Cell Motility and the Cytoskeleton 397390 1998
`
`Fluorescent Taxoids as Probes
`of the Microtubule Cytoskeleton
`
`Juan A Evangelio1 Miguel Aba11 Isabel Barasoain1 Andre A Souto2
`M Pilar Lillo3 A Ulises Acuria3 Francisco AmatGuen12 and Jose M Andreul
`
`1Centro de lnvestigaciones BiolOgicas CSIC Madrid Spain
`2Instituto de Quimica Organica CSIC Madrid Spain
`3Instituto de QuimicaFisica Rocasolano CSIC Madrid Spain
`
`taxoids
`
`to
`
`Microtubulcs arc specifically and efficiently visualized with thc ncw fluorescent
`70$14 fluoresceincarbony1falanylltaxol
`FLUTAX and 701114 tetramethyl
`rhodaminecarbony1falanylltaxol ROTAX Similarly to taxol FLUTAX and ROTAX
`tubulin into microtubule assembly One
`are able to drive inactive GDPliganded
`molecule of FLUTAX binds per 43tubulin dimer assembled competing with taxol for
`the same microtubule binding site with an eightfold smaller relative affinity FLUTAX
`induced microtubule elongation is markedly Mg2±dependent encompassing the bind
`tubulin dimer polymerized than in the case of taxol A
`ing of one Mg2+ ion more per
`small perturbation of the absorption spectrum of bound FLUTAX is consistent with a
`cationic microenvironment
`relative to the solution The fluorescence anisotropy of
`FLUTAX increases by an order of magnitude
`upon binding to microtubules and
`time resolved measurements
`indicate that the fluorescein moiety remains considerably
`mobile on a protein surface The rate of labeling suggests that this is the outer microtubule
`the microtubule lumen would be functional FLUTAX and
`wall Alternatively
`ROTAXinduced microtubules radial structures and organized microtubule bundles are
`readily observed under the fluorescence microscope Rapid and accurate visualization of
`native or very mildly fixed cytoplasmic
`and spindle microtubules of a variety of
`permeabilized cells is simply obtained with micromolar FLUTAX with an advantage
`immunofluorescence In addition FLUTAX labels the centrosomes of PtK2 cells
`over
`to x or 3tubulin and co localizing with antibodies
`more intensely
`than antibodies
`ytubulin Two brightly fluorescent spots probably separating or duplicating
`can be resolved
`in the centrosomes of interphase cells This finding indicates
`centrosomes may well be additional targets of action oftaxoids FLUTAX strongly labels
`microtubules near
`the spindle poles as well as microtubules at the telophase spindle
`equator and the central part of the midbody in cytokinesis
`instead of the dark zone
`frequently observed with immunofluorescence suggesting a predominant interaction of
`FLUTAX with sites at which tubulin is newly polymerized Nanomolar concentrations of
`FLUTAX also permit specific imaging of centrosomes
`halfspindles and midbodies in
`growing U937 cells Cell Motil Cytoskeleton 3973901998
`
`centrioles
`
`that
`
`1998 Wiley Liss Inc
`
`Key words microtubules centrosomes mitosis Taxol paclitaxel
`
`fluorescent probes
`
`Abbreviations GDP guanosine diphosphate PBS phosphate buffered
`saline PEDTA 10 mM sodium phosphate buffer 1 mM EDTA pH 70
`1 mM EDTA 1 mM GDP
`PEGM 10 mM sodium phosphate buffer
`with 7 mM MgCl2 and at pH 66 except otherwise indicated Pipes
`piperazineNN bis2ethanesulfonic acid PEM 100 mM Pipes
`mM EGTA 2 mM MgCl2 pH 68 SDS sodium dodecyl sulfate
`Trivial names of the fluorescent derivatives of taxol CUTAX 704N
`FLUTAX 704N4 fluores
`4coumarincarbony1Lalanylltaxol
`2 AcFLUTAX
`2 0acetyl70
`N4 fluoresceincarbony1Lalanyltaxol ROTAX 70N4
`
`ceincarbony1Lalanylltaxol
`
`1
`
`Contract grant sponsor Fundaci6n Cientifica de la Asociacion Espa
`nola Contra el Cancer Contract grant number Direcci6n General de
`Ensenanza Superior Contract grant numbers PB950116 PB90126
`APC960071 Contract grant sponsor CSIC Accion Especial
`to JM Andreu Centro de Investigaciones Biolog
`Correspondence
`cas CSIC Velazquez 144 28006 Madrid Spain
`Email cibjm07cccsices
`JAE and MA contributed equally to this work
`
`tetramethylrhodaminecarbony1Lalanyltaxol
`
`Received 25 June 1997 accepted
`
`13 October 1997
`
`1998 Wiley Liss Inc
`
`Abraxis EX2061
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`74
`
`Evangelio et al
`
`INTRODUCTION
`
`tubulincolchicine
`
`Microtubules
`are essential
`for
`the organization
`cytoplasmic transport motility and division of eukary
`otic cells In addition they are targets of antimitotic
`drugs including colchicine vinblastine and taxol Wil
`son and Jordan 19941 Tubulin was discovered
`as a
`colchicine binding protein that assembled to form micro
`tubules Taylor 1965 Wilson and Friedkin 1966 Weisen
`berg et al 1968 Weisenberg 1972 Lee and Timasheff
`19751 However
`the use of colchicine derivatives to
`visualize microtubules has been very limited Clark and
`Garland 1978 Moll et al 19821 since the
`complex inhibits the assembly and dynamics
`of microtubules by binding to their ends Panda et al
`19951 On the contrary taxol an antitumor drug is an
`inducer of microtubule assembly Schiff et al 1979
`19801 and a stabilizing inhibitor of microtubule dynamics
`that binds to the microtubule wall Parness and Horwitz
`1981 Horwitz
`1992 Derry
`et al 19951 Taxol
`is
`considered to bind to cellular microtubules Manfredi et
`targets Wolf
`al 19821 although it may have additional
`son et al 19971 C De Ines and I Barasoain unpub
`lished observations Until now taxol binding has not
`been efficiently visualized
`Taxol
`induces microtubule formation by binding
`preferentially to assembled rather than to unassembled
`tubulin Parness and Horwitz 1981 Carlier and Panta
`loni 19831 The binding of exactly one molecule of taxol
`per o43tubulin dimer polymerized completely bypasses
`the requirement of the y phosphate of bound GTP for
`assembly Diaz and Andreu 19931 Specific
`tubulin
`labeling with azidotaxol derivatives has
`photoaffinity
`the N terminal 31
`mapped
`taxolbinding sequences
`residues and at positions 217231 of the 13 subunit Rao
`et al 1994 19951 The low resolution structures of
`microtubules in solution show that taxolinduced microtu
`bules have on average one protofilament
`less than is
`found in microtubules assembled with glycerol microtu
`buleassociated proteins or docetaxel Andreu et al
`1992 19941 Taxol binding also induces a 35 lengthen
`ing of the 4nm axial spacing between tubulin monomers
`along protofilaments Vale et al 1994 Arnal and Wade
`19951 and modifies the flexibility of microtubules Dye et
`al 1993 Venier et al 1994 Mickey et al 19951 From
`structural Andreu et al 19921 thermodynamic Diaz et
`and kinetic studies of
`the intermediates of
`al 19931
`taxoidinduced microtubule assembly Diaz et al 19961
`we have proposed that taxol binding 1 modifies the
`bules possibly by binding between protofilaments 2
`switches GDPtubulin from the inactive to the assembling
`conformation and 3 probably induces the lateral accre
`tion of tubulin oligomers transforming them into active
`
`at
`
`lateral interactions between tubulin molecules in microtu
`
`microtubule nucleating species The mid resolution struc
`ture of tubulin in zinc induced sheets has shown a single
`located
`taxol binding site per dimer
`approximately
`between the protofilaments Nogales et al 1995 Wolff et
`al 19961 however
`the antiparallel orientation of these
`protofilaments relative to microtubules precludes deduc
`in microtubules
`ing from these data the position of taxol
`It can be expected that
`in analogy to fluorescent
`derivatives of phalloidin Wulf et al 1979 widely
`employed to study actin microfilaments fluorescent taxol
`derivatives could be extremely useful
`to study cellular
`as well as the mechanisms of microtubule
`microtubules
`assembly and stabilization by taxol The complex chemis
`try of taxol Kingston 1994 Nicolaou and Guy 19951
`made difficult obtaining watersoluble significantly active
`derivatives labeled with appropriate fluorescent groups
`but this was recently accomplished Souto et al 19951 In
`the present work we have used fluorescent taxoids with
`and selectivity
`to probe the taxol
`adequate
`affinity
`binding site of in vitro assembled and cellular microtu
`bules Cytoplasmic microtubules mitotic spindles and
`centrosomes
`are directly and specifically visualized and
`
`studied with fluorescent taxoids
`
`MATERIALS AND METHODS
`Fluorescent Taxoids and Other Materials
`
`70 N4 fluoresceincarbonyl Lalanylltaxol
`FLUTAX and 704N4coumarincarbony1Lalanyll
`taxol CUTAX were obtained by the reaction of 70L
`alanyltaxol with the corresponding amine reactive fluo
`rescent dye as described Souto et al 19951 Other
`fluorescent taxoids were obtained by this general method
`and purified by preparative Thin layer chromatography
`TLC They gave a single spot on TLC plates with
`several eluents 2 OAcetyl7O N4 fluoresceincar
`2 AcFLUTAX was obtained
`bony1Lalanylltaxol
`from 2 0acetyltaxol Mellado et al
`19841 by its
`and sub
`conversion into 2 0acetyl70Lalanyltaxol
`sequent reaction with 4 carboxyfluorescein
`ester Molecular Probes C2210 Eugene OR N4
`methyl ester was prepared
`fluoresceincarbony1Lalanine
`ester with
`the same succinimidyl
`by the reaction of
`Lalanine methyl ester hydrochloride Fluka 05200 Buchs
`Switzerland
`70F4 tetramethylrhodaminecarbony1Lalanyll
`taxol ROTAX was synthesized from 70Lalany1
`taxol and 4 carboxytetramethylrhodamine
`succinimidyl
`ester Molecular Probes C2211 and its structure Fig 1
`was confirmed as follows HRFAB+MS mNBA miz
`13375141 MH± calculated for C83H85N401913375182
`1H NMR 300 MHz CDC13 298 K TMS 8 = 838 s
`1H H3 R 820 d 1H H5 R 807 d 2H o H of
`Pha 769 d 2H oH of Phc 769719 band 11H
`
`succinimidyl
`
`

`

`MicrotubuleFluorescent
`
`Taxoid Interactions
`
`75
`
`trile
`
`gradient
`
`600M11 €555 = 80000 M1 cm
`
`s 3H 10CH3C0 209 m 1H H6b 179 s 6H
`H18 H19 134 d 3H CH3 of Ala 118 s 3H H17
`116 s 3H H16
`FLUTAX CUTAX ROTAX and 2 AcFLUTAX
`had ultraviolet UV visible absorbance and fluorescence
`emission spectra in accordance with the respective chro
`mophores Highperformance
`liquid chromatography
`HPLC purity 228 nm was FLUTAX 91 03
`taxol 2 AcFLUTAX 89 undetectable taxol 08
`FLUTAX C18 25 X 04 cm column 2080 acetoni
`in 005 trifluoroacetic
`acid in water
`ROTAX 95 undetectable taxol C4 column in the
`of FLUTAX ROTAX
`same eluent The concentrations
`and 2 AcFLUTAX were determined spectrophotometri
`cally in 05 sodium dodecyl sulfate SDS 50 mM
`sodium phosphate buffer pH 70 respectively employing
`€458€458 =
`the practical extinction coefficients €458 = = 23100 ±
`16600 ± 600 M1 cm 1 obtained by weighing and by
`SDS solubilization of the taxoids Similar to taxol Song
`et al 19961 FLUTAX partially
`adsorbs to glass and
`plastic containers from which it can be recovered with
`05 SDS Without SDS about 1525 of 2 to 25µM
`FLUTAX solutions would adsorb
`to the walls of a
`spectrophotometer cell Ligand solubilities were deter
`mined by ultracentrifugation of 02 ml aliquots at 100000g
`and 25°C during 10 min and measuring the concentration
`in the upper and lower half of the tubes
`Taxol Paclitaxel was in part a gift
`Myers Squibb Princeton NJ the owner of the regis
`from
`tered trademark Docetaxel Taxotere was a gift
`RhonePoulenc Rorer Autong France SDS was from
`BioRad Hercules CA Other chemicals were as de
`scribed Diaz and Andreu 1993 De Ines et al 19941
`unless otherwise indicated
`
`from Bristol
`
`Proteins Microtubule Assembly Binding of
`Ligands and Spectroscopic Measurements
`
`diphosphate GDPtubulin was
`Purified guanosine
`prepared from calf brain as described elsewhere Diaz
`
`and Andreu 19931 GDP from Sigma St Louis MO
`sodium salt type I undetectable GTP in HPLC was now
`employed giving 95 ± 3 GDPliganded tubulin in the
`
`HPLC nucleotide analysis GDPtubulin was assembled
`with a 121 taxoidtubulin molar ratio except otherwise
`indicated in PEGM buffer pH 66 at 37°C for 2 hr
`followed by 10 min centrifugation of 02 ml aliquots at
`100000g 37°C Pellets were resuspended in cold PEDTA
`buffer or in 10 mM sodium phosphate buffer 025 SDS
`pH 70 FLUTAX and tubulin concentrations
`in pellets
`and supernatants were determined fluorometrically in
`aliquots diluted in 025 SDS with the appropriate
`standards and taxol was determined by HPLC with
`as an internal standard Bovine microtubule
`docetaxel
`
`H
`OAc 2
`OH oCOPha
`
`cPhCONFI 0
`bPhN1
`H
`
`zO
`
`taxol
`
`FLUTAX
`
`COCHMeNFICO
`
`CO2H
`
`OH
`
`ROTAX
`
`COCHMeNFICO
`
`5R
`
`40
`
`1R
`
`3R
`3R
`
`CO2
`
`8R7 7R
`
`Me2N
`
`0
`
`4R
`
`NMe2
`
`5R
`
`Fig 1 Chemical structures of the fluorescent
`ployed in this work
`
`taxol derivatives em
`
`m and pH of Pha o m and pH of Phb m and pH
`of Phc 700 d 1H NH 651648 band 2H H 2R
`H7R 643 and 642 two s 2H H4R H5R 637628
`band 3H H 1R H 7R H6 R 627 s 1H H10 612
`t 1H H13 575563 band 3H H2 H7 H3
`490
`m 2H H5 CH of Ala 474 d 1H H2
`425 d 1H
`H20a 416 d 1H H 20b 388 d 1H H3 357
`broad s 2 OH 292 s 12H 2xNCH32 252 m 1H
`H6a 234 s 3H 4CH3C0 230 m 2H H14 224
`
`

`

`76
`
`Evangelio et al
`
`2 uranyl
`
`protein was prepared as described de Pereda et al 19951
`and assembled 4 mgml in 100 mM Pipes 1 mM EGTA
`1 mM MgC12 1 mM GTP pH 69 at 37°C Aliquots of
`noncentrifuged microtubules were examined by fluores
`cence microscopy see below or adsorbed to Formvar
`and carbon coated copper grids negatively stained with
`and photographed with a Philips
`acetate
`EM400 electron microscope
`Absorption spectra were recorded with Varian Cary
`3E and Hitachi U2000 spectrophotometers Light scatter
`ing by microtubules was corrected by double logarithmic
`back extrapolation from non absorbing spectral
`Andreu and Timasheff
`19841
`Fluorescence
`spectra
`were acquired with Shimadzu RF540 and SLM 8000 D
`lifetime steadystate
`Fluorescence
`spectrofluorimeters
`anisotropy measurements were per
`and time resolved
`formed as described elsewhere Mateo et al 19911
`
`regions
`
`Cell Lines Cytoskeletons and Fluorescence
`Microscopy
`
`PtK2 potoroo epithelial like kidney cells U937
`monocytic human leukemia and K562 human myelocytic
`leukemia cells were grown as described previously De
`Ines et al 1994 Neuro 2A mouse neuroblastoma was
`grown in DME nutrient mixture F12 Ham Sigma with
`10 fetal calf serum FCS 2 mM glutamine nonessen
`and antibiotics
`tial amino acids
`Trypanosoma cruzi
`strain Tulahuen Tagliaferro and Pizzi 19551 was cul
`tured as described Baum et al 19811 Coverslip
`attached PtK2 cytoskeletons were prepared
`as described
`De Ines et al 19941 They were either directly observed
`fixed with 02 glutaraldehyde in PEM at
`room
`or
`temperature for 15 mM and then for 15 mM in 2 mgml
`NaBH4 These mildly fixed cytoskeletons were sequen
`tially incubated with DM1A monoclonal antibody Sigma
`diluted 1400 in PBS 10 mgml BSA 1 hr at 37°C
`biotinylated sheep antimouse immunoglobulins Amer
`sham diluted 1400 45 mM and streptavidinTexas Red
`Amersham Amersham Buckinghamshire UK di
`luted 1400 30 min 1 µM FLUTAX was added into the
`In other experiments unfixed PtK2
`three incubations
`labeled with FLUTAX were photographed
`prior to fixation with methanol 10 min at 20°C and
`acetone 6 min at 20°C incubated with TU30 mouse
`monoclonal antibody to ytubulin diluted 18 NovakOva
`et al 19961 and C85 purified rabbit monospecific antibod
`ies to utubulin Arevalo et al 19901 40 µgm1 for 1 hr at
`37°C followed by incubation with biotinylated sheep
`antimouse immunoglobulins 11000 for 45 min and
`red 11000 and fluores
`finally with streptavidinTexas
`immunoglobulins Sigma 140
`ceinated goat antirabbit
`for 30 min Cells and cytoskeletons were mounted in 013
`M glycine buffer pH 86 containing 020 M NaC1 70
`glycerol and 005 µgm1 Hoescht 33342 Sigma Living
`
`cytoskeletons
`
`cells were mounted in fluorescent
`taxoid containing
`culture medium to which propidium iodide 1 µgm1
`Sigma and Hoescht 33342 005 µgm1 had been added
`just before observation living cells excluded propidium
`iodide and their nuclei were stained with Hoescht 33342
`through 63X
`were observed
`Cells and cytoskeletons
`PlanApochromat and PlanNeofluar objectives with Zeiss
`Axioplan epifluorescence microscopes with appropriate
`filter combinations for Hoescht 33342 fluorescein rhoda
`mine and Texas red their images were recorded
`sepa
`rately either on Kodak TMAX film and scanned with an
`Ektron 1412 digital
`imaging camera system or with a
`Photometrics 200 KAF1400 cooled CCD camera and
`software and were printed with Adobe
`IPLab Spectrum
`Photo shop
`
`RESULTS
`
`The chemical structures of fluorescein and rhoda
`mine labeled taxoids prepared by conjugation through the
`free amino group of 70Lalanyltaxol with amine
`dyes Souto et al 19951 Materials and Meth
`reactive
`ods are shown in Figure 1 FLUTAX the main probe
`employed in this work is fully >02 mM soluble in 10
`mM sodium phosphate buffer at pH 85 and insoluble at
`pH 40 The solubility of FLUTAX and 2 AcFLUTAX
`at pH 70 is approximately 80 of the initial
`concentra
`tion <01 mM 50 at pH 66 and further reduced by 7
`mM MgC12 The solubility of FLUTAX increases in the
`presence of
`tubulin and has been employed without
`problems in cell culture media containing 10 FCS The
`solubility of ROTAX in aqueous buffers at neutral pH is
`approximately 1 µM
`
`Microtubule Assembling and Binding Activities of
`Fluorescent Taxoids
`
`test
`
`A stringent biochemical
`for the activity of
`taxoids is their ability to induce the assembly of microtu
`bules from the otherwise inactive GDPliganded form of
`tubulin Diaz and Andreu 19931 Figure 2A shows the
`time courses of assembly of microtubules induced by
`line 1 FLUTAX line 2 and the rhodamine
`taxol
`analogue ROTAX line 3 indicating that the fluorescent
`
`slower assembly inducers
`than
`are slightly
`analogues
`taxol As negative controls we employed the fluorescein
`moiety N4 fluoresceincarbony1Lalanine methyl es
`ter line 4 and 2 AcFLUTAX line 5 which lacks the
`essential 2 hydroxy group of
`the taxol
`side chain
`Williams et al 1996 JimenezBarbero
`et al submit
`ted The polymers induced by FLUTAX and ROTAX are
`microtubules as shown in Figure 2BC although possible
`modifications in their substructure as those induced by
`to be ad
`the parent drug see Introduction are still
`dressed Sedimentation measurements of assembled tubu
`
`

`

`MicrotubuleFluorescent
`
`Taxoid Interactions
`
`77
`
`two and fourfold larger than that of
`are respectively
`taxol Comparative critical concentration measurements
`analyzed with the theory of linked functions Wyman and
`Gill 1990 Diaz et al 19931
`have indicated that
`the
`magnesium dependence of the FLUTAXinduced poly
`merization is more pronounced
`than that of taxol with
`29 instead of 19 additional Mg2+
`ions bound per
`tubulin polymerized Fig 3B The pH dependence
`is
`slightly less pronounced 02 instead of 05 additional H±
`ions per tubulin and the effect of temperature is more
`for FLUTAX than for taxol the value of the
`pronounced
`apparent vant Hoff enthalpy change was 123 instead of
`64 kJ mo11 not shown
`Spectrofluorometric measurements of ligand bound
`to the microtubule pellets confirmed that each assembled
`tubulin dimer binds 097 ± 008 FLUTAX molecules not
`shown the same as taxol Diaz and Andreu 19931
`FLUTAX was also bound by preassembled microtubules
`containing microtubule associated proteins in an approxi
`mately unitary ratio with the tubulin dimers although
`FLUTAX precipitation in Pipes buffer prevented accurate
`quantitation not shown Measurements of competition
`between FLUTAX and taxol for binding to microtubules
`Fig 3C points are quantitatively described by a simple
`model of binding to the same microtubule
`site solid
`lines with an apparent dissociation constant
`8 times
`larger for FLUTAX than for taxol
`
`Spectroscopic Changes in Fluorescent Taxoid
`upon Binding to Microtubules
`
`The fluorescence
`spectrum and lifetime of
`the
`fluorescein chromophore covalently bound to 7alanyl
`taxol as well as the chromophore ionization constant are
`very similar to those of
`free fluorescein in solution
`Although FLUTAX was initially
`as a taxol
`designed
`derivative substituted at nonessential position 7 Kings
`ton 19941 very probably out of the binding site it has
`to be a microenvironmentsensitive
`probe
`turned out
`Interestingly when FLUTAX is bound to microtubules
`there is a 3nm red shift
`in the absorption band of the
`fluorescein dianion 493 nm and an increase relative to
`the absorption of the shoulder at 464 nm Fig 4A and
`Table I These changes could be determined with accu
`racy by comparison with the inactive controls N4
`methyl ester and 2 Ac
`
`fluoresceincarbony1Lalanine
`FLUTAX showing that
`the fluorescein dianion the
`emitting species in FLUTAX Fig 4B is preferentially
`bound by microtubules This suggests a cationic microen
`vironment around the fluorescent probe in microtubules
`Comparative titrations have indicated a small but signifi
`cant down shift of 03 ± 01 pH units in the apparent pKa
`of FLUTAX upon binding to microtubules relative to its
`value in free solution 654 ± 003 MP Lillo et al
`
`unpublished observations
`
`r
`
`080
`
`060
`
`nm
`
`A
`
`040
`
`020
`
`000
`
`4
`
`5
`
`2
`
`3
`
`1
`
`I
`
`I
`
`1
`
`I
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100 120 140 200
`
`Time min
`
`bsorbance4
`
`00
`
`AA
`
`taxoids A
`Fig 2 Microtubule
`assembly induced by fluorescent
`records of 25 itM purified GDPtubulin in PEGM buffer
`Turbidimetric
`pH 66 at 37°C assembled with 30 itM taxol 1 FLUTAX 2
`ROTAX 3 N4 fluoresceincarbony1ralanine methyl ester 4 or
`2 AcFLUTAX 5 B Electron micrograph of microtubules
`as
`stained with uranyl acetate C
`sembled with FLUTAX negatively
`Microtubules assembled with ROTAX Bar = 100 nm
`
`lin at varying total FLUTAX concentrations
`indicated an
`apparent stoichiometry of one FLUTAX bound per tubu
`lin not shown
`Taxoidinduced assembly of GDPtubulin is a rigor
`ous equilibrium protein condensation polymerization in
`which the critical protein concentration the minimal
`required for assembly is to a good approxi
`concentration
`to the apparent equilibrium constant
`mation equivalent
`of dissociation of one aBtubulin molecule from the
`microtubule end Oosawa and Asakura 1975 see Diaz et
`al 19931 The measurements of Figure 3A indicate
`of FLUTAX and ROTAX which
`critical concentrations
`
`

`

`1
`
`1 6
`
`1
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`10
`
`08
`
`06
`
`04
`
`02
`
`00
`
`LigandpProtein1
`
`14
`
`a 13
`
`12
`
`11
`
`10
`
`62 60 58 56
`
`54 52
`
`78
`
`Evangelio et al
`
`A
`
`A
`
`rad
`rEi not E
`9YWIC
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`30
`
`25
`
`20
`
`15
`
`10
`
`5
`
`0 L
`
`0
`
`pM
`
`TUBULIN
`
`TUBULINo
`
`µN1
`
`Ln a
`
`Mg2+
`
`Ln Flutaxs
`
`Faxogs
`
`Fig 3 A Critical concentration Cr plots for GDPtubulin assembly with
`taxol drdea c 15 µM FLUTAX triangle Cr 26 µM and ROTAX
`scluaresl Cr 55 µM under same conditions as Figure 2 The concentration
`of pelleted protein filled symbols and protein in the supernatant open
`symbols is plotted as a function of total tubulin concentration B Linkage
`constant Km
`graphs Wyman and Gill 1990 of the apparent elongation
`Cril of FLUTAX filled d rd es and taxolinduced open d rd es polymer
`ization of tubulin versus activity of Mg2+ ions The slopes 29 and 19
`to the increment of Mg2+ ions preferentially bound
`respectively are equal
`the protomer C Competition of FLUTAX
`by the tubulin polymer over
`filled d rd es and taxol open d rd es for binding to microtubules assembled
`
`06
`
`0 c
`
`o 04
`
`02
`
`00
`300
`
`and 50 µM total
`from 45 µM GDPtubulin
`performed essentially as described for docetaxel and taxol Diaz and Andreu
`1993 in PEGM buffer at pH 70 and 37°C 7582 of total tubulin was
`assembled The ratio of free FLUTAX to free taxol concentration
`
`taxoid The experiment was
`
`abscissa
`
`was measured in the supematant It was modified by varying the ratio total
`taxol The extents of binding of FLUTAX filled circles and
`FLUTAXtotal
`taxol empty el roles change whereas their sum squares remains practi
`cally constant The lines are the best fit
`to a simple model of competition for
`the same site in assembled tubulin Diaz and Andreu 1993 which indicates
`a ratio of apparent affinities of FLUTAX to taxol of 012
`
`10
`
`08
`
`cd
`
`71
`
`g 04
`
`cn0
`
`02= 02
`
`LL
`
`I
`
`I
`
`350
`
`400 450 500 550
`
`Wavelength
`
`nm
`
`520
`560
`600
`Wavelength nm
`
`Fig 4 A Absorption
`spectra of free FLUTAX 2 µM in PEGM buffer pH 66 line 1 absorbance
`multiplied by 10 FLUTAX bound to microtubules 20 µM line 2 same conditions plus 25 µM tubulin
`notice the light scattering due to microtubules and free FLUTAX 20 µ111
`in the same buffer dashed line 3
`due to FLUTAX aggregation B Corrected
`shown for comparison
`notice the absorption changes
`fluorescence spectrum of FLUTAX in microtubules Xex = 470 nm slit 2 nm The normalized spectrum of
`free FLUTAX in buffer was marginally wider at the red side
`
`anisotropy r of
`The
`fluorescence
`stationary
`FLUTAX increased by an order of magnitude to 02
`when bound to microtubules compared with the value in
`solution Table I nevertheless this is still onehalf of
`
`what would be observed for the completely immobilized
`dye ro470 = 038 Chen and Bowman 19651 The
`lifetime of FLUTAX in solution and bound
`fluorescence
`to microtubules was the same 45 nsec The time
`
`

`

`MicrotubuleFluorescent
`
`Taxoid Interactions
`
`79
`
`TABLE I Spectroscopic Parameters of Free and MicrotubuleBound FLUTAX in PEMG Buffer at 37°C
`b M1 cm 1 0D4930D464
`xr
`nm
`
`maxnm
`
`X
`
`Concn
`11A
`
`FLUTAX
`
`Ea41a6r
`
`i3000
`
`±005
`
`QtFel
`
`nsd
`±02
`
`re
`±0004
`
`nsr
`
`18
`21
`21
`
`08
`10
`08
`
`45
`
`0028
`0030
`020
`
`4 it
`
`1
`
`006
`
`33000
`43000
`
`46000
`
`492
`
`493
`
`496
`
`522
`
`522
`
`522
`
`2 2
`
`20
`
`Free PEGM pH 66
`Free PEGM pH 70
`Bound PEGM pH 66
`Corrected emission spectra Xex = 470 nm slit 2 nm
`bEffective molar absorption coefficient
`fluorescence yield Xex = 470 nm bandwidth 2 nm reference QF = 10 for FLUTAX 2 itM in PEGM buffer pH 70
`lifetime model
`decays were best
`fitted to a single exponential
`Xex = 470 nm >kern = 520 nm slit 2 nm
`rAnisotropy relaxation time di and residual anisotropy r Xex = 380 nm >kern > 500 nm cutoff
`
`eRelative
`
`dFluorescence
`
`eSteadystate fluorescence anisotropy
`
`filter
`
`restrictions
`
`resolved analysis of rt contains
`a very fast hundreds of
`picoseconds initial depolarization from r0380 = 024
`to 018 that could not be resolved with our instrumenta
`tion This is followed by a slower depolarization process
`with a time constant of 4 ± 1 nsec to finally attain a
`residual value roo = 006 Table I The biphasic depolar
`ization results from the very fast unrestricted angular
`motions of the dye moiety followed by a slower motion
`with its supporting molecular structures while the non
`roo value is characteristic of
`zero residual
`the angular
`imposed to probe rotation by the protein
`matrix Note that application of the diffusion inacone
`results in wobbling semi
`model Kinosita et al 19771
`angles of 66° and 80°
`for the fast and slow motions
`respectively A possible alternative to account
`for the
`depolarization might be the resonant
`time dependent
`transfer of energy between identical chromophores homo
`transfer Van der Meer et al 19941 However
`the
`contribution of homotransfer to the depolarization in the
`present case is considered to be less than 20 This upper
`limit was obtained from the changes observed in anisot
`ropy by FLUTAX excitation in the red edge of
`absorption spectra Valeur and Weber 19781 In addition
`numerical modelling of the transfer rate in a microtubule
`lattice excludes homotransfer as a cause of the very fast
`initial depolarization
`
`its
`
`Observation and Self organization of Microtubules
`With Fluorescent Taxoids
`
`assembled
`from GDPtubulin with
`Microtubules
`FLUTAX and ROTAX are readily visualized under the
`fluorescence microscope either as individual microtu
`bules or with different degrees of entanglement Fig
`5AB Radial structures resembling asters could be
`assembled
`between
`observed
`samples
`in microtubule
`glass slide and coverslip at room temperature inset Fig
`5B Dense microtubule bundles Fig 5C consistently
`formed irrespective of microtubules being polymerized
`either in a tube at 37°C or directly between glasses When
`from tubulin and MAPs
`microtubules were assembled
`the further addition of FLUTAX resulted in rapid <1
`
`mm labeling with the fluorescent
`taxoid as shown in
`Figure 5D This confirmed
`that FLUTAX binds to
`irrespective of MAPs see above
`and
`microtubules
`taxoid would also bind to
`the fluorescent
`
`suggested that
`cellular microtubules
`
`Specific Labeling of Microtubules and
`Centrosomes of Cytoskeletons With FLUTAX
`Comparison With Antitubulin Antibodies
`FLUTAX 1 itM was
`found to bind to and
`stabilize microtubules of unfixed PtK2 cytoskeletons
`in
`PBS making microtubules readily observable under the
`fluorescence microscope not shown Fixation of cyto
`skeletons with formaldehyde or methanol destroyed the
`FLUTAX binding sites fixation of cells incubated with
`20 itM FLUTAX with 37 formaldehyde and methanol
`fixation with 02
`gave the same result However
`glutaraldehyde permitted the simultaneous visualization
`of microtubules with both FLUTAX and monoclonal
`antibodies to tubulin As shown in Figure 6ABCD the
`same individual cytoplasmic microtubules can be stained
`by FLUTAX and the DM1A antibody although the taxoid
`detected somewhat weakly the fixed microtubules Bind
`ing of FLUTAX is specific since 1 it
`is displaced by
`excess docetaxel 50 itM not shown and 2 most of the
`
`microtubules disappear by previous cell
`treatment with
`inhibitor CI980025 itM De Ines et al
`the powerful
`19941 Fig 6CD There
`are several
`important differ
`to antibodies FLUTAX stained centro
`ences with respect
`somes more accurately
`and brightly than the antibody
`both in interphase Fig 6CD and in mitotic cells Fig
`6EJ and stained astral microtubules less intensely than
`Two bright
`spots could be frequently
`the antibody
`resolved with FLUTAX in the centrosomes of interphase
`cells The anaphase and telophase
`spindles frequently
`the equator corre
`showed a characteristic dark line at
`sponding to the midzone of the cleavage furrow when
`fixed with 02 glutaraldehyde were visu
`alized by immunofluorescence with DM1A Fig 6H and
`other antibodies to tubulin not shown note that this zone
`may be stained after methanol fixation However micro
`
`cytoskeletons
`
`

`

`80
`
`Evangelio et al
`
`taxoids under the epifluorescence microscope A Microtu
`Fig 5 Microtubules
`imaged with fluorescent
`bules assembled with 25 gM tubulin and FLUTAX B Another view of FLUTAX microtubules
`of FLUTAX microtubules C Bundles of ROTAX microtubules D
`radial
`asterlike formation
`Microtubules assembled with MAPs and visualized immediately after addition of FLUTAX and dilution to
`microtubule protein Bar = 10 gm
`04 mg
`
`inset a
`
`

`

`MicrotubuleFluorescent
`
`Taxoid Interactions
`
`81
`
`indirect
`
`Fig 6 Simultaneous binding of FLUTAX ACEGI and the DM1A monoclonal antibody to tubulin
`immunofluorescence BDFHJ to mildly fixed PtK2 cytoskeletons AB Interphase cells CD
`Cells treated with 025 gM CI980 for 4 hr to depolymeryze microtubules EF Anaphase spindle GH
`spindle Lb Cytokinesis Bars = 10 gm Bar in C for C and D bar in I for all other panels
`
`Telophase
`
`

`

`82
`
`Evangelio et al
`
`tubules were clearly labeled in the midzone by FLUTAX
`Fig 6EG Consequently the central zone of midbodies
`was much more strongly stained by the taxoid Fig 61 It
`should be noted that marked staining of the central zone
`of midbodies was also observed with ROTAX in unfixed
`not shown and with FLUTAX in living
`cells see Fig 9
`
`cytoskeletons
`
`To further characterize
`the centrosomal staining
`with FLUTAX it was compared to detection with TU30
`antibody to ytubulin Novakova
`et al
`monoclonal
`19961 after recording the corresponding FLUTAX im
`ages of cytoskeletons The two bright spots labeled by
`FLUTAX in interphase cells presumably corresponding
`to two centrioles Fig 7A coincide with the zone
`detected by the antibody to ytubulin Fig 7B Co
`localization was equally observed after centrosomal divi
`sion in prophase Fig 7EF and in anaphase cells Fig
`7IJ and only one FLUTAX spot per centrosome was
`distinguished in these cases
`
`Observation of Unfixed Cellular Microtubules and
`Living Cells With FLUTAX
`
`Optimal microtubule visualization was obtained
`shortly after <1mM FLUTAX addition to unfixed Ptk2
`in PEM microtubulestabilizing buffer
`cytoskeletons
`ROTAX gave microtubular
`images but also nonmicrotu
`bular vesicular staining possibly due to the binding of
`rhodamine to other organelles and its use with cells was
`not further pursued FLUTAX is a specific microtubule
`visualization probe since the microtubule images were
`and a few microtubules of
`reduced to the centrosomes
`Ptk2 cells by pretreatment with 10 laM colchicine not
`shown and were erased by an excess of nonfluorescent
`taxoid in a few minutes or with 2 AcFLUTAX instead
`of FLUTAX Fig 8AB note that the nucleolar staining
`by FLUTAX in cytoskeletons is nonspecific
`since it
`is
`not displaced by an excess of nonfluorescent taxoid On
`the other hand treatment of a variety of cells with 1
`laM
`FLUTAX during 054 hr or culturing the cells with
`of the probe followed sim
`subinhibitory concentrations
`ply by removal of excess medium and directly mounting
`the coverslips with 70 glycerol containing buffer gave
`good images of the cellular microtubule network
`typi
`than immunofluorescence of
`cally with less background
`examples of Ptk2 epithelial
`fixed cells Representative
`cells U937 leukemia cells neuro2A neuroblastoma cells
`and Trypanosoma cruzi directly stained with FLUTAX
`are presented in Figure 8CF respectively Each of these
`images disappeared when 50 laM docetaxel was added to
`the cultures simultaneously with FLUTAX not shown
`The images shown in Figure 8CF do not result
`from FLUTAX internalized and bound by living cells but
`its intensity is mai