CEP295 interacts with microtubules and is required for centriole elongation.

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules.

Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly.

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel-shaped structure. Recent studies have proposed a structural model of the SAS-6-based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS-6 via its carboxyl-terminus and with MTs via its amino-terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP-induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS-6 had a dominant-negative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS-6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP-mediated centriole elongation.

The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.

Assessment of autonomic dysfunction in patients with type 2 diabetes using reactive hyperemia.

It is known that aging and type 2 diabetes mellitus contribute to atherosclerosis and autonomic dysfunction. By using the air pressure sensing system (APSS), peak-peak intervals (PPIs) of wrist arterial waveforms from baseline and reactive hyperemia (RH) were obtained. Through frequency domain analysis of heart rate variability (HRV) and nonlinear Poincaré method, the HRV of healthy young individuals (Group 1, n=25), healthy upper middle-aged individuals (Group 2, n=22), and patients with type 2 diabetes (Group 3, n=28) were assessed. By using the standard deviation (SD) of the instantaneous PPI variability (SD1)/the SD of the long PPI variability (SD2) ratio (SSR), PPIs of the same individuals before and after RH induction were compared. Reduced SSR1₋10 was noted only in patients with diabetes. Moreover, a significient correlation between SSR1₋10 and endothelial function was observed in all subjects (r=0.290, p=0.033) after RH. However, no correlation with low-frequency to high-frequency power ratio (LHR) was noted before and after RH. In conclusion, according to our results, campared to the baseline, there were more significant changes of SSR1₋10 after RH in patients with diabetes; and, a significient correlation between SSR1₋10 and endothelial function at the moment of RH was noted.

Six-channel ECG-based pulse wave velocity for assessing whole-body arterial stiffness.

BACKGROUND: Despite the proposal of different means of non-invasive arterial stiffness assessment, none offers simultaneous information on whole-body peripheral arterial condition. We investigated the validity of applying a six-channel electrocardiogram-based pulse wave velocity (ECG-PWV) measurement system for this purpose. METHODS: The study consisted of two parts. Part One enrolled hypertensive (Group 1, n = 32) and normal (Group 2, n = 32) subjects, whereas Part Two recruited diabetic (Group 3, n = 50) and normal (Group 4, n = 50) subjects. To validate the application of ECG-PWV in assessing peripheral arterial stiffness in different parts of body, ECG-PWV data were compared with three other parameters including the cardio-ankle vascular index (CAVI), pulse wave velocity-digital volume pulse (PWV-DVP) and intima-media thickness (IMT). RESULTS: ECG-PWV in healthy subjects in Part One correlated significantly with CAVI and PWV-DVP (p < 0.05), whereas ECG-PWV and CAVI were significantly different between the hypertensive and normal subjects. Moreover, comparison of IMT and ECG-PWV from different sites showed significant correlation only between IMT and ECG-PWV from earlobe (r = 0.495, p = 0.004). No significant association, however, was noted between IMT and CAVI. For Part Two, significant differences existed between diabetic and normal subjects in body weight, waist circumference, level of HbA1c, fasting blood sugar, serum creatinine and ECG-PWV from the foot. However, no significant difference was noted in PWV-DVP between two groups. CONCLUSIONS: Six-channel ECG-PWV measurement system showed remarkable correlation with IMT in hypertensive subjects and with key anthropometric and biochemical parameters in diabetic patients, suggesting its validity in assessing whole-body arterial stiffness in subjects with peripheral arterial diseases within 10 min.

Measurement of local pulse wave velocity for carotid artery by using an ultrasound-based method.

Currently, pulse wave velocity (PWV) is an important physical index for characterizing the mechanical properties of arteries. Carotid-femoral PWV (cfPWV) is a clinically-approved parameter for evaluating the cardiovascular risk and therapeutic efficacy. However, cfPWV only provides global information about vessel properties. Many recent studies have indicated that local PWV measurements provide precise evaluation of artery conditions. Here, an ultrasound (US) method based on a novel vessel displacement waveform correction, is proposed for improving the accuracy of local carotid PWV measurement. A programmable US device and a commercial array transducer were used, which allow a user to excite transducer and receive US signals arbitrarily with different beam settings. The local PWV measurement accuracy was verified using a phantom. The number of US beams used for PWV measurements was also considered, which indicates that eight elements is the acceptable setting. Subsequently, local carotid PWV and cfPWV were measured in 35 healthy human subjects (age: 21.9 ± 2.4 years) by using the US method and SphygmoCor device, respectively. The cfPWV and local carotid PWV were 6.65 ± 0.74 and 4.63 ± 0.57 m/s, respectively. A good linear correlation was observed between the two aforementioned methods (r = 0.8) for the subjects. All the results indicated that when few US beams were used, the proposed method exhibited a reliable measurement of local PWV.

Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-SAS-4.

We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423-607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311-422). Site-specific mutagenesis revealed that the mutations that either disrupt the alpha-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved approximately 20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4's MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis.

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

Possible Role of Aurora-C in Meiosis.

The meiotic generation of haploid gametes with equal contents of genetic material is important for sexual reproduction in mammals. Errors in the transmission of chromosomes during meiosis may lead to aneuploidy, which is the leading cause of miscarriage and congenital birth defects in humans. The Aurora kinases, which include Aurora-A, Aurora-B, and Aurora-C, are highly conserved serine-threonine kinases that play essential roles in centrosome function, chromosome segregation, and cytokinesis during mitosis and meiosis. While Aurora-A and Aurora-B have been extensively studied in mitosis, the role of Aurora-C in meiosis is only now starting to be revealed. For example, the perturbation of Aurora-C kinase activity by microinjection of Aurora-C-kinase-dead mutant mRNAs into mouse oocytes induced multiple defects, including chromosome misalignment, abnormal kinetochore-microtubule attachment, premature chromosome segregation, and failure of cytokinesis during meiotic division. However, the analysis of such defects is complicated by the possibility that Aurora-B may be present in mammalian germ cells. Interestingly, a homozygous mutation of Aurora-C in humans leads to the production of large-headed polyploid spermatozoa and causes male infertility, but homozygous females are fertile. Mouse studies regarding the roles of Aurora-B and Aurora-C in female meiotic divisions have yielded inconsistent results, and it has proven difficult to explain why homozygous human females have no significant clinical phenotype. In this review, we will discuss the controversial status of Aurora-B in oocytes and the possible role of Aurora-C during meiotic division.

Application of multiscale entropy in arterial waveform contour analysis in healthy and diabetic subjects.

We applied multiscale entropy (MSE) to assess variation in crest time (CT), a parameter in arterial waveform analysis, in diagnosing patients with diabetes. Data on digital volume pulse were obtained from 93 individuals in three groups [Healthy young (Group 1, 20 < age ≤ 40, n = 30), healthy upper-middle-aged (Group 2, age > 40, n = 30), and diabetic (Group 3, n = 33) subjects]. Crest time, normalized crest time, crest time ratio (CTR), small- and large-scale MSE on CT [MSESS(CT) and MSELS(CT), respectively] were computed and correlated with anthropometric (i.e., body weight/height, waist circumference), hemodynamic (i.e., blood pressure), and biochemical parameters (i.e., serum triglyceride, high-density lipoprotein, fasting blood sugar, and glycosylated hemoglobin). The results demonstrated higher variability in CT in healthy subjects (Groups 1 and 2) compared with that in diabetic patients (Group 3) as reflected in significantly elevated MSESS(CT) and MSELS(CT) in the former (p < 0.003 and p < 0.001, respectively). MSELS(CT) also showed significant association with waist circumference and fasting blood sugar (i.e., two diagnostic criteria of metabolic syndrome) as well as glycosylated hemoglobin concentration. In conclusion, using MSE analysis for assessing CT variation successfully distinguished diabetic patients from healthy subjects. MSESS(CT) and MSELS(CT) therefore may serve as noninvasive tools for identifying subjects with diabetes and those at risk.

Multiscale cross-approximate entropy analysis as a measure of complexity among the aged and diabetic.

Complex fluctuations within physiological signals can be used to evaluate the health of the human body. This study recruited four groups of subjects: young healthy subjects (Group 1, n = 32), healthy upper middle-aged subjects (Group 2, n = 36), subjects with well-controlled type 2 diabetes (Group 3, n = 31), and subjects with poorly controlled type 2 diabetes (Group 4, n = 24). Data acquisition for each participant lasted 30 minutes. We obtained data related to consecutive time series with R-R interval (RRI) and pulse transit time (PTT). Using multiscale cross-approximate entropy (MCE), we quantified the complexity between the two series and thereby differentiated the influence of age and diabetes on the complexity of physiological signals. This study used MCE in the quantification of complexity between RRI and PTT time series. We observed changes in the influences of age and disease on the coupling effects between the heart and blood vessels in the cardiovascular system, which reduced the complexity between RRI and PTT series.

CEP120 interacts with CPAP and positively regulates centriole elongation.

Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1-associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle-regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding-defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.

Assessing spontaneous baroreflex in aged with pulse-pulse intervals and pulse amplitudes.

Cardiac autonomic dysfunction is a serious condition in the elder subjects. Baroreflex sensitivity (BRS) by measuring pulse intervals and blood pressure has been proven as an effective indicator. This paper proposes a novel index by substitution blood pressure with amplitudes of pressure pulse. We recruited 61 subjects divided into two groups: healthy young subjects (Group1, n=33), healthy elders (Group2, n=28). The wrist pulse pressures of each subject were measured for 5 minutes to obtain pulse-pulse intervals and amplitudes then applied within the spontaneous sequence technique to calculate the pulse-pulse interval and amplitude ratio (PAR). We verified the reproducibility of PAR and agreement with spectral analysis of heart rate variability in group1 participants. We discovered significant differences between different groups in PAR (Group1 vs. Group2: 0.90 ± 0.42 vs. 0.62 ± 0.27, P=0.010). In contrast with measurements of BRS, this study proposes a simple approach without the necessity of blood pressure calibration or professional expertise to conduct measurements, thereby providing a convenient method for assessing autonomic function at home.

Arterial stiffness using radial arterial waveforms measured at the wrist as an indicator of diabetic control in the elderly.

Although current technique of photoplethysmography (PPG) is a popular noninvasive method of waveform contour analysis in assessing arterial stiffness, data obtained are frequently affected by various environmental and physiological factors. We proposed an easily operable air pressure sensing system (APSS) for radial arterial signal capturing. Totally, 108 subjects (young, the aged with or without diabetes) were recruited from July 2009 to May 2010. Arterial waveform signals from the wrist were obtained and analyzed using Hilbert-Huang transformation (HHT). Through ensemble empirical mode decomposition (EEMD), the signals were decomposed into eight intrinsic mode functions (IMF1-8) of which IMF5 was found to be the desired signal with a discernible diastolic peak. The results showed significant differences in reflection index (RI) and stiffness index (SI) from the young subjects and those from the aged participants with or without diabetes. Significant differences in RI and SI were also noted between subjects with well-controlled diabetes and those without. Good reproducibility and correlation were demonstrated. In conclusion, the present study proposed the application of radial arterial signal capturing subsystem and HHT in acquiring more reliable data on RI and SI compared with the conventional PPG method.

Multiscale entropy analysis of pulse wave velocity for assessing atherosclerosis in the aged and diabetic.

This study proposed a dynamic pulse wave velocity (PWV)-based biomedical parameter in assessing the degree of atherosclerosis for the aged and diabetic populations. Totally, 91 subjects were recruited from a single medical institution between July 2009 and October 2010. The subjects were divided into four groups: young healthy adults (Group 1, n = 22), healthy upper middle-aged adults (Group 2, n = 28), type 2 diabetics with satisfactory blood sugar control (Group 3, n = 21), and unsatisfactory blood sugar control (Group 4, n = 20). A self-developed six-channel electrocardiography (ECG)-PWV-based equipment was used to acquire 1000 successive recordings of PWV(foot) values within 30 min. The data, thus, obtained were analyzed with multiscale entropy (MSE). Large-scale MSE index (MEI(LS)) was chosen as the assessment parameter. Not only did MEI(LS) successfully differentiate between subjects in Groups 1 and 2, but it also showed a significant difference between Groups 3 and 4. Compared with the conventional parameter of PWV(foot) and MEI on R-R interval [i.e., MEI(RRI)] in evaluating the degree of atherosclerotic change, the dynamic parameter, MEI(LS) (PWV), could better reflect the impact of age and blood sugar control on the progression of atherosclerosis.

Aurora-C kinase deficiency causes cytokinesis failure in meiosis I and production of large polyploid oocytes in mice.

We previously isolated Aurora-C/Aie1 in a screen for kinases expressed in mouse sperm and eggs. Here, we show the localization of endogenous Aurora-C and examine its roles during female mouse meiosis. Aurora-C was detected at the centromeres and along the chromosome arms in prometaphase I-metaphase I and was concentrated at centromeres at metaphase II, in which Aurora-C also was phosphorylated at Thr171. During the anaphase I-telophase I transition, Aurora-C was dephosphorylated and relocalized to the midzone and midbody. Microinjection of the kinase-deficient Aurora-C (AurC-KD) mRNA into mouse oocytes significantly inhibited Aurora-C activity and caused multiple defects, including chromosome misalignment, abnormal kinetochore-microtubule attachment, premature chromosome segregation, and cytokinesis failure in meiosis I. Furthermore, AurC-KD reduced Aurora-C and histone H3 phosphorylation and inhibited kinetochore localization of Bub1 and BubR1. Similar effects also were observed in the oocytes injected with INCNEP-delIN mRNAs, in which the Aurora-C binding motif was removed. The most dramatic effect observed in AurC-KD-injected oocytes is cytokinesis failure in meiosis I, resulting in producing large polyploid oocytes, a pattern similar to Aurora-C deficiency human spermatozoa. Surprisingly, we detected no Aurora-B protein in mouse oocytes. We propose that Aurora-C, but not Aurora-B, plays essential roles in female mouse meiosis.

CPAP is a cell-cycle regulated protein that controls centriole length.

Centriole duplication involves the growing of a procentriole (progeny centriole) next to the proximal end of each pre-existing centriole (parental centriole). The molecular mechanisms that regulate procentriole elongation remain obscure. We show here that expression of the centriolar protein CPAP (centrosomal P4.1-associated protein) is carefully regulated during the cell cycle, with the protein being degraded in late mitosis. Depletion of CPAP inhibited centrosome duplication, whereas excess CPAP induced the formation of elongated procentriole-like structures (PLSs), which contain stable microtubules and several centriolar proteins. Ultrastructural analysis revealed that these structures are similar to procentrioles with elongated microtubules. Overexpression of a CPAP mutant (CPAP-377EE) that does not bind to tubulin dimers significantly inhibited the formation of CPAP-induced PLSs. Together, these results suggest that CPAP is a new regulator of centriole length and its intrinsic tubulin-dimer binding activity is required for procentriole elongation.

Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis.

Aurora-C was first identified during screening for kinases expressed in mouse sperm and eggs. Herein, we report for the first time the precise subcellular localization of endogenous Aurora-C during male meiotic division. The localization of Aurora-C was analyzed by immunofluorescence staining on chromosome spreads of mouse spermatocytes or in squashed seminiferous tubules. Aurora-C was first detected at clusters of chromocenters in diplotene spermatocytes and was concentrated at centromeres in metaphase I and II. Interestingly, Aurora-C was also found along the chromosome axes, including both the regions of centromeres and the chromosome arms in diakinesis. During the anaphase I/telophase I and anaphase II/telophase II transitions, Aurora-C was relocalized to the spindle midzone and midbody. A similar distribution pattern was also observed for Aurora-B during male meiotic divisions. Surprisingly, we detected no Aurora-C in mitotic spermatogonia. Furthermore, immunoprecipitation analyses revealed that INCENP associated with Aurora-C in the male testis. We propose that INCENP recruits Aurora-C (or some other factor(s) recruit INCENP and Aurora-C) to meiotic chromosomes, while Aurora-C may either work alone or cooperate with Aurora-B to regulate chromosome segregation during male meiosis.

Overexpression of an Aurora-C kinase-deficient mutant disrupts the Aurora-B/INCENP complex and induces polyploidy.

Aurora kinases are emerging as key regulators of centrosome function, chromosome segregation and cytokinesis. We previously isolated Aurora-C (Aie1), a third type of Aurora kinase, in a screen for kinases expressed in mouse sperm and eggs. Currently, we know very little about the precise localization and function of Aurora-C. Immunofluorescence analysis of ectopically expressed GFP-Aurora-C has revealed that Aurora-C is a new member of the chromosomal passenger proteins localizing first to the centromeres and then to the central spindles during cytokinesis. In order to study the potential role of Aurora-C, we examined the effects of a kinase-deficient (KD) mutant (AurC-KD) in HeLa Tet-Off cells under tetracycline control. Our results showed that overexpression of AurC-KD causes defects in cell division and induces polyploidy and apoptosis. Interestingly, AurC-KD overexpression also inhibits centromere/kinetochore localization of Aurora-B, Bub1, and BubR1, reduces histone H3 phosphorylation, and disrupts the association of INCENP with Aurora-B. Together, our results showed that Aurora-C is a chromosomal passenger protein, which may serve as a key regulator in cell division.