Uttam Surana - Academia.edu (original) (raw)

Papers by Uttam Surana

Research paper thumbnail of Faculty of 1000 evaluation for Extensive DNA damage-induced sumoylation contributes to replication and repair and acts in addition to the mec1 checkpoint

F1000 - Post-publication peer review of the biomedical literature, 2012

Research paper thumbnail of Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast

The EMBO Journal, 1993

It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 ... more It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomcyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B-type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of espi mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.

Research paper thumbnail of Faculty of 1000 evaluation for Kinetochore geometry defined by cohesion within the centromere

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Faculty Opinions recommendation of Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2022

Research paper thumbnail of Faculty Opinions recommendation of Aurora B switches relative strength of kinetochore-microtubule attachment modes for error correction

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

Research paper thumbnail of Faculty Opinions recommendation of The Negative Cross-Talk between SAG/RBX2/ROC2 and APC/C E3 Ligases in Regulation of Cell Cycle Progression and Drug Resistance

Anaphase-promoting complex/cyclosome (APC/C) is a well-characterized E3 ligase that couples with ... more Anaphase-promoting complex/cyclosome (APC/C) is a well-characterized E3 ligase that couples with UBE2C and UBE2S E2s for substrate ubiquitylation by the K11 linkage. Our recent data show that SAG/RBX2/ROC2, a RING component of Cullin-RING E3 ligase, also complexes with these E2s for K11-linked substrate polyubiquitylation. Whether these two E3s cross-talk with each other was previously unknown. Here, we report that SAG competes with APC2 for UBE2C/ UBE2S binding to act as a potential endogenous inhibitor of APC/C, thereby regulating the G2-to-M progression. As such, SAG knockdown triggers premature activation of APC/C, leading to mitotic slippage and resistance to anti-microtubule drugs. On the other hand, SAG itself is a substrate of APC/C CDH1 for targeted degradation at the G1 phase. The degradation-resistant mutant of SAG-R98A/L101A accelerates the G1-to-S progression. Our study reveals that the negative cross-talk between SAG and APC/C is likely a mechanism to ensure the fidelity of cell cycle progression.

Research paper thumbnail of Additional file 3: Figure S3. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

AML mice developed myeloid sarcoma. a Representative images of multiple organs from CD34+ engraft... more AML mice developed myeloid sarcoma. a Representative images of multiple organs from CD34+ engrafted mice were shown (scale bar: 1 cm) and b analyzed using H&E and immunohistochemical stain for human CD45, CD117, and MPO. Representative images of multiple organs were shown; scale bar: 1 cm or 100 μm as indicated. (TIFF 13606 kb)

Research paper thumbnail of Additional file 1: Figure S1. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Immune profile of BM mononuclear cells from AML patients. a Mononuclear cells isolated from AML p... more Immune profile of BM mononuclear cells from AML patients. a Mononuclear cells isolated from AML patients were immunolabeled with human CD45, CD34, CD38, CD33, and CD117 and analyzed using flow cytometry. Gating using CD45 and CD34 showed three subsets indicating of (i) CD45hiCD34− non-blast cells, (ii) CD45loCD34− blast cells, and (iii) CD45loCD34+ blast cells. Expression of CD38, CD33, and CD117 for each subset was shown. Frequency of CD33+CD117+ relative to total human CD45+ cells in each subset was shown. b Level of primary engraftment from poor responders. Newborn NSG pups were injected intrahepatically with 8.7 × 104–7.9 × 105 cells after sublethal irradiation. Level of engraftment in peripheral blood was determined at specified weeks post-engraftment and in spleen and BM at endpoint using event number of human CD45+ cells divided by the sum of human CD45+ cells and mouse CD45.1+ cells. Data are presented as mean % human CD45+ cells relative to total CD45+ cells ± SEM. (TIFF 32...

Research paper thumbnail of Content Alerts

This article cites 51 articles, 20 of which can be accessed free

Research paper thumbnail of CHILD syndrome in a Malaysian adult with identification of a novel heterozygous missense mutation NSDHL c.602A>G

International Journal of Dermatology, 2020

CHILD syndrome in a Malaysian adult with identification of a novel heterozygous missense mutation... more CHILD syndrome in a Malaysian adult with identification of a novel heterozygous missense mutation NSDHL c.602A>G Dear Editor, Congenital Hemidysplasia with Ichthyosiform nevus and Limb Defects (CHILD) syndrome (OMIM #308050) is a rare Xlinked dominant disorder with male-lethal trait, resulting from the loss of function mutations in the NSDHL gene located at chromosome Xq28, which encodes the NAD(P)H steroid dehydrogenase-like protein (NSDHL). 1-3 The enzyme is vital for the synthesis of cholesterol. It presents at or shortly after birth with unilateral ichthyosiform nevus, unilateral skeletal aplasia/hypoplasia, and/or defects in the cardiovascular, renal, central nervous system, genitourinary, pulmonary, or endocrine systems. 1-3 Here, we describe the first confirmed case of CHILD of Chinese ethnicity in Malaysia.

Research paper thumbnail of Faculty of 1000 evaluation for Bub1 kinase and sgo1 modulate pericentric chromatin in response to altered microtubule dynamics

F1000 - Post-publication peer review of the biomedical literature, 2012

Background-Tension sensing of bi-oriented chromosomes is essential for the fidelity of chromosome... more Background-Tension sensing of bi-oriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1) to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known. Results-We have identified a Bub1/Sgo1 dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract while the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics. Conclusions-Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through Histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.

Research paper thumbnail of Faculty of 1000 evaluation for A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint

F1000 - Post-publication peer review of the biomedical literature, 2015

The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment du... more The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such subfunctionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

Research paper thumbnail of Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

Journal of Cell Biology, 2018

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinet... more In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore m...

Research paper thumbnail of DNA damage checkpoint execution and the rules of its disengagement

Frontiers in Cell and Developmental Biology

Chromosomes are susceptible to damage during their duplication and segregation or when exposed to... more Chromosomes are susceptible to damage during their duplication and segregation or when exposed to genotoxic stresses. Left uncorrected, these lesions can result in genomic instability, leading to cells’ diminished fitness, unbridled proliferation or death. To prevent such fates, checkpoint controls transiently halt cell cycle progression to allow time for the implementation of corrective measures. Prominent among these is the DNA damage checkpoint which operates at G2/M transition to ensure that cells with damaged chromosomes do not enter the mitotic phase. The execution and maintenance of cell cycle arrest are essential aspects of G2/M checkpoint and have been studied in detail. Equally critical is cells’ ability to switch-off the checkpoint controls after a successful completion of corrective actions and to recommence cell cycle progression. Interestingly, when corrective measures fail, cells can mount an unusual cellular response, termed adaptation, where they escape checkpoint a...

Research paper thumbnail of Mechanical properties of Bacillus subtilis cell walls: effects of removing residual culture medium

Journal of Bacteriology, 1991

Experiments are described in which the tensile strength, the initial (Youngs') modulus, and o... more Experiments are described in which the tensile strength, the initial (Youngs') modulus, and other mechanical properties of the bacterial cell wall were obtained as functions of relative humidity (RH) in the range of 20 to 95%. These properties were deduced from tensile tests on bacterial thread, a fiber consisting of many highly aligned cells of Bacillus subtilis, from which residual culture medium had been removed by immersion in water. Reasons are given to support the idea that the mechanical properties of bacterial thread relate directly to those of the cylinder wall and that they are not influenced by septa, cytoplasm, or the thread assembly. The data show that the cell wall, like many other heteropolymers, is visco-elastic. When dry, it behaves like a glassy polymer with a tensile strength of about 300 MPa and a modulus of about 13 GPa. When wet, its behavior is more like a rubbery polymer with a tensile strength of about 13 MPa and a modulus of about 30 MPa. Thus, the cell...

Research paper thumbnail of Additional file 6: Figure S6. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Sorafenib and Regorafenib treatment has no impact on the frequency of CD34+ and CD34+CD117+ AML c... more Sorafenib and Regorafenib treatment has no impact on the frequency of CD34+ and CD34+CD117+ AML cells in mice. Magnetically sorted CD34+ pooled BM cells and splenocytes from secondary-engrafted NSG mice were injected intrahepatically in newborn NSG pups after sublethal irradiation (1 × 105 cells per pup). Successfully engrafted mice with more than 30 human CD45+ cells per microliter of blood (between week 12 to 16 post-engraftment) were randomly assigned to either untreated (n = 3), Regorafenib (n = 6; 5 mg/kg body weight; gavage-fed once daily), or Sorafenib (n = 6; 10 mg/kg body weight; gavage-fed once daily) treatment groups and monitored for 1 month. a Representative flow cytometry plots illustrating the expression of CD34 and CD117 in human CD45 cells. b Comparison of the frequencies of total CD34+ (B, above) and CD34+CD117+ (B, below) cells relative to human CD45 cells in peripheral blood, spleen, and BM of different treatment groups after 4 weeks post-drug treatment. Data are...

Research paper thumbnail of Additional file 4: Figure S4. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Engraftment of AML cells is highest in the BM at week 4 post-engraftment. Magnetically sorted CD3... more Engraftment of AML cells is highest in the BM at week 4 post-engraftment. Magnetically sorted CD34+ pooled BM cells and splenocytes from primary engrafted NSG mice were injected intrahepatically in NSG newborn pups (n = 4) after sublethal irradiation (1 × 105 cells per pup). a Frequencies of mouse CD45.1+ cells and human CD45+ cells in peripheral blood, BM, and spleen were determined at week 4 post-engraftment. Frequency of human CD45+ cells and mouse CD45.1+ cells are calculated by normalizing the event number of human CD45+ cells or mouse CD45.1+ cells over the sum of human CD45+ cells and mouse CD45.1+ cells event numbers. b Frequency and c absolute count of human CD45+ cells in peripheral blood, spleen, and BM. Data are presented as mean frequencies or absolute count per organ ± SEM. Two-tailed Mann Whitney U test; *; p

Research paper thumbnail of Supplemental Experimental Procedures

The cell monolayers and frozen sections or paraffin embedded sections from tumors were fixed in 4... more The cell monolayers and frozen sections or paraffin embedded sections from tumors were fixed in 4% paraformaldehyde for 10 min, washed with PBS and permeabilized with 0.2 % Triton-X in PBS 30 min. After blocking in PBS containing 10 % FBS for 20 min, samples were incubated 1 h at 37°C with anti-LANA 1:1000, or

Research paper thumbnail of Faculty of 1000 evaluation for Aurora kinase A localises to mitochondria to control organelle dynamics and energy production

F1000 - Post-publication peer review of the biomedical literature, 2018

Research paper thumbnail of Faculty of 1000 evaluation for Inner centromere localization of the CPC maintains centromere cohesion and allows mitotic checkpoint silencing

F1000 - Post-publication peer review of the biomedical literature, 2017

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chrom... more Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation.

Research paper thumbnail of Faculty of 1000 evaluation for Extensive DNA damage-induced sumoylation contributes to replication and repair and acts in addition to the mec1 checkpoint

F1000 - Post-publication peer review of the biomedical literature, 2012

Research paper thumbnail of Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast

The EMBO Journal, 1993

It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 ... more It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomcyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B-type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of espi mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.

Research paper thumbnail of Faculty of 1000 evaluation for Kinetochore geometry defined by cohesion within the centromere

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Faculty Opinions recommendation of Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2022

Research paper thumbnail of Faculty Opinions recommendation of Aurora B switches relative strength of kinetochore-microtubule attachment modes for error correction

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

Research paper thumbnail of Faculty Opinions recommendation of The Negative Cross-Talk between SAG/RBX2/ROC2 and APC/C E3 Ligases in Regulation of Cell Cycle Progression and Drug Resistance

Anaphase-promoting complex/cyclosome (APC/C) is a well-characterized E3 ligase that couples with ... more Anaphase-promoting complex/cyclosome (APC/C) is a well-characterized E3 ligase that couples with UBE2C and UBE2S E2s for substrate ubiquitylation by the K11 linkage. Our recent data show that SAG/RBX2/ROC2, a RING component of Cullin-RING E3 ligase, also complexes with these E2s for K11-linked substrate polyubiquitylation. Whether these two E3s cross-talk with each other was previously unknown. Here, we report that SAG competes with APC2 for UBE2C/ UBE2S binding to act as a potential endogenous inhibitor of APC/C, thereby regulating the G2-to-M progression. As such, SAG knockdown triggers premature activation of APC/C, leading to mitotic slippage and resistance to anti-microtubule drugs. On the other hand, SAG itself is a substrate of APC/C CDH1 for targeted degradation at the G1 phase. The degradation-resistant mutant of SAG-R98A/L101A accelerates the G1-to-S progression. Our study reveals that the negative cross-talk between SAG and APC/C is likely a mechanism to ensure the fidelity of cell cycle progression.

Research paper thumbnail of Additional file 3: Figure S3. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

AML mice developed myeloid sarcoma. a Representative images of multiple organs from CD34+ engraft... more AML mice developed myeloid sarcoma. a Representative images of multiple organs from CD34+ engrafted mice were shown (scale bar: 1 cm) and b analyzed using H&E and immunohistochemical stain for human CD45, CD117, and MPO. Representative images of multiple organs were shown; scale bar: 1 cm or 100 μm as indicated. (TIFF 13606 kb)

Research paper thumbnail of Additional file 1: Figure S1. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Immune profile of BM mononuclear cells from AML patients. a Mononuclear cells isolated from AML p... more Immune profile of BM mononuclear cells from AML patients. a Mononuclear cells isolated from AML patients were immunolabeled with human CD45, CD34, CD38, CD33, and CD117 and analyzed using flow cytometry. Gating using CD45 and CD34 showed three subsets indicating of (i) CD45hiCD34− non-blast cells, (ii) CD45loCD34− blast cells, and (iii) CD45loCD34+ blast cells. Expression of CD38, CD33, and CD117 for each subset was shown. Frequency of CD33+CD117+ relative to total human CD45+ cells in each subset was shown. b Level of primary engraftment from poor responders. Newborn NSG pups were injected intrahepatically with 8.7 × 104–7.9 × 105 cells after sublethal irradiation. Level of engraftment in peripheral blood was determined at specified weeks post-engraftment and in spleen and BM at endpoint using event number of human CD45+ cells divided by the sum of human CD45+ cells and mouse CD45.1+ cells. Data are presented as mean % human CD45+ cells relative to total CD45+ cells ± SEM. (TIFF 32...

Research paper thumbnail of Content Alerts

This article cites 51 articles, 20 of which can be accessed free

Research paper thumbnail of CHILD syndrome in a Malaysian adult with identification of a novel heterozygous missense mutation NSDHL c.602A>G

International Journal of Dermatology, 2020

CHILD syndrome in a Malaysian adult with identification of a novel heterozygous missense mutation... more CHILD syndrome in a Malaysian adult with identification of a novel heterozygous missense mutation NSDHL c.602A>G Dear Editor, Congenital Hemidysplasia with Ichthyosiform nevus and Limb Defects (CHILD) syndrome (OMIM #308050) is a rare Xlinked dominant disorder with male-lethal trait, resulting from the loss of function mutations in the NSDHL gene located at chromosome Xq28, which encodes the NAD(P)H steroid dehydrogenase-like protein (NSDHL). 1-3 The enzyme is vital for the synthesis of cholesterol. It presents at or shortly after birth with unilateral ichthyosiform nevus, unilateral skeletal aplasia/hypoplasia, and/or defects in the cardiovascular, renal, central nervous system, genitourinary, pulmonary, or endocrine systems. 1-3 Here, we describe the first confirmed case of CHILD of Chinese ethnicity in Malaysia.

Research paper thumbnail of Faculty of 1000 evaluation for Bub1 kinase and sgo1 modulate pericentric chromatin in response to altered microtubule dynamics

F1000 - Post-publication peer review of the biomedical literature, 2012

Background-Tension sensing of bi-oriented chromosomes is essential for the fidelity of chromosome... more Background-Tension sensing of bi-oriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1) to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known. Results-We have identified a Bub1/Sgo1 dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract while the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics. Conclusions-Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through Histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.

Research paper thumbnail of Faculty of 1000 evaluation for A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint

F1000 - Post-publication peer review of the biomedical literature, 2015

The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment du... more The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such subfunctionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

Research paper thumbnail of Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

Journal of Cell Biology, 2018

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinet... more In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore m...

Research paper thumbnail of DNA damage checkpoint execution and the rules of its disengagement

Frontiers in Cell and Developmental Biology

Chromosomes are susceptible to damage during their duplication and segregation or when exposed to... more Chromosomes are susceptible to damage during their duplication and segregation or when exposed to genotoxic stresses. Left uncorrected, these lesions can result in genomic instability, leading to cells’ diminished fitness, unbridled proliferation or death. To prevent such fates, checkpoint controls transiently halt cell cycle progression to allow time for the implementation of corrective measures. Prominent among these is the DNA damage checkpoint which operates at G2/M transition to ensure that cells with damaged chromosomes do not enter the mitotic phase. The execution and maintenance of cell cycle arrest are essential aspects of G2/M checkpoint and have been studied in detail. Equally critical is cells’ ability to switch-off the checkpoint controls after a successful completion of corrective actions and to recommence cell cycle progression. Interestingly, when corrective measures fail, cells can mount an unusual cellular response, termed adaptation, where they escape checkpoint a...

Research paper thumbnail of Mechanical properties of Bacillus subtilis cell walls: effects of removing residual culture medium

Journal of Bacteriology, 1991

Experiments are described in which the tensile strength, the initial (Youngs') modulus, and o... more Experiments are described in which the tensile strength, the initial (Youngs') modulus, and other mechanical properties of the bacterial cell wall were obtained as functions of relative humidity (RH) in the range of 20 to 95%. These properties were deduced from tensile tests on bacterial thread, a fiber consisting of many highly aligned cells of Bacillus subtilis, from which residual culture medium had been removed by immersion in water. Reasons are given to support the idea that the mechanical properties of bacterial thread relate directly to those of the cylinder wall and that they are not influenced by septa, cytoplasm, or the thread assembly. The data show that the cell wall, like many other heteropolymers, is visco-elastic. When dry, it behaves like a glassy polymer with a tensile strength of about 300 MPa and a modulus of about 13 GPa. When wet, its behavior is more like a rubbery polymer with a tensile strength of about 13 MPa and a modulus of about 30 MPa. Thus, the cell...

Research paper thumbnail of Additional file 6: Figure S6. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Sorafenib and Regorafenib treatment has no impact on the frequency of CD34+ and CD34+CD117+ AML c... more Sorafenib and Regorafenib treatment has no impact on the frequency of CD34+ and CD34+CD117+ AML cells in mice. Magnetically sorted CD34+ pooled BM cells and splenocytes from secondary-engrafted NSG mice were injected intrahepatically in newborn NSG pups after sublethal irradiation (1 × 105 cells per pup). Successfully engrafted mice with more than 30 human CD45+ cells per microliter of blood (between week 12 to 16 post-engraftment) were randomly assigned to either untreated (n = 3), Regorafenib (n = 6; 5 mg/kg body weight; gavage-fed once daily), or Sorafenib (n = 6; 10 mg/kg body weight; gavage-fed once daily) treatment groups and monitored for 1 month. a Representative flow cytometry plots illustrating the expression of CD34 and CD117 in human CD45 cells. b Comparison of the frequencies of total CD34+ (B, above) and CD34+CD117+ (B, below) cells relative to human CD45 cells in peripheral blood, spleen, and BM of different treatment groups after 4 weeks post-drug treatment. Data are...

Research paper thumbnail of Additional file 4: Figure S4. of An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Engraftment of AML cells is highest in the BM at week 4 post-engraftment. Magnetically sorted CD3... more Engraftment of AML cells is highest in the BM at week 4 post-engraftment. Magnetically sorted CD34+ pooled BM cells and splenocytes from primary engrafted NSG mice were injected intrahepatically in NSG newborn pups (n = 4) after sublethal irradiation (1 × 105 cells per pup). a Frequencies of mouse CD45.1+ cells and human CD45+ cells in peripheral blood, BM, and spleen were determined at week 4 post-engraftment. Frequency of human CD45+ cells and mouse CD45.1+ cells are calculated by normalizing the event number of human CD45+ cells or mouse CD45.1+ cells over the sum of human CD45+ cells and mouse CD45.1+ cells event numbers. b Frequency and c absolute count of human CD45+ cells in peripheral blood, spleen, and BM. Data are presented as mean frequencies or absolute count per organ ± SEM. Two-tailed Mann Whitney U test; *; p

Research paper thumbnail of Supplemental Experimental Procedures

The cell monolayers and frozen sections or paraffin embedded sections from tumors were fixed in 4... more The cell monolayers and frozen sections or paraffin embedded sections from tumors were fixed in 4% paraformaldehyde for 10 min, washed with PBS and permeabilized with 0.2 % Triton-X in PBS 30 min. After blocking in PBS containing 10 % FBS for 20 min, samples were incubated 1 h at 37°C with anti-LANA 1:1000, or

Research paper thumbnail of Faculty of 1000 evaluation for Aurora kinase A localises to mitochondria to control organelle dynamics and energy production

F1000 - Post-publication peer review of the biomedical literature, 2018

Research paper thumbnail of Faculty of 1000 evaluation for Inner centromere localization of the CPC maintains centromere cohesion and allows mitotic checkpoint silencing

F1000 - Post-publication peer review of the biomedical literature, 2017

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chrom... more Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation.