The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural o... more The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural organization of the major proteins composing the lamina remains poorly defined. Using three-dimensional Structured Illumination Microscopy and computational image analysis, we have characterized the supramolecular structures of lamin A, C, B1 and B2 in mouse embryo fibroblast nuclei. Each isoform forms a distinct fiber meshwork, having comparable physical characteristics with respect to mesh edge length, mesh face area and shape, and edge connectivity to form faces. Some differences were found in face areas between isoforms due to variation in the edge lengths and number of edges per face, suggesting that each meshwork has somewhat unique assembly characteristics. In fibroblasts null for the expression of either lamins A/C or lamin B1, the remaining lamin meshworks are altered compared with the lamin meshworks in wild type nuclei or nuclei lacking lamin B2. Nuclei lacking LA/C exhibit sli...
Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved... more Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence o...
More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature... more More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor re...
Advances in experimental medicine and biology, 2014
In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with... more In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with the inner membrane of the nuclear envelope. The lamina provides mechanical support for the nucleus and functions as the major determinant of its size and shape. At its innermost aspect it associates with peripheral components of chromatin and thereby contributes to the organization of interphase chromosomes. The A- and B-type lamins are the major structural components of the lamina, and numerous mutations in the A-type lamin gene have been shown to cause many types of human diseases collectively known as the laminopathies. These mutations have also been shown to cause a disruption in the normal interactions between the A and B lamin networks. The impact of these mutations on nuclear functions is related to the roles of lamins in regulating various essential processes including DNA synthesis and damage repair, transcription and the regulation of genes involved in the response to oxidativ...
The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuc... more The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuclear pore, embedded within the nuclear envelope. However, we found that for SV40, the nuclear envelope poses a major hurdle to infection: FISH analysis revealed that the majority of viral DNA remains trapped in the ER; silencing of Lamin A/C rendered the cells more susceptible to infection; and proliferating cells are more susceptible to infection than quiescent cells. Surprisingly, we observed that following SV40 infection the nuclear envelope, including lamins A/C, B1, B2 and the nuclear pore complex, was dramatically deformed, as seen by immunohistochemistry. The infection induced fluctuations in the level of lamin A/C, dephosphorylation of an unknown epitope and leakage to the cytoplasm just prior to and during nuclear entry. Deformations were transient, and the spherical structure of the nuclear envelope was restored subsequent to nuclear entry. Nuclear envelope deformations and lam...
The nuclear lamins are type V intermediate filament proteins that are critically important for th... more The nuclear lamins are type V intermediate filament proteins that are critically important for the structural properties of the nucleus. In addition, they are involved in the regulation of numerous nuclear processes, including DNA replication, transcription and chromatin organization. The developmentally regulated expression of lamins suggests that they are involved in cellular differentiation. Their assembly dynamic properties throughout the cell cycle, particularly in mitosis, are influenced by posttranslational modifications. Lamins may regulate nuclear functions by direct interactions with chromatin and determining the spatial organization of chromosomes within the nuclear space. They may also regulate chromatin functions by interacting with factors that epigenetically modify the chromatin or directly regulate replication or transcription.
This study provides insights into the role of nuclear lamins in DNA replication. Our data demonst... more This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insight...
Telomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated wit... more Telomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated with cellular ageing. Telomere protection involves the insertion of the 3' overhang facilitated by telomere repeat-binding factor 2 (TRF2) into telomeric DNA, forming t-loops. We present evidence suggesting that t-loops can also form at interstitial telomeric sequences in a TRF2-dependent manner, forming an interstitial t-loop (ITL). We demonstrate that TRF2 association with interstitial telomeric sequences is stabilized by co-localization with A-type lamins (lamin A/C). We also find that lamin A/C interacts with TRF2 and that reduction in levels of lamin A/C or mutations in LMNA that cause an autosomal dominant premature ageing disorder--Hutchinson Gilford Progeria Syndrome (HGPS)-lead to reduced ITL formation and telomere loss. We propose that cellular and organismal ageing are intertwined through the effects of the interaction between TRF2 and lamin A/C on chromosome structure.
Over the past few years it has become evident that the intermediate filament proteins, the types ... more Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remark- ably large number of disease-causing mutations in
Proceedings of the National Academy of Sciences, 2007
Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-... more Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LADelta50/progerin). These modifications cause an abnormal association of LADelta50/progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G(1) is impaired in cells expressing LADelta50/progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.
Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re... more Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re-establish a functional nucleus. Time-lapse, FRAP and FRET analyses in human cells showed that barrier-to-autointegration factor (BAF), a DNA-binding protein, assembled first at the distinct ;core' region of the telophase chromosome and formed an immobile complex by directly binding with other core-localizing NE proteins, such as lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an electron-dense structure on the chromosome surface of the core, close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate core assembly of BAF. Disruption of the spindle MTs consistently abolished BAF accumulation at the core. In addition, RNAi of BAF eliminated the core assembly of lamin A and emerin, caused abnormal cytoplasmic accumulation of precursor nuclear membranes and resulted in a significant delay of NE assembly. These results suggest that the MT-mediated BAF accumulation at the core facilitates NE assembly at the end of mitosis.
Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nucle... more Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells.
Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved... more Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence of WI-38 cells. This overexpression eventually leads to cell cycle arrest at the G1/S boundary. These results demonstrate the importance of LB1 in regulating the proliferation and senescence of human diploid cells through a ROS signaling pathway.
The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural o... more The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural organization of the major proteins composing the lamina remains poorly defined. Using three-dimensional Structured Illumination Microscopy and computational image analysis, we have characterized the supramolecular structures of lamin A, C, B1 and B2 in mouse embryo fibroblast nuclei. Each isoform forms a distinct fiber meshwork, having comparable physical characteristics with respect to mesh edge length, mesh face area and shape, and edge connectivity to form faces. Some differences were found in face areas between isoforms due to variation in the edge lengths and number of edges per face, suggesting that each meshwork has somewhat unique assembly characteristics. In fibroblasts null for the expression of either lamins A/C or lamin B1, the remaining lamin meshworks are altered compared with the lamin meshworks in wild type nuclei or nuclei lacking lamin B2. Nuclei lacking LA/C exhibit sli...
Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved... more Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence o...
More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature... more More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor re...
Advances in experimental medicine and biology, 2014
In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with... more In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with the inner membrane of the nuclear envelope. The lamina provides mechanical support for the nucleus and functions as the major determinant of its size and shape. At its innermost aspect it associates with peripheral components of chromatin and thereby contributes to the organization of interphase chromosomes. The A- and B-type lamins are the major structural components of the lamina, and numerous mutations in the A-type lamin gene have been shown to cause many types of human diseases collectively known as the laminopathies. These mutations have also been shown to cause a disruption in the normal interactions between the A and B lamin networks. The impact of these mutations on nuclear functions is related to the roles of lamins in regulating various essential processes including DNA synthesis and damage repair, transcription and the regulation of genes involved in the response to oxidativ...
The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuc... more The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuclear pore, embedded within the nuclear envelope. However, we found that for SV40, the nuclear envelope poses a major hurdle to infection: FISH analysis revealed that the majority of viral DNA remains trapped in the ER; silencing of Lamin A/C rendered the cells more susceptible to infection; and proliferating cells are more susceptible to infection than quiescent cells. Surprisingly, we observed that following SV40 infection the nuclear envelope, including lamins A/C, B1, B2 and the nuclear pore complex, was dramatically deformed, as seen by immunohistochemistry. The infection induced fluctuations in the level of lamin A/C, dephosphorylation of an unknown epitope and leakage to the cytoplasm just prior to and during nuclear entry. Deformations were transient, and the spherical structure of the nuclear envelope was restored subsequent to nuclear entry. Nuclear envelope deformations and lam...
The nuclear lamins are type V intermediate filament proteins that are critically important for th... more The nuclear lamins are type V intermediate filament proteins that are critically important for the structural properties of the nucleus. In addition, they are involved in the regulation of numerous nuclear processes, including DNA replication, transcription and chromatin organization. The developmentally regulated expression of lamins suggests that they are involved in cellular differentiation. Their assembly dynamic properties throughout the cell cycle, particularly in mitosis, are influenced by posttranslational modifications. Lamins may regulate nuclear functions by direct interactions with chromatin and determining the spatial organization of chromosomes within the nuclear space. They may also regulate chromatin functions by interacting with factors that epigenetically modify the chromatin or directly regulate replication or transcription.
This study provides insights into the role of nuclear lamins in DNA replication. Our data demonst... more This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insight...
Telomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated wit... more Telomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated with cellular ageing. Telomere protection involves the insertion of the 3' overhang facilitated by telomere repeat-binding factor 2 (TRF2) into telomeric DNA, forming t-loops. We present evidence suggesting that t-loops can also form at interstitial telomeric sequences in a TRF2-dependent manner, forming an interstitial t-loop (ITL). We demonstrate that TRF2 association with interstitial telomeric sequences is stabilized by co-localization with A-type lamins (lamin A/C). We also find that lamin A/C interacts with TRF2 and that reduction in levels of lamin A/C or mutations in LMNA that cause an autosomal dominant premature ageing disorder--Hutchinson Gilford Progeria Syndrome (HGPS)-lead to reduced ITL formation and telomere loss. We propose that cellular and organismal ageing are intertwined through the effects of the interaction between TRF2 and lamin A/C on chromosome structure.
Over the past few years it has become evident that the intermediate filament proteins, the types ... more Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remark- ably large number of disease-causing mutations in
Proceedings of the National Academy of Sciences, 2007
Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-... more Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LADelta50/progerin). These modifications cause an abnormal association of LADelta50/progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G(1) is impaired in cells expressing LADelta50/progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.
Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re... more Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re-establish a functional nucleus. Time-lapse, FRAP and FRET analyses in human cells showed that barrier-to-autointegration factor (BAF), a DNA-binding protein, assembled first at the distinct ;core' region of the telophase chromosome and formed an immobile complex by directly binding with other core-localizing NE proteins, such as lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an electron-dense structure on the chromosome surface of the core, close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate core assembly of BAF. Disruption of the spindle MTs consistently abolished BAF accumulation at the core. In addition, RNAi of BAF eliminated the core assembly of lamin A and emerin, caused abnormal cytoplasmic accumulation of precursor nuclear membranes and resulted in a significant delay of NE assembly. These results suggest that the MT-mediated BAF accumulation at the core facilitates NE assembly at the end of mitosis.
Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nucle... more Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells.
Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved... more Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence of WI-38 cells. This overexpression eventually leads to cell cycle arrest at the G1/S boundary. These results demonstrate the importance of LB1 in regulating the proliferation and senescence of human diploid cells through a ROS signaling pathway.
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