[Comparative analysis of the brain distribution of [18F]FDG in populations of patients with Alzheimer's disease with or without family history of dementia]. / Analyse comparative de la distribution cérébrale du [18F]FDG dans une population de patients souffrant de maladie d'Alzheimer présentant ou non des antécédents familiaux de démence.

Hereditary forms of Alzheimer's disease (AD) and early-onset forms have more brain damage than sporadic or late-onset forms at the time of diagnosis (1, 2). Data in the literature are contradictory concerning familial forms without known heredity or mutation. The aim of this study was to compare the brain distribution of FDG between two populations of patients with a clinical diagnosis of sporadic AD according to the presence or not of a first degree family history of dementia. We retrospectively included 243 patients with clinical diagnosis of AD who underwent brain FDG PET imaging between 2012 and 2017. SPM12 was used to compare the FDG brain distribution in 199 patients with AD and no familial history of dementia and 43 patients with AD and first degree familial history of dementia. Compared to a database of 22 healthy control subjects, both groups of AD patients showed a significant decrease of FDG distribution in temporo-parietal, posterior cingulate and posterior left frontal cortex with respect to the controls (p inferior to 0.05 corrected for the family-wise error, pFWE-corr). There were no significant differences between the two AD groups (pFWE-corr superior to 0.05 and p superior to 0.001 uncorrected for multiple comparisons) that present the same brain metabolic pathology.

Les formes héréditaires de la maladie d'Alzheimer (MA) et les formes à début précoce présentent une atteinte cérébrale plus importante que les formes sporadiques ou celles à début tardif au moment du diagnostic (1, 2). Les données de la littérature sont contradictoires en ce qui concerne les formes familiales sans hérédité ni mutation connue. L'objectif de cette étude était de comparer la distribution cérébrale du [18F]fluoro-2-deoxy-D-glucose ([18F]FDG) entre deux populations de patients présentant un diagnostic clinique de la MA sporadique selon la présence, ou non, d'une histoire familiale de démence au premier degré. Dans cette étude rétrospective, nous avons inclus 243 patients vus entre 2012 et 2017. Le logiciel SPM12 a été utilisé pour comparer la distribution cérébrale du FDG entre 199 patients souffrant de MA, sans histoire familiale et 43 patients souffrant de MA avec une histoire familiale de démence au premier degré. Comparés à une base de données de 22 sujets contrôles sains, chacun des deux groupes de patients présentait une réduction significative de la distribution du FDG au niveau du cortex temporo-pariétal, cingulaire postérieur et frontal postérieur gauche (p inf�rieur a 0,05 corrigé pour le family-wise error, pFWE-corr), caractéristique de la maladie. Il n'y avait pas de différence significative entre les deux groupes MA (pFWE-corr sup�rieur a 0,05 et p sup�rieur a 0,001 non corrigé, pour des comparaisons multiples) qui présentent donc la même altération métabolique cérébrale.

[Lacunar stroke as a very late complication of radiotherapy: value of nuclear medicine techniques]. / Ictus lacunar como complicación muy tardía de la radioterapia: valor de las técnicas de medicina nuclear.

INTRODUCTION: Radiation therapy is a very useful treatment for central nervous systemS neoplasms. The time range of its complications is very wide; they appear even many years after its completion. These late complications behave clinically and radiologically similar to a relapse; a functional diagnostic study with radioactive isotopes can help to make a therapeutic decision. CASE REPORT: A male suddenly presented deficient neurological symptoms in the same site where he received radiation therapy 25 years earlier for a pilocytic astrocytoma. The MRI findings suggested a lacunar stroke but a finding in the perfusion sequence forced us to be more precise in the diagnosis. A PET-CT 11C-methionine was performed which showed an increased uptake compatible with neoplasia. The spontaneous regressive evolution of the symptoms inclined us to take a conservative attitude. Lacunar ictus was confirmed on MRI three months later. CONCLUSIONS: The reappearance of neurological symptoms years after radiotherapy of a brain neoplasm poses a diagnostic dilemma. Current diagnostic techniques are very accurate but present false positives. The various nuclear medicine techniques, in particular PET-CT 11C-methionine, are a diagnostic aid. With the presentation of this case we intend to draw attention to one of the late complications of radiation therapy and the various differential diagnoses. Diagnostic and therapeutic advances have increased the life expectancy of cancer patients, so these late complications are expected to be more frequent.

TITLE: Ictus lacunar como complicación muy tardía de la radioterapia: valor de las técnicas de medicina nuclear.Introducción. La radioterapia es un tratamiento de gran utilidad en las neoplasias del sistema nervioso central. El rango temporal de sus complicaciones es muy amplio, ya que aparecen incluso muchos años más tarde de haberla finalizado. Estas complicaciones tardías se comportan clínica y radiológicamente de forma similar a una recidiva; un estudio funcional diagnóstico con isótopos radiactivos puede ayudar a tomar una decisión terapéutica. Caso clínico. Varón que presentó de forma brusca sintomatología neurológica deficitaria en la misma localización donde 25 años antes había recibido radioterapia por un astrocitoma pilocítico. La resonancia magnética sugería un ictus lacunar, pero un hallazgo en la secuencia de perfusión obligaba a ser más preciso en el diagnóstico. Una tomografía por emisión de positrones-tomografía computarizada (PET-TC) con C11-metionina mostró un aumento de captación compatible con neoplasia. La evolución espontánea regresiva de los síntomas inclinó a tomar una actitud conservadora. Una resonancia magnética realizada tres meses más tarde confirmó el ictus lacunar. Conclusiones. La reaparición de síntomas neurológicos años más tarde de la radioterapia de una neoplasia cerebral supone un dilema diagnóstico. Las técnicas diagnósticas actuales son muy precisas, pero presentan falsos positivos. Las distintas técnicas de medicina nuclear, en concreto la PET-TC con C11-metionina, suponen una ayuda diagnóstica. Con este caso se pretende llamar la atención sobre una de las complicaciones tardías de la radioterapia y los distintos diagnósticos diferenciales. Los avances diagnósticos y terapéuticos han aumentado la esperanza de vida de los pacientes oncológicos, con lo que estas complicaciones tardías se prevén más frecuentes.

[Conversion disorder : functional neuroimaging and neurobiological mechanisms]. / Vignette diagnostique de l'étudiant. Trouble de conversion : neuroimagerie fonctionnelle et mécanismes neurobiologiques.

Conversion disorder is a psychiatric disorder often encountered in neurology services. This condition without organic lesions was and still is sometimes referred as an imaginary illness or feigning. However, the absence of organic lesions does not exclude the possibility of cerebral dysfunction. The etiologic mechanisms underlying this disorder remain uncertain even today.The advent of cognitive and functional imaging opens up a field of exploration for psychiatry in understanding the neurobiological mechanisms underlying mental disorders and especially the conversion disorder. This article reports several neuroimaging studies of conversion disorder and attempts to generate hypotheses about neurobiological mechanisms.

Le trouble de conversion est une pathologie psychiatrique fréquemment rencontrée dans les services de neurologie. Cette pathologie «sans substrat¼ a été et est encore parfois qualifiée de maladie imaginaire ou de simulation. Cependant, l'absence de substrat «organique¼ n'exclut pas la possibilité d'un dysfonctionnement cérébral. Les mécanismes étiopathogéniques qui sous-tendent ce trouble sont longtemps restés incertains, encore aujourd'hui. L'avènement des sciences cognitives et de l'imagerie fonctionnelle ouvre un champ d'exploration pour la psychiatrie dans la compréhension des mécanismes neurobiologiques qui sous-tendent les troubles mentaux et, en particulier, le trouble de conversion. Cet article reprend plusieurs études de neuroimagerie sur le trouble de conversion et tente d'en dégager des hypothèses sur ses mécanismes neurobiologiques.

Data set of healthy old people assessed for three walking conditions using accelerometric and opto-electronic methods.

BACKGROUND: Gait patterns of healthy aging are needed to allow a comparison with pathological situations. However, little data is available. OBJECTIVE: To present gait pattern of healthy older specially selected to be "healthy walkers". METHOD: Fifty-seven older people benefited from a geriatric assessment including clinical and functional evaluations to include only those without gait disorders. Gait data were simultaneously recorded using a tri-axial accelerometer placed on the waist and four 3D position markers placed on the feet at the level of the heel and the toe. Volunteers walked at comfortable self-selected speed (CW), fast self-selected speed (FW), and finally in dual task walking condition (DTW). The extracted gait parameters were: gait speed, stride length, stride frequency, regularity and symmetry, swing, stance and double support time and ratio and minimum toe clearance. Gait speed and stride length were normalized to the right leg length. RESULTS: Fifty-seven older people with a mean age of 69.7 ± 4.2 years old (range from 65 to 82 years) were included. Data were analyzed according to the gender and according to the age (<70 or ≥70 years old). After normalization to leg length, the main significant differences were shown for stride length and minimum toe clearance in CW, FW and in DTW that were shorter in women. The regularity in FW was significantly lower among older volunteers. CONCLUSIONS: This work provides a data set considering 14 gait parameters obtained from 57 healthy old people strictly selected and assessed for three walking conditions and shows that GS, SL and MTC have to be related to the gender. The age-related impact on gait performances appears reduced in this cohort.

Gait speed or gait variability, which one to use as a marker of risk to develop Alzheimer disease? A pilot study.

BACKGROUND: Previous literature demonstrates the interest of gait analysis to predict cognitive decline in old people. AIMS: This pilot study aims to determine if gait speed or gait variability is a marker able to early identify, among mild cognitive impairment (MCI) subjects, those at risk to develop Alzheimer's disease (AD) in the future. METHODS: 13 MCI subjects were included in 2007. Their gait parameters (walking speed, stride length and gait frequency, regularity and symmetry) were measured in 2007 and 2008 in simple task (ST) and in dual task (DT) using a triaxial accelerometer (Locometrix(®)). Among the 13 MCI subjects included in 2007, 10 were assessed in 2008. So, 23 (13 in 2007 + 10 in 2008) gait tests were collected. In 2011, MCI people were considered as "MCI+" when they developed AD (between baseline and 2011) and as "MCI-" if they did not. Among the 23 gait tests, 15 were from MCI+ (9 gait tests in 2007 and 6 in 2008) and 8 from MCI- (4 gait tests in 2007 and 4 gait tests in 2008). Mann-Whitney non-parametric U test was used to compare gait parameters of MCI+ and MCI-. RESULTS: Gait speed, symmetry and regularity were lower in MCI+ than in MCI-. DISCUSSION: Despite the small sample size, the results presented in this original pilot study are in line as the infrequent previous literature related to this topic. The authors discuss lacks and strengths of this work. CONCLUSIONS: These results suggest that both gait speed and gait variability could be markers to early identify MCI at risk to develop AD.

Biodistribution and Radiation Dosimetry for the Novel SV2A Radiotracer [(18)F]UCB-H: First-in-Human Study.

PURPOSE: [(18)F]UCB-H is a novel radiotracer with a high affinity for synaptic vesicle glycoprotein 2A (SV2A), a protein expressed in synaptic vesicles. SV2A is the binding site of levetiracetam, a "first-in-class" antiepileptic drug with a distinct but still poorly understood mechanism of action. The objective of this study was to determine the biodistribution and radiation dosimetry of [(18)F]UCB-H in a human clinical trial and to establish injection limits according to biomedical research guidelines. Additionally, the clinical radiation dosimetry results were compared to estimations in previously published preclinical data. PROCEDURES: Dynamic whole body positron emission tomography/X-ray computed tomography (PET/CT) imaging was performed over approximately 110 min on five healthy male volunteers after injection of 144.5 ± 7.1 MBq (range, 139.1-156.5 MBq) of [(18)F]UCB-H. Major organs were delineated on CT images, and time-activity curves were obtained from co-registered dynamic PET emission scans. The bladder could only be delineated on PET images. Time-integrated activity coefficients were calculated as area under the curve using trapezoidal numerical integration. Urinary excretion data based on PET activities including voiding was also simulated using the dynamic bladder module of OLINDA/EXM. The radiation dosimetry was calculated using OLINDA/EXM. RESULTS: The effective dose to the OLINDA/EXM 70-kg standard male was 1.54 × 10(-2) ± 6.84 × 10(-4) millisieverts (mSv)/MBq, with urinary bladder wall, gallbladder wall, and the liver receiving the highest absorbed dose. The brain, the tracer's main organ of interest, received an absorbed dose of 1.89 × 10(-2) ± 2.32 × 10(-3) mGy/MBq. CONCLUSIONS: This first human dosimetry study of [(18)F]UCB-H indicated that the tracer shows similar radiation burdens to widely used common clinical tracers. Single injections of at maximum 672 MBq for US practice and 649 MBq for European practice keep radiation exposure below recommended limits. Recently published preclinical dosimetry data extrapolated from mice provided satisfactory prediction of total body and effective dose but showed significant differences in organ absorbed doses compared to human data.

Brain Metabolic Dysfunction in Capgras Delusion During Alzheimer's Disease: A Positron Emission Tomography Study.

Capgras delusion is characterized by the misidentification of people and by the delusional belief that the misidentified persons have been replaced by impostors, generally perceived as persecutors. Since little is known regarding the neural correlates of Capgras syndrome, the cerebral metabolic pattern of a patient with probable Alzheimer's disease (AD) and Capgras syndrome was compared with those of 24-healthy elderly participants and 26 patients with AD without delusional syndrome. Comparing the healthy group with the AD group, the patient with AD had significant hypometabolism in frontal and posterior midline structures. In the light of current neural models of face perception, our patients with Capgras syndrome may be related to impaired recognition of a familiar face, subserved by the posterior cingulate/precuneus cortex, and impaired reflection about personally relevant knowledge related to a face, subserved by the dorsomedial prefrontal cortex.

Early neuropsychological detection of Alzheimer's disease.

Lifestyle modification offers a promising way of preventing or delaying Alzheimer's disease (AD). In particular, nutritional interventions can contribute to decrease the risk of dementia. The efficacy of such interventions should be assessed in individuals thought to be prone to AD. It is therefore necessary to identify markers that may help detecting AD as early as possible. This review will focus on subtle neuropsychological changes that may already exist in the predementia phase, and that could point to individuals at risk of dementia. Episodic memory decline appears consistently as the earliest sign of incipient typical AD. An episodic memory test that ensures deep encoding of information and assesses retrieval with free as well as cued recall appears as a useful tool to detect patients at an early stage of AD. Beyond the memory domain, category verbal fluency has been shown to decline early and to predict progression to AD. Moreover, in line with current diagnosis criteria for prodromal AD, combining neuropsychological scores and neuroimaging data allows a better discrimination of future AD patients than neuroimaging or neuropsychological data alone. Altogether, the detection of cognitive changes that are predictive of the typical form of probable AD already in the predementia stage points to at risk people who are the best target for therapeutic interventions, such as nutrition or physical exercise counseling or dietary interventions.

[The effect of normal and pathological aging on cognition]. / Les effets du vieillissement normal et pathologique sur la cognition.

Cognitive deficits in the executive and memory domains are observed in normal aging and Alzheimer's disease (AD). These deficits are associated with changes at the brain activity level. However, a series of factors are prone to delay the occurrence of cognitive deficits, such as mental stimulation or physical activity. Similarly, cognitive rehabilitation allows improving the daily life functioning of patients with AD. The identification of factors and techniques that contribute to maintain cognitive efficiency and/or counteract the effects of AD will allow optimizing quality of life of older people.

[Clinical study of the month: depressive pseudo-dementia]. / Le cas clinique du mois. Pseudo-démence dépressive.

We report the case of a man aged 62 suffering from a known type I bipolar disorder and referred by his attending psychiatrist because of a state of spatiotemporal disorientation, confusion and prostration evoking significant neurologic impairment. The interest of this case report is in the use of the 18-FDG PET-Scanner, which is increasingly widespread in clinical psychiatry, to support the differential diagnosis between a psycho-organic pathology like dementia or a functional psychiatric pathology like depressive pseudo-dementia (also named melancholic dementia), in which some patterns of dysfunction can now be identified by functional imaging.

[Dementia, end of life and euthanasia]. / Troubles cognitifs, fin de vie et euthanasia.

Among legislative criteria granting the right to practice euthanasia or assisted suicide, there are systematically four major elements. Precisely, any request must be voluntary, persistent, to be well thought and well informed. Such euthanasia raises numerous difficult questions in case of dementia. It also justifies thinking about possibilities that can offer specific arrangements of anticipated demands in such peculiar cases. Empirical experiences show us that it applies with difficulties in practice. Finally, to avoid that a big majority of these demands would find themselves not applied in practice, it would certainly be necessary to add to it structural valuation of advance care planning, and assure its recognition and development. These should not be limited to a single pathological target but would address all of us to increase advance care planning initiation, which remains the most limiting factor of such any early but continuous procedure.

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores.

Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1-Mis12-Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1-Zwint1 complex is required to recruit the Rod-Zwilch-Zw10 (RZZ) and Mad1-Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1-Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Progression of Alzheimer disease in Europe: data from the European ICTUS study.

The clinical progression of Alzheimer disease (AD) was studied in European subjects under treatment with AChE inhibitors (AChE-I) in relation to geographical location over a 2-years period. One thousand three hundred and six subjects from 11 European countries were clustered into 3 regions (North, South, West) and investigated with biannual follow-up over 2 years. Primary outcomes were cognitive, functional and behavioral measures. Caregiver burden, hospital admission and admission to nursing home were also recorded. Participant cognitive function declined non-linearly over time (MMSE: -1.5 pts/first year, -2.5 pts/second year; ADAScog: + 3.5 pts/first year, + 4.8 pts/second year), while the progression of behavioral disturbances (NPI scale) was linear. Neither scale showed regional differences, and progression of the disease was similar across Europe despite different health care systems. Functional decline (ADL, IADL) tended to progress more rapidly in Southern Europe (p=0.09), while progression of caregiver burden (Zarit Burden Interview) was most rapid in Northern Europe (5.6 pts/y, p=0.04). Incidences of hospital admission (10.44, 95%CI: 8.13-12.75, p < 0.001) and admission to nursing home (2.97, 95%CI: 1.83-4.11, p < 0.001) were lowest in Southern Europe. In general cognitive and functional decline was slower than in former cohorts. European geographical location reflecting differences in culture and in health care system does not impact on the progression of AD but does influence the management of AD subjects and caregiver burden.

Deformations within moving kinetochores reveal different sites of active and passive force generation.

Kinetochores mediate chromosome segregation at mitosis. They are thought to contain both active, force-producing and passive, frictional interfaces with microtubules whose relative locations have been unclear. We inferred mechanical deformation within single kinetochores during metaphase oscillations by measuring average separations between fluorescently labeled kinetochore subunits in living cells undergoing mitosis. Inter-subunit distances were shorter in kinetochores moving toward poles than in those moving away. Inter-subunit separation decreased abruptly when kinetochores switched to poleward movement and decreased further when pulling force increased, suggesting that active force generation during poleward movement compresses kinetochores. The data revealed an active force-generating interface within kinetochores and a separate passive frictional interface located at least 20 nanometers away poleward. Together, these interfaces allow persistent attachment with intermittent active force generation.

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore-microtubule attachment.

Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.

The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells.

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.

The KMN protein network--chief conductors of the kinetochore orchestra.

Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming kinetochore microtubules (kMTs) that tether one sister to one spindle pole and the other sister to the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generation that can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint (SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring a full complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highly conserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between the kinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the key kinetochore functions, with a focus on human cells.

How we discovered fluorescent speckle microscopy.

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ~10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual "speckled" appearance-there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome.

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.