CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

Summary: The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-β1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking. : Mönnich et al. show that CEP128 localizes to the subdistal appendages of the mother centriole and basal body of the primary cilium. CEP128 regulates vesicular trafficking and targeting of RAB11 to the primary cilium. CEP128 loss leads to impaired TGF-β/BMP signaling, which, in zebrafish, is associated with defective organ development. Keywords: primary cilium, basal body, centriole, subdistal appendage, centrosome, transforming growth factor β, TGF-β, bone morphogenetic protein, BMP, zebrafish, phosphoproteomics, CEP12

The primary cilium as a regulator of cellular senescence in human fibroblasts

Somatic cells senesce in culture after a finite number of divisions, indefinitely arresting their proliferation. Among the major causes of senescence is persistent DNA damage signalling. DNA damage and senescence increase the cellular number of centrosomes, the two microtubule organising centres that ensure bipolar mitotic spindles. Centrosomes also provide the basal body, a foundation for the formation of primary cilia, microtubule-based organelles that extend from the surface of most human cell types to sense and transduce various extracellular signals. Primary cilium formation is facilitated by cellular quiescence, a temporary exit from the cell cycle, but the impact of senescence on cilia has not been described. In this project we show that increased cilium frequency and length accompanies senescence in primary human fibroblasts and that ciliation induced by depletion of the centriolar protein CP110 causes senescence. A higher frequency of senescent BJ, MRC5 and NHDF cells had a primary cilium compared to proliferating controls. Cilia were significantly longer on senescent cells. Senescent BJ fibroblasts have elevated numbers of centrioles and this correlates with an increase in ciliary abnormality. Senescent cells showed reduced expression levels of components of the Hedgehog signalling pathway. Inhibition of Hedgehog signalling with cyclopamine reduced proliferation in young cell populations, with increased cilium length accompanying the induction of cell cycle arrest. Ciliary regrowth experiments demonstrated that cilium length is independent of the growth arrest period and that it is intrinsic to the cell. Senescent cells showed reduced levels of the negative ciliary length regulator, CP110. siRNA-mediated depletion of CP110 in young populations increased ciliation, reduced proliferation and elevated cellular senescence. These data demonstrate that primary cilium length regulation through CP110 is a potential novel determinant of cellular proliferative capacity

Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity

Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence

Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity

<div><p>Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence.</p></div

The proteome speciation of an immortalized cystic fibrosis cell line: New perspectives on the pathophysiology of the disease

Cystic Fibrosis (CF) is a recessively inherited disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR has a pivotal role in the onset of CF, and several proteins are involved in its homeostasis. To study CFTR interactors at protein species level, we used a functional proteomics approach combining 2D-DIGE, mass spectrometry and enrichment analysis. A human bronchial epithelial cell line with cystic fibrosis (CFBE41o-) and the control (16HBE14o-) were used for the comparison. 73 differentially abundant spots were identified and some validated by western-blot. Enrichment analysis highlighted molecular pathways in which ezrin, HSP70, endoplasmin and lamin A/C, in addition to CFTR, were considered central hubs in CFTR homeostasis. These proteins acquire different functions through post-translational modifications, emphasizing the importance of studying the CF proteome at protein species level. Moreover, serpin H1, prelamin A/C, protein-SET and cystatin-B were associated to CF, demonstrating the importance of heat shock response, cross-talk between the cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating in the pathophysiology of the disease. These results open new perspectives for the understanding of the proteostasis network, characteristic of CF pathology, and could provide a springboard for new therapeutic strategies. Biological significance: Homeostasis of CFTR is a dynamic process managed by multiple proteostatic pathways. The used gel-based proteomic approach and enrichment analysis pointed out protein species variations among Human Bronchial (16HBE14o-) and Cystic Fibrosis Bronchial Epithelial cell lines (CFBE41o-) and specific molecular mechanisms involved in CF. In particular, we have highlighted HSP70 (HSP7C), HSP90 (endoplasmin), ERM proteins (ezrin), and lamin-A/C as central hubs of the functional analysis. Moreover, for the first time we consider serpin H1, lamin A/C, protein-SET and cystatin-B important player in CF, affecting acute exacerbation, cytoskeleton reorganization, CFTR gating and chronic inflammation in CF. Due to the presence of different spots corresponding to the same protein, we focalize our attention on the idea that a "protein species discourse" is mandatory to well-define functional roles of proteins.Our approach has permitted to pay attention to the molecular mechanisms which regulate pathways directly or indirectly involved with CFTR defects: heat shock response, cross-talk between cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating. Our data could open new perspectives into the understanding of CF, identifying potential targets for drug treatments in order to alleviate δ508CFTR membrane instability and consequently increase life expectancy for CF patients

Time to Adjuvant Chemotherapy for Breast Cancer in National Comprehensive Cancer Network Institutions

BackgroundHigh-quality care must be not only appropriate but also timely. We assessed time to initiation of adjuvant chemotherapy for breast cancer as well as factors associated with delay to help identify targets for future efforts to reduce unnecessary delays.MethodsUsing data from the National Comprehensive Cancer Network (NCCN) Outcomes Database, we assessed the time from pathological diagnosis to initiation of chemotherapy (TTC) among 6622 women with stage I to stage III breast cancer diagnosed from 2003 through 2009 and treated with adjuvant chemotherapy in nine NCCN centers. Multivariable models were constructed to examine factors associated with TTC. All statistical tests were two-sided.ResultsMean TTC was 12.0 weeks overall and increased over the study period. A number of factors were associated with a longer TTC. The largest effects were associated with therapeutic factors, including immediate postmastectomy reconstruction (2.7 weeks; P &lt; .001), re-excision (2.1 weeks; P &lt; .001), and use of the 21-gene reverse-transcription polymerase chain reaction assay (2.2 weeks; P &lt; .001). In comparison with white women, a longer TTC was observed among black (1.5 weeks; P &lt; .001) and Hispanic (0.8 weeks; P &lt; .001) women. For black women, the observed disparity was greater among women who transferred their care to the NCCN center after diagnosis (P (interaction) = .008) and among women with Medicare vs commercial insurance (P (interaction) &lt; .001).ConclusionsMost observed variation in TTC was related to use of appropriate therapeutic interventions. This suggests the importance of targeted efforts to minimize potentially preventable causes of delay, including inefficient transfers in care or prolonged appointment wait times

Time to Adjuvant Chemotherapy for Breast Cancer in National Comprehensive Cancer Network Institutions

BACKGROUND: High-quality care must be not only appropriate but also timely. We assessed time to initiation of adjuvant chemotherapy for breast cancer as well as factors associated with delay to help identify targets for future efforts to reduce unnecessary delays. METHODS: Using data from the National Comprehensive Cancer Network (NCCN) Outcomes Database, we assessed the time from pathological diagnosis to initiation of chemotherapy (TTC) among 6622 women with stage I to stage III breast cancer diagnosed from 2003 through 2009 and treated with adjuvant chemotherapy in nine NCCN centers. Multivariable models were constructed to examine factors associated with TTC. All statistical tests were two-sided. RESULTS: Mean TTC was 12.0 weeks overall and increased over the study period. A number of factors were associated with a longer TTC. The largest effects were associated with therapeutic factors, including immediate postmastectomy reconstruction (2.7 weeks; P < .001), re-excision (2.1 weeks; P < .001), and use of the 21-gene reverse-transcription polymerase chain reaction assay (2.2 weeks; P < .001). In comparison with white women, a longer TTC was observed among black (1.5 weeks; P < .001) and Hispanic (0.8 weeks; P < .001) women. For black women, the observed disparity was greater among women who transferred their care to the NCCN center after diagnosis (P (interaction) = .008) and among women with Medicare vs commercial insurance (P (interaction) < .001). CONCLUSIONS: Most observed variation in TTC was related to use of appropriate therapeutic interventions. This suggests the importance of targeted efforts to minimize potentially preventable causes of delay, including inefficient transfers in care or prolonged appointment wait times