
Paula A. CoelhoCalifornia Institute of Technology | CIT
Paula A. Coelho
PhD in Biomedical Sciences
About
43
Publications
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Introduction
I am interested in understanding fundamental cell biological processes, and in particular their connection with development, disease and cancer.
I am currently investigating the mechanisms underlying the role of supranumerary centrosomes on cancer development. I am using a transgenic mouse to induce centrosome amplification by Plk4 over-expression, which results in severe tissue hyperplasia in the absence of the tumour suppressor protein p53. My focus is now to understand the abnormalities resulting from too many centrosomes. I aim to mechanistically address the connection between centrosome number, tumorigenesis or differentiation.
Additional affiliations
January 1996 - December 2005
January 2009 - present
January 2008 - present
Publications
Publications (43)
During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle a...
Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interf...
To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the...
There is remarkable redundancy between the Cyclin–Cdk complexes that comprise the cell cycle machinery. None of the mammalian A-, D-, or E-type cyclins are required in development until implantation, and only Cdk1 is essential for early cell divisions. Cyclin B1 is essential for development, but whether it is required for cell division is contentio...
Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified fa...
Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified fa...
The execution of female meiosis and the establishment of the zygote is arguably the most critical stage of mammalian development. The egg can be arrested in the prophase of meiosis I for decades, and when it is activated, the spindle is assembled de novo. This spindle must function with the highest of fidelity and yet its assembly is unusually achi...
Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosoph...
Time-Lapse Confocal Microscopy of S2 Cells Stably Expressing CID-GFP and RFP-H2B
Z-stacks were acquired every 30 s (time is shown in seconds). Each Z-stack is 10 μm and composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merge color images of RFP-H2B (red) and CID-GFP (green) channels are shown on the left. The CID-GFP (white)...
Time-Lapse Microscopy of S2 Cells Stably Expressing CID-GFP and RFP-H2B and Depleted for TOPO II, 72 h after the Addition of the dsRNA
Z-stacks are composed of ten optical sections covering 10 μm and were acquired every 20 s. Each Z-stack is 10 μm and composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merge color images of RF...
Quantification of Sister Centromere Distance during Progression through Mitosis in TOPO II–Depleted Cells
(A) Images from time-lapse recording of S2 cells stably expressing the centromere marker CID-mCherry (red and individual channel on the right) and GFP-α-tubulin. Z-stacks were collected in both control and TOPO II–depleted cells.
(B) Quantifica...
Characterization of Mitotic Exit after Depletion of TOPO II and RAD21
Progression through mitosis was determined using cyclin B to clearly determine exit from mitosis and also the earlier stages, such as prometaphase (72 h of treatment). Either (A) control or (B) TOPO II– and DRAD21-depleted cells were immunostained for cyclin B (green), CID (red),...
Characterization of Mitotic Exit of TOPO II–Depleted Cells
(A and B) Immunolocalization of cyclin B and CID in (A) control or (B) TOPO II–depleted cells. In both control and TOPO II–depleted cells, cyclin B localizes to the spindle, centromeres, and poles during prometaphase and metaphase. During anaphase, the overall level of cyclin B falls and is...
Time-Lapse Microscopy of S2 cells Stably Expressing CID-GFP and RFP-H2B and Depleted for TOPO II, 96 h after the Addition of the dsRNA
Z-stacks are composed of ten optical sections covering 10 μm and were acquired every 15 s. Anaphase onset corresponds to time 0 s. Merge color images for H2BRFP (red) and CID-GFP (green) is shown on the left. The se...
Time-Lapse Microscopy of S2 Cells Stably Expressing GFP-α-Tubulin and CID-mCherry
Z-stacks were acquired every 40 s. Each Z-stack is 10 μm and composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merge color images for CID-mCherry (red) and GFP-α-tubulin (green) are shown on the left. On the right, the separated channel for CID...
Time-Lapse Microscopy of S2 Cells Stably Expressing GFP-α-Tubulin and CID-mCherry That Were Depleted for TOPO II, 72 h after the Addition of the dsRNA
Z-stacks were acquired every 40 s. Each Z-stack is 10 μm and is composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merged color images for Cherry-CID (red) and GFP-α-tubulin (g...
Time-Lapse Microscopy of S2 Cells Stably Expressing GFP-α-Tubulin and CID-mCherry
Z-stacks were acquired every 30 s. Each Z-stack is 10 μm and composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merge color images for CID-mCherry (red) and GFP-α-tubulin (green) is shown on the left. Separated channels for CID-GFP (black) and G...
Time-Lapse Microscopy of S2 Cells Stably Expressing CID-GFP and RFP-H2B That Were Simultaneously Depleted of TOPO II and RAD21, 96 h after the Addition of the dsRNA
Z-stacks were acquired every 15 s. Each Z-stack is 10 μm and is composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merged color images for H2B- RFP (red) and CID-...
In Vivo Analysis of Mitotic Progression after Depletion of RAD21 in S2 Cells
(A and B) Progression through mitosis was determined using cyclin B (red) and α-tubulin and DNA (blue) for either (A) control or (B) RAD21-depleted cells. Scale bar represents 5 μm. (B) RAD21-depleted cells are delayed in mitosis, exhibiting separated sister chromatids.
(C...
Immunolocalization of PH3 and TOPO II in ICRF-187–Treated Cells
(A) Control and (B) cells treated for 2 h with 10 μM ICRF-187, after which they were immunostained for Phospho-S10-histone H3 (green) and for TOPO II (blue). TOPO II and PH3 localized to the chromosomes both in control and treated cells, although a reduction in the levels of PH3 was de...
Time-Lapse Microscopy of S2 Cells Stably Expressing CID-GFP and RFP-H2B and Depleted for TOPO II, 72 h after the Addition of the dsRNA
Z-stacks are composed of ten optical sections covering 10 μm and were acquired every 15 s. Anaphase onset corresponds to time 0 s. Merge color images for H2B- RFP (red) and CID-GFP (green).
(806 KB MOV)
Analysis of Centromere Organization after Depletion of TOPO II. Immunolocalization of CID in S2 Cells Treated with Hypotonic Shock prior to Cell Fixation with two CID dots per chromosome (individual channel on the right).
(B) In TOPO II–depleted cells, chromosomes are observed as larger clusters of chromatids with interconnecting chromatin and broa...
Microtubule–Kinetochore Interaction in TOPO II–Depleted Cells
(A and B) Immunofluorescence for α-tubulin (green), CID (red), and DNA (blue) in (A) control and (B) TOPO II–depleted cells subjected to the MG132-Taxol assay.
(C) Quantification shows that a few chromosomes (≤3%; control, n = 35 cells; TOPO II dsRNAi, n = 38 cells), either in control or...
Time-Lapse Microscopy of S2 Cells Stably Expressing GFP-α-Tubulin and CID-mCherry That Were Incubated with 50 μg/ml ICRF-187 after the Establishment of a Bipolar Attachment
Z-stacks were acquired every 30 s. Each Z-stack is 10 μm and composed of ten optical sections. Merge color images for CID-mCherry (red) and GFP-α-tubulin (green) are shown on th...
Time-Lapse Microscopy of S2 Cells Stably Expressing GFP-α-Tubulin and CID-mCherry That Were Incubated for 1 h with 50 μg/ml ICRF-187 before the Recording
Z-stacks were acquired every 30 s. Each Z-stack is 10 μm and composed of ten optical sections. Anaphase onset corresponds to time 0 sec. Merge color images for CID-mCherry (red) and GFP-α-tubulin...
Time-Lapse Microscopy of S2 Cells Stably Expressing CID-GFP and RFP-H2B That Were Simultaneously Depleted of TOPO II and RAD21, 96 h after the Addition of the dsRNA
Z-stacks were acquired every 15 s. Each Z-stack is 10 μm and is composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merged color images for H2B- RFP (red) and CID-...
Time-Lapse Microscopy of S2 Cells Stably Expressing CID-GFP and RFP-H2B That Were Depleted of RAD21, 96 h after the Addition of the dsRNA
Each Z-stack is 10 μm and is composed of ten optical sections. Anaphase onset corresponds to time 0 s. Merged color images for H2B-RFP (red) and CID-GFP (green) are shown on the left. The separated channel for CI...
Regulation of gene expression occurs at different levels, from DNA to protein, and through various mechanisms. One of them is modification of the chromatin structure, which is involved in the definition of transcriptional active and inactive regions of the chromosomes. These phenomena are associated with reversible chemical modifications of the gen...
During cell division, chromatin undergoes structural changes essential to ensure faithful segregation of the genome. Condensins, abundant components of mitotic chromosomes, are known to form two different complexes, condensins I and II. To further examine the role of condensin I in chromosome structure and in particular in centromere organization,...
One of the most remarkable and yet poorly understood events during the cell cycle is how dispersed chromatin fragments are transformed into chromosomes every time cells undergo mitosis. It has been postulated that mitotic chromosomes might contain an axial scaffold that is involved in condensation but its molecules and structure have remained elusi...
Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sist...
In Drosophila there is limited evidence on the nature of evolutionary forces affecting chromosomal arrangements other than inversions. The study of the X/4 fusion polymorphism of Drosophila americana is thus of interest. Polymorphism patterns at the paralytic (para) gene, located at the base of the X chromosome, suggest that there is suppressed cro...
Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells ha...
'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prop...
We have isolated a Hoppel-like transposon from heterochromatin of the second chromosome of Drosophila melanogaster and used a conserved DNA sequence between the different elements of this family to determine their distribution in both mitotic and polytene chromosomes. The hybridization pattern of polytene chromosomes extends throughout the entire c...
We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of speci...
. We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in α-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specifi...
The centromere is an essential cis-acting structure present in the chromosomes of all eukaryotes, central to the mechanism that ensures proper segregation during meiosis and mitosis. Molecular characterization of centromeres in the budding and fission yeasts has advanced significantly over the last few years due to their relatively small size and t...