slow or did not
occur in theseexperiments. At similar concentrations, vitamin C has
previouslybeen shown to stop radical-induced damage in
human-serumsamples (27). The MT-degradation rates measured for
systemsincluding the two membrane-soluble vitamins (E and K)
wereidentical within experimental uncertainty (�0.034 � 0.003
�m�secand �0.036 � 0.006 �m�sec, respectively), and both slowed
MTdegradation relative to control by over an order of magnitude
underthe given lipid, probe, and scavenger concentrations. We
alsoconsidered the possibility that the decreases in
MT-degradationrates observed upon addition of the antioxidants were
due tospectral overlap between the vitamins and the 6-AS
fluorescentprobe; however, no spectral overlap with 6-AS is
observed forvitamins C or E, and the overlap for vitamin K appears
to be small.�Thus, we conclude that the observed reduction in MT
degradationis due to free-radical scavenging.
Varying Lipid Saturation. Another key parameter in our
modelsystem is the degree of lipid saturation. Unsaturated lipids,
whichare more susceptible to peroxidation than saturated
lipids,produce 4-hydroxynonenal upon peroxidation (28), which
maycause MT breakdown. We investigated the influence of
lipidsaturation by varying the composition of the synthetic
vesicles;typical results are shown in Fig. 6. The observed MT
degradationrate was highest for vesicles composed of �60%
unsaturated
�When we extrapolated down to the concentration of vitamin K
actually used in degrada-tion experiments, we found the absorption
to be 2.98 � 10�3 at 362 nm, the peak for 6-AS,yielding an
extinction coefficient of �1,000. This is lower than the extinction
coefficient of6-AS. Additionally, 6-AS was present in the membrane
at 10-fold higher concentrationsthan vitamin K. In our vesicle
preparations, the absorption of vitamin K was lower than thatof
6-AS by a factor of 27. Thus, spectral overlap should not have
significantly influenced theresults obtained for vesicles doped
with vitamin K.
Fig. 3. Increasing 6-AS concentration increases the rate of MT
degradation.Plot of MT degradation rate (or change in membrane
extension length) as afunction of the 6-AS concentration used to
prepare the MT-encapsulatinglipid vesicles. Error bars represent
standard errors of the mean for measure-ments of four, six, and
four vesicles (left to right; note that the error bars arepartially
occluded for the leftmost point because they are nearly the same
sizeas the diamond). The linear fit has R2 � 0.9926.
Fig. 4. The position of the free-radical source within the
membrane affectsthe degradation rate. Degradation rates observed
for incorporation of dif-ferent fluorescent lipid probes in
vesicles having lipid compositions of 5:2DLPC:DOPS and 1800:1
lipid:probe. The anthracene moiety is closer to thesolvent–membrane
interface in the 2-AS probe and farthest (most buried)using 16-AP.
The degradation rates showed a striking decrease with distanceof
the anthracene moiety from the interface; the fastest rate was
observed for2-AS, and degradation was substantially slower when
16-AP was used. Thestandard error of the mean was used for 6-AS
(calculated from measurementsof six vesicles). For 2-AS and 16-AP,
the standard error of the slope from themeasurement of the
degradation rate of one vesicle was used to calculate theerror bar
for each (note that the error bars for 16-AP are partially
occludedbecause they are nearly the same size as the diamond).
Fig. 5. Incorporating free-radical scavengers inhibits MT
degradation. Typ-ical plots of MT-extension length as a function of
time after UV excitation forvesicles containing various
antioxidants are shown. All data were recorded forvesicles having
lipid compositions of 5:2 DLPC:DOPS and 1,800:1 lipid:6-AS.Vitamins
E and K, which are membrane-soluble, were studied at
concentra-tions of 11,000:1 lipid:vitamin (data represented by
purple squares and bluetriangles, respectively); water-soluble
vitamin C was located in the buffer at aconcentration of 5.7 � 10�3
M (data represented by green circles). Data for thecontrol (without
antioxidant present) are represented by dark-blue diamonds.For each
antioxidant, data from more than one degradation curve are
super-imposed to provide a representation of the range of
degradation ratesobserved; each data set is distinguished by a
different symbol grayscale. Lowconcentrations of antioxidants
greatly reduced the degradation rate.
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brain polar lipid extract. At the concentration of 6-AS
used(14,000:1 lipid:probe), collapse of membrane extensions for
thislipid composition was very fast; collapse was typically
completein �10 sec (depending on the initial length of the
membraneextension). The MT-degradation rate was more than an order
ofmagnitude lower for liposomes composed solely of fully satu-rated
lipids [DLPC and 1,2-dilauroyl-sn-glycero-3-phosphatidyl-glycerol
(DLPG)].
DiscussionThe simple model system that we have demonstrated
hereprovides a ‘‘bottom-up’’ approach to the study of damage to
MTsin a cell-mimetic lipid container. This type of system, in which
thecytoskeletal proteins and components of the lipid membrane
andfluid volume can be well controlled, provides a means
ofquantitatively examining factors that influence MT
degradation.
We have characterized the response of MTs in this modelsystem
with respect to several factors. Specifically, decreasing
theconcentration of the free-radical source (6-AS), increasing
thelipid-saturation level, or increasing the concentration of
free-radical scavengers led to decreased rates of MT
degradation.Positioning the free-radical source (in our studies,
the anthra-cene moiety of the UV-excitable fluorophore) toward the
centerof the lipid bilayer also led to decreased degradation
rates.
A key aspect of the model system we have developed is
theincorporation of the lipid membrane. In contrast with studies
ofMTs in solution, this allows us to examine effects arising from
theinteraction of cytoskeletal proteins with
membrane-associatedspecies. Increasing the complexity of the model
in a stepwisefashion by adding cellular components will allow us to
mimicprocesses involved in neuronal dystrophies more closely.
MethodsVesicle Preparation. Vesicles were prepared by using the
gentlehydration method of Evans and Needham (29). Lipids were
pro-cured from Avanti Polar Lipids. Lipid purity was verified by
usingthin-layer chromatography on activated silica plates
(KeystoneScientific, Bellefonte, PA), with a
chloroform:methanol:water(64:25:4) (vol:vol:vol) solution or a
chloroform:methanol:ammo-nium hydroxide (64:25:4) (vol:vol:vol)
solution and developed withmolybdenum blue solution
(Sigma-Aldrich). Lipids and fluorescentprobes dissolved in
chloroform were mixed and deposited on ascored Teflon disk in a
beaker. The chloroform was allowed toevaporate, and the resulting
film was dried in a vacuum desiccatorfor 6 h. Upon removal from the
desiccator, the film was hydratedwith 10 ml of Pipes buffer (pH
6.9; containing 1 mM EGTA, 2 mMMgSO4, and 100 mM Pipes) that had
been warmed to at least 55°C.The solution was blanketed with argon,
and the beaker was sealedwith aluminum foil. The system was then
incubated at 55°C for 4 hand allowed to cool slowly in an insulated
box.
Tubulin Purification. Tubulin was purified from bovine brain by
usinga protocol provided by W. O. Hancock (Department of
Bioengi-neering, Pennsylvania State University). Briefly, we
extracted thecerebrums from fresh bovine brains by dissection on
ice. Thecerebrums were processed in a chilled blender in the
presence ofprotease inhibitors; the homogenate was clarified in a
centrifuge at54,000 � g and 4°C. All subsequent spins were
performed at 300,000� g (achieved by using a Beckman Ultra 50.2 Ti
rotor). Thesupernatant was collected and warmed to 37°C to induce
tubulinpolymerization. This solution was clarified in a centrifuge,
and theMT pellet was retained. The MTs were depolymerized on ice
andclarified again by centrifugation at 4°C. This
polymerization�depolymerization cycle was repeated. Finally, the
resulting super-natant was passed through a phosphocellulose column
to concen-trate MTs and remove MT-associated proteins.
Microscopy Methods. For the MT degradation experiments, 20 �l
ofvesicles was combined with 10 �l of 10 mM GTP
(Sigma-Aldrich)solution and 10 �l of 100 mM tubulin dimer into a
cryovial(66008-251; VWR Scientific). We encapsulated tubulin inside
ves-icles with a freeze–thaw cycle; the cryovial containing
vesicles andtubulin was dipped into liquid nitrogen, and the
mixture was thawedon ice. During inspection by using optical
microscopy, samples wereheated with an objective heater (Bioptechs,
Butler, PA) set to 40°Cto induce tubulin polymerization.
Experiments were performed on a Nikon Eclipse TE300inverted
microscope that was equipped for differential interfer-ence
contrast and epifluorescence. A mercury bulb and along-pass filter
cube (XF02; Omega Optical, Brattleboro, VT)were used for
fluorescence excitation, and an ORCA 100 camera(Hamamatsu
Photonics) was used for detection. A 50-msecexposure to UV light
was used to initiate MT degradation;progress of the
depolymerization was monitored by collectingdifferential
interference contrast micrographs.
We thank Profs. Will Hancock and Donald Schmechel for
insightfuldiscussions, Prof. Hancock for the use of his laboratory
for purificationof tubulin, Dr. Anat Hatzor for preliminary studies
on liposome-encapsulated MTs in our laboratory, and Dr. Michael
Elbaum for initialguidance in the encapsulation protocol. This work
was supported by theNational Science Foundation. A.E.C. was
supported by a Ruth L.Kirschstein National Research Service Award
from the National Insti-tutes of Health.
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