centromeres were classically defined as the primary constriction of chromosomes  a region where paired sister chromatids are held tightly together until the onset of anaphase .
classic microscopic observations revealed that this was the region where mitotic chromosomes attached to the spindle microtubules . in the 1960s , electron microscopy first recognized that the surface of the centromere was the site of assembly of a button - like structure  the kinetochore  which formed the actual point of microtubule attachment ( luykx 1965 ; brinkley and stubblefield 1966 ; jokelainen 1967 ) . in subsequent years , studies by a great many laboratories have developed a still emergent picture of the kinetochore as one of the most wonderfully complex supramolecular structures found in cells ( maiato et al .
2004 ; cheeseman and desai 2008 ; hori and fukagawa 2012 ; westhorpe and straight 2013 ) . for years
, there was an ongoing debate about the distinction ( if any ) between centromeres and kinetochores .
both terms were initially proposed to describe essentially the same aspect of chromosome structure and function , so this seemed liked a semantic discussion . however , in the post - genomic era of cloned and sequenced genomes , a meaningful distinction has emerged .
it is now generally accepted that the underlying dna sequence that defines the primary constriction of mitotic chromosomes is the centromere , whereas the proteinaceous structure that assembles on the surface of the centromeric chromatin is the kinetochore .
the histone h3 variant cenp - a is a specific protein marker for centromeres ( earnshaw and rothfield 1985 ; earnshaw et al .
2013 ) , whereas kinetochores are characterized by the presence of > 100 different proteins , the earliest described being cenp - c ( earnshaw and rothfield 1985 ; saitoh et al .
studies of the centromere have been more limited , in part due to its complex repetitive substructure . in budding yeast , the point centromere ( pluta et al .
1995 ) is defined by a 125  bp dna sequence ( hegemann and fleig 1993 ) . in fission yeast and vertebrates
, there is no single sequence that defines the centromere , with cenp - a being distributed across wide regions of dna .
we therefore coined the term  regional centromeres  to describe most centromeres outside budding yeast ( pluta et al . 1995 ) . with the advent of increasingly sophisticated molecular approaches , and particularly deep sequencing
, it is now possible to begin to define the centromeric dna in vertebrates by chip - seq in a few specialized instances .
detailed cenp - a mapping is not possible for most metazoan centromeres because they are comprised of hugely redundant families of repetitive dna that have foiled efforts to create contiguous linear maps .
1997 ; barry et al . 1999 ; burrack and berman 2012 ) , chromosome 11 of the horse ( wade et al .
2009 ) , and chromosomes z , 5 , and 27 in chicken ( shang et al .
all of those centromeres are assembled on unique sequence dna , so that it is possible to exactly map the extent of the cenp - a domain .
even though the cenp - a domain can not be mapped precisely in humans , analysis of extended chromatin fibers has revealed that centromeres are comprised of intercalated domains of cenp - a chromatin and chromatin containing centromeric canonical histone h3 ( blower et al .
2002 ; sullivan and karpen 2004 ; ribeiro et al . 2010 ; sullivan et al . 2011 ) .
this observation lead to a working model in which the centromeric chromatin formed an amphipathic - like solenoid , with the cenp - a - containing surface at the chromosome exterior abutting the kinetochore and a h3-containing surface facing the interior of the chromosome ( sullivan and karpen 2004 ) .
more recent super - resolution mapping of various kinetochore protein pairs ( joglekar et al . 2009 ; wan et al .
2013 ) and observation of antibody - labeled chromatin fibers led to the proposal of a boustrophedon model of the kinetochore chromatin as multi - layered sinusoidally folded patch on the chromosome surface , rather than a helix embedded in it ( ribeiro et al .
one parameter that is essential in establishing detailed models for the folding of the centromeric chromatin fiber in the inner kinetochore is the amount of dna in the assembled kinetochore . by this , we do not mean the amount of dna occupied by cenp - a , as determined by chip - seq , but instead the amount of dna that is folded into the three - dimensional functional mitotic kinetochore .
this has not been measured , largely because the inner kinetochore is embedded in the centromeric chromatin , and can not be distinguished from the surrounding dna in compact mitotic chromosomes by conventional microscopy methods .
the aim of the present study was to take advantage of a chicken dt40 condensin mutant in which kinetochores undergo  excursions  during which they are occasionally stretched considerable distances from the surface of the centromeric chromatin ( ribeiro et al .
2009 ) . under those circumstances , the kinetochore is spatially isolated away from the rest of the underlying centromeric chromatin .
we have used quantitative fluorescence to estimate the amount of dna within the folded kinetochore domain as defined by cenp - h - gfp , expressed from its own promoter and exogenous cenp - a - gfp .
this analysis revealed that dt40 kinetochores in their native conformation contain ~5060  kbp of dna , a number that is remarkably close to 50150  kbp size of the cenp - a domain measured in human neocentromeres ( barry et al .
1999 ; alonso et al . 2007 ) and the 41    6  kbp size of the cenp - a domain measured by chip - seq for 12 chicken centromeres and neocentromeres ( shang et al .
thus , when kinetochores assemble on mitotic chromosomes , they do not incorporate significant amounts of pericentromeric dna surrounding the core cenp - a domain .
the distribution of kinetochore proteins along the dna has been mapped at vertebrate neocentromeres by a number of methods ( alonso et al . 2007 ; shang et al . 2013 ; saffery et al . 2003 ; capozzi et al .
2008 ) , but it is not known how much dna is folded into a functional mitotic kinetochore .
our published studies have established that kinetochores of smc2-depleted chicken dt40 cells are structurally and functionally normal ( ribeiro et al .
it is , however , required to regulate the compliance ( stretchiness ) of the pericentromeric heterochromatin , which acts as a spring linking the two sister kinetochores ( ribeiro et al .
therefore , because condensin - depleted kinetochores are spatially well resolved from the surface of the primary constriction when undergoing  excursions 
1a , b ) , they can be used to determine the dna content of a functioning kinetochore in situ by quantifying the dna within the compact cenp - h and cenp - a domains.fig .
1quantification of dna in a functional vertebrate kinetochore . a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna .
inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5  m ) quantification of dna in a functional vertebrate kinetochore .
a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna .
inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5  m ) the strategy adopted here was to image metaphase kinetochores of smc2-depleted cells that were expressing either cenp - h - gfp or cenp - a - gfp as three - dimensional image stacks , identify the volume occupied by cenp - h and cenp - a , and then ask how much dna ( stained with dapi ) was present within that volume ( fig . 
, the dapi staining was measured relative to a standard present in the same image .
cenp - h was used in these experiments because the single copy cenp - h gene on the chicken z chromosome had been labeled by knock - in of a gfp cdna ( fukagawa et al .
thus , the fusion protein was expressed from its native promoter and was able to support life of the cells . in the case of gfp - cenp - a , the fusion protein was a transgene expressed in a stable cell line . other work in human ( foltz et al .
2013 ) cells has shown that there appears to be a homeostatic regulation of cenp - a levels , such that when exogenous cenp - a is expressed , levels of the endogenous protein fall , so that overall cenp - a levels remain similar ( foltz et al .
furthermore , chip - seq analysis in chicken has shown that the size of cenp - a chromatin domains is not significantly increased following expression of exogenous cenp - a - gfp ( shang et al .
, we initially used s. cerevisiae cells in g1 phase , but rapidly realized that the amounts of dna in chicken mitotic kinetochores were far smaller than this .
we therefore switched to the use of three bacteriophages , namely : t4 ( genome size 168  kb ) , p1 ( 90  kb ) , and  ( 48  kb ) to calibrate the dna quantification since their genome size is within the range of the dna amounts in the kinetochore . when these bacteriophages were fixed , stained with dapi , and mixed and imaged with the deltavision microscope ( fig . 
2a ) , a linear correlation between dna amount and intensity was obtained ( fig . 
2three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5  m ) .
b standard curve of fluorescence intensity ( dapi  y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5  m ) .
b standard curve of fluorescence intensity ( dapi  y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) this calibration allowed us to quantify the amount of dna in functional mitotic kinetochores .
smc2 cells expressing tagged endogenous ( knock - in ) cenp - h - gfp or exogenous gfp - cenp - a were mixed with bacteriophages and processed for image acquisition .
we looked for cells in which one or more kinetochores had stretched polewards , away from the body of its chromosome and where we could also acquire the three different bacteriophages in the same field of view . in each case , the gfp - labeled protein was used to define a volume envelope for the kinetochore , and the amount of dapi staining within that envelope was measured .
standard curves determined for the bacteriophages in each experiment were used to calibrate the dna intensity values for the cenp - a or cenp - h - containing chromatin ( fig . 
it is worth emphasizing that these gfp - labeled kinetochores were functional at the time of fixation , as they were attached to microtubule bundles and exerting force towards the spindle poles .
furthermore , it is important to note that while the chromatin beneath the kinetochore was abnormally stretched , the kinetochores themselves appeared structurally normal at both the light and electron microscope levels ( ribeiro et al .
2009).fig . 3 
 a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) .
these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna .
c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) .
d summary table showing values obtained from the quantification done in c 
 
 a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) .
these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna .
c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) .
d summary table showing values obtained from the quantification done in c 
  a measurement of 40 centromeres in each case revealed that compact cenp - a and cenp - h domains of functional smc2-depleted kinetochores contained an average of 61 and 54  kb of dna , respectively ( range28147  kb , fig . 
because the kinetochores measured come from a number of different dt40 chromosomes , the observed range of dna content could be in part due to size variation between kinetochores .
however , a recent study in the fukagawa lab has revealed that all of the kinetochores in dt40 cells are of similar size when measured with a variety of kinetochore markers ( t. hori and t. fukagawa , personnal communication ) .
importantly , their measurements included kinetochores assembled on repetitive centromeres as well as those on nonrepetitive centromeres and on both micro and macro chromosomes .
thus , the most likely explanation for the variation is that because kinetochores are pulled away from the primary constriction to differing degrees , the amount of pericentromeric chromatin immediately adjacent to the kinetochore may vary .
the distribution of kinetochore proteins along the dna has been mapped at vertebrate neocentromeres by a number of methods ( alonso et al . 2007 ; shang et al . 2013 ; saffery et al . 2003 ; capozzi et al .
2008 ) , but it is not known how much dna is folded into a functional mitotic kinetochore .
our published studies have established that kinetochores of smc2-depleted chicken dt40 cells are structurally and functionally normal ( ribeiro et al .
it is , however , required to regulate the compliance ( stretchiness ) of the pericentromeric heterochromatin , which acts as a spring linking the two sister kinetochores ( ribeiro et al .
therefore , because condensin - depleted kinetochores are spatially well resolved from the surface of the primary constriction when undergoing  excursions 
1a , b ) , they can be used to determine the dna content of a functioning kinetochore in situ by quantifying the dna within the compact cenp - h and cenp - a domains.fig .
1quantification of dna in a functional vertebrate kinetochore . a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna .
inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5  m ) quantification of dna in a functional vertebrate kinetochore .
a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna .
inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5  m ) the strategy adopted here was to image metaphase kinetochores of smc2-depleted cells that were expressing either cenp - h - gfp or cenp - a - gfp as three - dimensional image stacks , identify the volume occupied by cenp - h and cenp - a , and then ask how much dna ( stained with dapi ) was present within that volume ( fig . 
, the dapi staining was measured relative to a standard present in the same image .
cenp - h was used in these experiments because the single copy cenp - h gene on the chicken z chromosome had been labeled by knock - in of a gfp cdna ( fukagawa et al .
thus , the fusion protein was expressed from its native promoter and was able to support life of the cells . in the case of gfp - cenp - a , the fusion protein was a transgene expressed in a stable cell line . other work in human ( foltz et al .
2013 ) cells has shown that there appears to be a homeostatic regulation of cenp - a levels , such that when exogenous cenp - a is expressed , levels of the endogenous protein fall , so that overall cenp - a levels remain similar ( foltz et al .
furthermore , chip - seq analysis in chicken has shown that the size of cenp - a chromatin domains is not significantly increased following expression of exogenous cenp - a - gfp ( shang et al .
, we initially used s. cerevisiae cells in g1 phase , but rapidly realized that the amounts of dna in chicken mitotic kinetochores were far smaller than this .
we therefore switched to the use of three bacteriophages , namely : t4 ( genome size 168  kb ) , p1 ( 90  kb ) , and  ( 48  kb ) to calibrate the dna quantification since their genome size is within the range of the dna amounts in the kinetochore . when these bacteriophages were fixed , stained with dapi , and mixed and imaged with the deltavision microscope ( fig . 
2a ) , a linear correlation between dna amount and intensity was obtained ( fig . 
2three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5  m ) .
b standard curve of fluorescence intensity ( dapi  y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5  m ) .
b standard curve of fluorescence intensity ( dapi  y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) this calibration allowed us to quantify the amount of dna in functional mitotic kinetochores .
smc2 cells expressing tagged endogenous ( knock - in ) cenp - h - gfp or exogenous gfp - cenp - a were mixed with bacteriophages and processed for image acquisition .
we looked for cells in which one or more kinetochores had stretched polewards , away from the body of its chromosome and where we could also acquire the three different bacteriophages in the same field of view . in each case , the gfp - labeled protein was used to define a volume envelope for the kinetochore , and the amount of dapi staining within that envelope was measured .
standard curves determined for the bacteriophages in each experiment were used to calibrate the dna intensity values for the cenp - a or cenp - h - containing chromatin ( fig . 
it is worth emphasizing that these gfp - labeled kinetochores were functional at the time of fixation , as they were attached to microtubule bundles and exerting force towards the spindle poles .
furthermore , it is important to note that while the chromatin beneath the kinetochore was abnormally stretched , the kinetochores themselves appeared structurally normal at both the light and electron microscope levels ( ribeiro et al .
2009).fig . 3 
 a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) .
these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna .
c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) .
d summary table showing values obtained from the quantification done in c 
 
 a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) .
these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna .
c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) .
d summary table showing values obtained from the quantification done in c 
  a measurement of 40 centromeres in each case revealed that compact cenp - a and cenp - h domains of functional smc2-depleted kinetochores contained an average of 61 and 54  kb of dna , respectively ( range28147  kb , fig . 
because the kinetochores measured come from a number of different dt40 chromosomes , the observed range of dna content could be in part due to size variation between kinetochores .
however , a recent study in the fukagawa lab has revealed that all of the kinetochores in dt40 cells are of similar size when measured with a variety of kinetochore markers ( t. hori and t. fukagawa , personnal communication ) .
importantly , their measurements included kinetochores assembled on repetitive centromeres as well as those on nonrepetitive centromeres and on both micro and macro chromosomes .
thus , the most likely explanation for the variation is that because kinetochores are pulled away from the primary constriction to differing degrees , the amount of pericentromeric chromatin immediately adjacent to the kinetochore may vary .
in order to build a structural model of the vertebrate kinetochore , we need to know not only the length of chromosomal dna occupied by cenp - a but also the way in which that dna is folded in order to build a three - dimensional structure capable of withstanding the pulling forces generated by spindle microtubules .
the earliest model proposed was for one or more stretches of chromatin superhelix with cenp - a facing the chromosome exterior and histone h3 facing the chromosome interior ( sullivan and karpen 2004 ) .
we later proposed a multilayered patch - like model in which the chromatin fibers are folded in sinusoidal boustrophedons ( ribeiro et al .
importantly , neither of these models can exclude the possibility that stretches of chromatin not associated with cenp - a might be incorporated into the folded kinetochore - associated chromatin .
we have here combined gene targeting with deconvolution microscopy to estimate the amount of dna in a fully functional vertebrate kinetochore .
our data indicate that functional chicken kinetochores , defined by the three - dimensional volumes occupied by cenp - a and cenp - h , contain 58    23  kb of dna .
it is important to note that this amount is an estimate , as we have used a single dna - binding dye ( dapi ) , which typically shows a preference for at - rich dna . nonetheless , because the amount of dna contained within each stretched centromere is influenced by the degree of stretching , which can not be readily controlled ,
the values measured in the present experiments show a remarkable similarity to the 41    6  kb span of chromosomal dna occupied by cenp - a on 12 newly generated chicken neocentromeres as measured by chip - seq ( shang et al .
interestingly , the amount of dna in chicken kinetochores is close to values determined by chromatin immunoprecipitation ( chip ) for human neocentromeres , which range from 54 to 464  kb [ reviewed in ( marshall et al .
based on the present data as well as previous studies from our laboratory , we propose that the kinetochore is assembled from a single contiguous chromatin segment in which alternating cenp - a and h3 domains are folded into a planar sinusoidal patch , or boustrophedon . we have previously argued that the boustrophedon is composed of several layers held together by structural crosslinks that depend upon cenp - c for their formation and stability ( ribeiro et al .
present studies from our group aim to determine whether other kinetochore proteins are also required to stabilize the multilayered kinetochore structure and determine the number of nucleosomes per layer . in the future , other super - resolution microscopy approaches will hopefully combine with genome three - dimensional mapping approaches ( e.g. , 3c , 4c , etc .
2013 ) ) to permit the development of more accurate models for the folding of the chromatin fiber in centromeres .
the smc2 conditional knockout cell line , smc2:cenp - h : gfp and smc2:gfp : cenp - a were cultured as previously described ( ribeiro et al .
condensin was depleted from smc2   conditional knockout cells expressing cenp - h - gfp or gfp - cenp - a by 30-h exposure to doxycycline , following which , cells were fixed with paraformaldehyde at 4  % . t4 ,
fifty microliter of each phage were washed in sm buffer ( 5  m nacl , 1  m mgso4 , 1  m tris  hcl ph 7.5 ) , fixed in 4  % pfa - sm for 5  min , and resuspended in 30  l of vectashield with 1.5  g / ml dapi for 20  min . for the quantification analysis , 2  l of the stained phages in vectashield were used to mount the slides with smc2 cells .
this concentration was determined empirically to give a sufficient number of bacteriophages so that each field of view containing a kinetochore undergoing an excursion would have a few bacteriophages in the background .
smc2 immunostaining image stacks were acquired using a microscope ( ix-70 ; olympus ) with a charge - coupled device camera ( ch350 or hq ; photometrics ) controlled by deltavision softworx ( applied precision , llc ) and a 100 s plan apochromat na 1.4 objective using a sedat filter set ( chroma technology corp . ) and running at rt .
we selected for imaging metaphase aligned cells where kinetochores were clearly resolved from the rest of the chromatin ( images of 1,024    1,024 pixels in z - stacks with a 200  nm step size ) . in the same field of view , phages were also acquired and used to define a standard curve between florescence intensity and dna amount .
all files were saved as tiff files and exported to photoshop ( adobe ) for final presentation .
levels were adjusted similarly for each experimental dataset to lower nonspecific background haze using the standard photoshop adjust levels tool .
after deconvolution , we measured the dapi content in the volume occupied by cenp - a or cenp - h . image planes where kinetochore  excursions  were observed were sum - quick projected .
regions of interest ( roi ) covering the phage area were selected by hand , and sum intensity value was annotated .
the same roi was moved between 5 and 10 pixels , and the intensity was annotated as background .
the intensity values for all phages in each experiment were entered in graphpad prism ( graphpad software , inc .
ca , usa ) where a xy graph was plotted and the best - fit linear regression calculated representing the linear standard of amount of dna versus intensity . for the smc2 cells , separated tiff files of the 488 and 528 channels were opened in image - pro plus ( media cybernetics , inc . , bethesda , usa ) . using the 528-nm image , a roi covering the area covered by the pulled centromere was selected .
this roi was copied and loaded into the 488-nm file where the sum value of intensity was determined .
again , the background value was determined by moving the roi 510 pixels away from the metaphase plate .
the absolute amount of dna was calculated according to the equation of the previously determined best - fit linear regression .
the line profile was drawn in imagej ( research national institute of mental health , bethesda , usa ) values exported to excel ( microsoft , redmond , wa ) and graphs of each channel superimposed .
the smc2 conditional knockout cell line , smc2:cenp - h : gfp and smc2:gfp : cenp - a were cultured as previously described ( ribeiro et al .
condensin was depleted from smc2   conditional knockout cells expressing cenp - h - gfp or gfp - cenp - a by 30-h exposure to doxycycline , following which , cells were fixed with paraformaldehyde at 4  % .
fifty microliter of each phage were washed in sm buffer ( 5  m nacl , 1  m mgso4 , 1  m tris  hcl ph 7.5 ) , fixed in 4  % pfa - sm for 5  min , and resuspended in 30  l of vectashield with 1.5  g / ml dapi for 20  min . for the quantification analysis , 2  l of the stained phages in vectashield were used to mount the slides with smc2 cells .
this concentration was determined empirically to give a sufficient number of bacteriophages so that each field of view containing a kinetochore undergoing an excursion would have a few bacteriophages in the background .
smc2 immunostaining image stacks were acquired using a microscope ( ix-70 ; olympus ) with a charge - coupled device camera ( ch350 or hq ; photometrics ) controlled by deltavision softworx ( applied precision , llc ) and a 100 s plan apochromat na 1.4 objective using a sedat filter set ( chroma technology corp . ) and running at rt .
we selected for imaging metaphase aligned cells where kinetochores were clearly resolved from the rest of the chromatin ( images of 1,024    1,024 pixels in z - stacks with a 200  nm step size ) . in the same field of view , phages were also acquired and used to define a standard curve between florescence intensity and dna amount .
all files were saved as tiff files and exported to photoshop ( adobe ) for final presentation .
levels were adjusted similarly for each experimental dataset to lower nonspecific background haze using the standard photoshop adjust levels tool .
after deconvolution , we measured the dapi content in the volume occupied by cenp - a or cenp - h . image planes where kinetochore  excursions 
single planes where phages were observed were selected for each image acquired . for the phage , the area was defined in the dapi channel .
regions of interest ( roi ) covering the phage area were selected by hand , and sum intensity value was annotated .
the same roi was moved between 5 and 10 pixels , and the intensity was annotated as background .
the intensity values for all phages in each experiment were entered in graphpad prism ( graphpad software , inc .
ca , usa ) where a xy graph was plotted and the best - fit linear regression calculated representing the linear standard of amount of dna versus intensity . for the smc2 cells , separated tiff files of the 488 and 528 channels were opened in image - pro plus ( media cybernetics , inc . , bethesda , usa ) .
using the 528-nm image , a roi covering the area covered by the pulled centromere was selected .
this roi was copied and loaded into the 488-nm file where the sum value of intensity was determined .
again , the background value was determined by moving the roi 510 pixels away from the metaphase plate .
the absolute amount of dna was calculated according to the equation of the previously determined best - fit linear regression .
the line profile was drawn in imagej ( research national institute of mental health , bethesda , usa ) values exported to excel ( microsoft , redmond , wa ) and graphs of each channel superimposed .
 in order to understand the three - dimensional structure of the functional kinetochore in vertebrates , we require a complete list and stoichiometry for the protein components of the kinetochore , which can be provided by genetic and proteomic experiments . 
 we also need to know how the chromatin - containing cenp - a , which makes up the structural foundation for the kinetochore , is folded , and how much of that dna is involved in assembling the kinetochore . in this ms , we demonstrate that functioning metaphase kinetochores in chicken dt40 cells contain roughly 50  kb of dna , an amount that corresponds extremely closely to the length of chromosomal dna associated with cenp - a in chip - seq experiments . 
 thus , during kinetochore assembly , cenp - a chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin . 
Introduction
Results
DNA content of a functional kinetochore
Discussion
Material and methods
Cell culture
Cell and phage fixation and staining
Quantification of DNA using Image-Pro Plus