The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating... more The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.
Nuclear shape and size are cell type-specific. Change in nuclear shape is seen during cell divisi... more Nuclear shape and size are cell type-specific. Change in nuclear shape is seen during cell division, development, and pathology. The nucleus of S. cerevisiae is spherical in interphase and becomes dumb-bell shaped during mitotic division to facilitate the transfer of one nucleus to the daughter cell. As yeast cells undergo closed mitosis, the nuclear envelope remains intact throughout the cell cycle. The pathways that regulate nuclear shape are not well characterized. The nucleus is organized into various sub-compartments, with the nucleolus being the most prominent. We have initiated a candidate-based genetic screen for nuclear shape abnormalities in S. cerevisiae to ask if the nucleolus influences the nuclear shape. We find that increasing nucleolar volume triggers a non-isometric nuclear envelope expansion resulting in an abnormal nuclear envelope shape. We further show that the tethering of rDNA to the nuclear envelope is required for the appearance of these extensions.
Ultraviolet (UV) radiation is a major environmental mutagen. Exposure
to UV leads to a sharp peak... more Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of H2AX, the phosphorylated form of the histone variant H2AX, in the S phase within an asynchronous population of cells. H2AX is often considered a definitive marker of DNA damage inside a cell. In this report, we show that H2AX in the S-phase cells after UV irradiation reports neither on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent of its secondary manifestations in the form of DNA double-strand breaks or in the inhibition of global transcription. Instead, H2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation. This accumulation of H2AX at replication sites slows down the replication. However, the cells do complete the replication of their genomes and arrest within the G2 phase. Our study suggests that it is not DNA damage itself but rather the response elicited, which peaks in the S phase upon UV irradiation, which leads to this effect.
Nuclear architecture is the organization of the genome within a cell nucleus with respect to diff... more Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as nuclear lamina, matrix or nucleoli. Lately it has emerged as a major regulator of gene expression in mammalian cells. The studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or in gene expression within a population. In this report we present a method for combining 3D DNA Fluorescence in situ Hybridization (FISH) with single molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine it with an imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of Cyclin A2 (CCNA2) gene for its known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position-which usually lies close to nuclear lamina-nor on the expression from the other copies.
In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damag... more In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damage. Chromatin has to be remodeled in order to repair such damage efficiently. But compact chromatin may also be more refractory to damage. Chromatin responses during DNA double strand break (DSB) repair have been studied with biochemistry or as indirect readouts for the physical state of the chromatin at the site of damage. Direct measures of global chromatin compaction upon damage are lacking. We used fluorescence anisotropy imaging of histone H2B-EGFP to directly interrogate global chromatin compaction changes in response to localized DSBs. Anisotropy maps were preserved in fixation and reported on underlying chromatin compaction states. Laser induced clustered DSBs led to a global compaction of even the undamaged chromatin. Live cell dynamics could be coupled with fixed cell assays. Repair factors, PARP1 and PCNA, were immediately recruited to the site of damage, though the local enrichment PCNA persisted longer than PARP1. Subsequently nodes of PCNA that incorporated deoxynucleotide analogs were observed in regions of low anisotropy open chromatin, even away from the site of damage. Such fluorescence anisotropy-based readout of chromatin compaction may be used in the context of different forms of DNA damage.
Edited by Xiao-Fan Wang Proper cell division relies on the coordinated regulation between a struc... more Edited by Xiao-Fan Wang Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G 2 /M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20-mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G 2 /M transition and thereby controls mitosis and cell division.
Single molecule Fluorescence in situ Hybridization (smFISH) for mRNA provides a powerful quantita... more Single molecule Fluorescence in situ Hybridization (smFISH) for mRNA provides a powerful quantitative handle on expression from endogenous gene loci. While the method has been widely applied in cells in culture, applications to primary tissue samples remain fewer, and often use involved cryosectioning. Even apart from quantitative access to absolute transcript counts in specific tissue volumes, many other advantages of smFISH can be envisaged in tissue samples. Primary among these are the ability to report on subtle differences in expression among different cell types within a tissue, and the ability to correlate the expression from different target genes. Here, we present a modified method of smFISH applicable on various primary wholemount tissues from the fruit fly Drosophila melanogaster, and show the efficacy of the method in a variety of larval and adult tissue, and embryos. We also combine smFISH in tissue with immunofluorescence to demonstrate the possibility of capturing transcriptional and translational aspects of gene expression in the same tissue. Given the widespread use of Drosophila melanogaster as a model system in Developmental Biology and Genetics, such methods are likely to be of wide interest and could yield rich information about gene expression in tissues from this organism.
DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such... more DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such damage is associated with the emergence of different cancers, neurological disorders and aging. DNA damage responses (DDR) in cells are closely associated with the cell cycle. While most of our knowledge of DDR comes from bulk biochemistry, such methods require cells to be arrested at specific stages for cell cycle studies, potentially altering measured responses; nor is cell to cell variability in DDR or direct cell-level correlation of two response metrics measured in such methods. To overcome these limitations we developed a microscopy-based assay for determining cell cycle stages over large cell numbers. This method can be used to study cell-cycle-dependent DDR in cultured cells without the need for cell synchronization. Upon DNA damage γH2A.X induction was correlated to nuclear enrichment of p53 on a cell-by-cell basis and in a cell cycle dependent manner. Imaging-based cell cycle staging was combined with single molecule P53 mRNA detection and immunofluorescence for p53 protein in the very same cells to reveal an intriguing repression of P53 transcript numbers due to reduced transcription across different stages of the cell cycle during DNA damage. Our study hints at an unexplored mechanism for p53 regulation and underscores the importance of measuring single cell level responses to DNA damage.
Nuclear shape and size are emerging as mechanistic regulators of genome function. Yet, the coupli... more Nuclear shape and size are emerging as mechanistic regulators of genome function. Yet, the coupling between chromatin assembly and various nuclear and cytoplasmic scaffolds is poorly understood. The present work explores the structural organization of a prestressed nucleus in a variety of cellular systems ranging from cells in culture to those in an organism. A combination of laser ablation and cellular perturbations was used to decipher the dynamic nature of the nucleo-cytoplasmic contacts. In primary mouse embryonic fibroblasts, ablation of heterochromatin nodes caused an anisotropic shrinkage of the nucleus. Depolymerization of actin and microtubules, and inhibition of myosin motors, resulted in the differential stresses that these cytoplasmic systems exert on the nucleus. The onset of nuclear prestress was then mapped in two contexts--first, in the differentiation of embryonic stem cells, where signatures of prestress appeared with differentiation; second, at an organism level, where nuclear or cytoplasmic laser ablations of cells in the early Drosophila embryo induced a collapse of the nucleus only after cellularization. We thus show that the interplay of physical connections bridging the nucleus with the cytoplasm governs the size and shape of a prestressed eukaryotic nucleus.
The nucleus is maintained in a prestressed state within eukaryotic cells, stabilized mechanically... more The nucleus is maintained in a prestressed state within eukaryotic cells, stabilized mechanically by chromatin structure and other nuclear components on its inside, and cytoskeletal components on its outside. Nuclear architecture is emerging to be critical to the governance of chromatin assembly, regulation of genome function and cellular homeostasis. Elucidating the prestressed organization of the nucleus is thus important to understand how the nuclear architecture impinges on its function. In this chapter, various chemical and mechanical methods have been described to probe the prestressed organization of the nucleus.
The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating... more The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.
Nuclear shape and size are cell type-specific. Change in nuclear shape is seen during cell divisi... more Nuclear shape and size are cell type-specific. Change in nuclear shape is seen during cell division, development, and pathology. The nucleus of S. cerevisiae is spherical in interphase and becomes dumb-bell shaped during mitotic division to facilitate the transfer of one nucleus to the daughter cell. As yeast cells undergo closed mitosis, the nuclear envelope remains intact throughout the cell cycle. The pathways that regulate nuclear shape are not well characterized. The nucleus is organized into various sub-compartments, with the nucleolus being the most prominent. We have initiated a candidate-based genetic screen for nuclear shape abnormalities in S. cerevisiae to ask if the nucleolus influences the nuclear shape. We find that increasing nucleolar volume triggers a non-isometric nuclear envelope expansion resulting in an abnormal nuclear envelope shape. We further show that the tethering of rDNA to the nuclear envelope is required for the appearance of these extensions.
Ultraviolet (UV) radiation is a major environmental mutagen. Exposure
to UV leads to a sharp peak... more Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of H2AX, the phosphorylated form of the histone variant H2AX, in the S phase within an asynchronous population of cells. H2AX is often considered a definitive marker of DNA damage inside a cell. In this report, we show that H2AX in the S-phase cells after UV irradiation reports neither on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent of its secondary manifestations in the form of DNA double-strand breaks or in the inhibition of global transcription. Instead, H2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation. This accumulation of H2AX at replication sites slows down the replication. However, the cells do complete the replication of their genomes and arrest within the G2 phase. Our study suggests that it is not DNA damage itself but rather the response elicited, which peaks in the S phase upon UV irradiation, which leads to this effect.
Nuclear architecture is the organization of the genome within a cell nucleus with respect to diff... more Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as nuclear lamina, matrix or nucleoli. Lately it has emerged as a major regulator of gene expression in mammalian cells. The studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or in gene expression within a population. In this report we present a method for combining 3D DNA Fluorescence in situ Hybridization (FISH) with single molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine it with an imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of Cyclin A2 (CCNA2) gene for its known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position-which usually lies close to nuclear lamina-nor on the expression from the other copies.
In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damag... more In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damage. Chromatin has to be remodeled in order to repair such damage efficiently. But compact chromatin may also be more refractory to damage. Chromatin responses during DNA double strand break (DSB) repair have been studied with biochemistry or as indirect readouts for the physical state of the chromatin at the site of damage. Direct measures of global chromatin compaction upon damage are lacking. We used fluorescence anisotropy imaging of histone H2B-EGFP to directly interrogate global chromatin compaction changes in response to localized DSBs. Anisotropy maps were preserved in fixation and reported on underlying chromatin compaction states. Laser induced clustered DSBs led to a global compaction of even the undamaged chromatin. Live cell dynamics could be coupled with fixed cell assays. Repair factors, PARP1 and PCNA, were immediately recruited to the site of damage, though the local enrichment PCNA persisted longer than PARP1. Subsequently nodes of PCNA that incorporated deoxynucleotide analogs were observed in regions of low anisotropy open chromatin, even away from the site of damage. Such fluorescence anisotropy-based readout of chromatin compaction may be used in the context of different forms of DNA damage.
Edited by Xiao-Fan Wang Proper cell division relies on the coordinated regulation between a struc... more Edited by Xiao-Fan Wang Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G 2 /M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20-mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G 2 /M transition and thereby controls mitosis and cell division.
Single molecule Fluorescence in situ Hybridization (smFISH) for mRNA provides a powerful quantita... more Single molecule Fluorescence in situ Hybridization (smFISH) for mRNA provides a powerful quantitative handle on expression from endogenous gene loci. While the method has been widely applied in cells in culture, applications to primary tissue samples remain fewer, and often use involved cryosectioning. Even apart from quantitative access to absolute transcript counts in specific tissue volumes, many other advantages of smFISH can be envisaged in tissue samples. Primary among these are the ability to report on subtle differences in expression among different cell types within a tissue, and the ability to correlate the expression from different target genes. Here, we present a modified method of smFISH applicable on various primary wholemount tissues from the fruit fly Drosophila melanogaster, and show the efficacy of the method in a variety of larval and adult tissue, and embryos. We also combine smFISH in tissue with immunofluorescence to demonstrate the possibility of capturing transcriptional and translational aspects of gene expression in the same tissue. Given the widespread use of Drosophila melanogaster as a model system in Developmental Biology and Genetics, such methods are likely to be of wide interest and could yield rich information about gene expression in tissues from this organism.
DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such... more DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such damage is associated with the emergence of different cancers, neurological disorders and aging. DNA damage responses (DDR) in cells are closely associated with the cell cycle. While most of our knowledge of DDR comes from bulk biochemistry, such methods require cells to be arrested at specific stages for cell cycle studies, potentially altering measured responses; nor is cell to cell variability in DDR or direct cell-level correlation of two response metrics measured in such methods. To overcome these limitations we developed a microscopy-based assay for determining cell cycle stages over large cell numbers. This method can be used to study cell-cycle-dependent DDR in cultured cells without the need for cell synchronization. Upon DNA damage γH2A.X induction was correlated to nuclear enrichment of p53 on a cell-by-cell basis and in a cell cycle dependent manner. Imaging-based cell cycle staging was combined with single molecule P53 mRNA detection and immunofluorescence for p53 protein in the very same cells to reveal an intriguing repression of P53 transcript numbers due to reduced transcription across different stages of the cell cycle during DNA damage. Our study hints at an unexplored mechanism for p53 regulation and underscores the importance of measuring single cell level responses to DNA damage.
Nuclear shape and size are emerging as mechanistic regulators of genome function. Yet, the coupli... more Nuclear shape and size are emerging as mechanistic regulators of genome function. Yet, the coupling between chromatin assembly and various nuclear and cytoplasmic scaffolds is poorly understood. The present work explores the structural organization of a prestressed nucleus in a variety of cellular systems ranging from cells in culture to those in an organism. A combination of laser ablation and cellular perturbations was used to decipher the dynamic nature of the nucleo-cytoplasmic contacts. In primary mouse embryonic fibroblasts, ablation of heterochromatin nodes caused an anisotropic shrinkage of the nucleus. Depolymerization of actin and microtubules, and inhibition of myosin motors, resulted in the differential stresses that these cytoplasmic systems exert on the nucleus. The onset of nuclear prestress was then mapped in two contexts--first, in the differentiation of embryonic stem cells, where signatures of prestress appeared with differentiation; second, at an organism level, where nuclear or cytoplasmic laser ablations of cells in the early Drosophila embryo induced a collapse of the nucleus only after cellularization. We thus show that the interplay of physical connections bridging the nucleus with the cytoplasm governs the size and shape of a prestressed eukaryotic nucleus.
The nucleus is maintained in a prestressed state within eukaryotic cells, stabilized mechanically... more The nucleus is maintained in a prestressed state within eukaryotic cells, stabilized mechanically by chromatin structure and other nuclear components on its inside, and cytoskeletal components on its outside. Nuclear architecture is emerging to be critical to the governance of chromatin assembly, regulation of genome function and cellular homeostasis. Elucidating the prestressed organization of the nucleus is thus important to understand how the nuclear architecture impinges on its function. In this chapter, various chemical and mechanical methods have been described to probe the prestressed organization of the nucleus.
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Papers by Aprotim Mazumder
to UV leads to a sharp peak of H2AX, the phosphorylated form of the histone variant
H2AX, in the S phase within an asynchronous population of cells. H2AX is often
considered a definitive marker of DNA damage inside a cell. In this report, we show
that H2AX in the S-phase cells after UV irradiation reports neither on the extent of
primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent
of its secondary manifestations in the form of DNA double-strand breaks or in
the inhibition of global transcription. Instead, H2AX in the S phase corresponds to
the sites of active replication at the time of UV irradiation. This accumulation of
H2AX at replication sites slows down the replication. However, the cells do complete
the replication of their genomes and arrest within the G2 phase. Our study
suggests that it is not DNA damage itself but rather the response elicited, which
peaks in the S phase upon UV irradiation, which leads to this effect.
to UV leads to a sharp peak of H2AX, the phosphorylated form of the histone variant
H2AX, in the S phase within an asynchronous population of cells. H2AX is often
considered a definitive marker of DNA damage inside a cell. In this report, we show
that H2AX in the S-phase cells after UV irradiation reports neither on the extent of
primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent
of its secondary manifestations in the form of DNA double-strand breaks or in
the inhibition of global transcription. Instead, H2AX in the S phase corresponds to
the sites of active replication at the time of UV irradiation. This accumulation of
H2AX at replication sites slows down the replication. However, the cells do complete
the replication of their genomes and arrest within the G2 phase. Our study
suggests that it is not DNA damage itself but rather the response elicited, which
peaks in the S phase upon UV irradiation, which leads to this effect.