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Find video protocols related to scientific articles indexed in Pubmed.
Phosphorylation-dephosphorylation cycle of HP1? governs accurate mitotic progression.
Cell Cycle
PUBLISHED: 04-30-2014
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Heterochromatin protein 1? (HP1?), a bona fide factor of silent chromatin, is required for establishing as well as maintaining the higher-order chromatin structure in eukaryotes. HP1? is decorated with several post-translational modifications, and many of these are critical for its cellular functions. HP1? is heavily phosphorylated; however, its physiological relevance had remained to be completely understood. We have recently demonstrated that human HP1? is a mitotic target for NDR kinase, and the phosphorylation at the hinge region of HP1? at the G 2/M phase of the cell cycle is crucial for mitotic progression and Sgo1 loading at mitotic centromeres (Chakraborty et al., 2014). We now demonstrate that the dephosphorylation of HP1? within its hinge domain occurs during mitosis, specifically soon after prometaphase. In the absence of the hinge-specific HP1? phosphorylation, either as a consequence of depleting NDR1 or in cells expressing a non-phosphorylatable HP1? mutant, the cells arrest in prometaphase with several mitotic defects. In this study we show that NDR1-depleted cells expressing hinge-specific phosphomimetic HP1? mutant rescues the prometaphase arrest but displays defects in mitotic exit, suggesting that the dephosphorylation of HP1? is required for the completion of cytokinesis. Taken together, our results reveal that the phosphorylation-dephosphorylation cycle of HP1? orchestrates accurate progression of cells through mitosis.
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Dynamic phosphorylation of HP1? regulates mitotic progression in human cells.
Nat Commun
PUBLISHED: 02-12-2014
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Heterochromatin protein 1? (HP1?), a key player in the establishment and maintenance of higher-order chromatin regulates key cellular processes, including metaphase chromatid cohesion and centromere organization. However, how HP1? controls these processes is not well understood. Here we demonstrate that post-translational modifications of HP1? dictate its mitotic functions. HP1? is constitutively phosphorylated within its amino terminus, whereas phosphorylation within the hinge domain occurs preferentially at G2/M phase of the cell cycle. The hinge-phosphorylated form of HP1? specifically localizes to kinetochores during early mitosis and this phosphorylation mediated by NDR1 kinase is required for mitotic progression and for Sgo1 binding to mitotic centromeres. Cells lacking NDR kinase show loss of mitosis-specific phosphorylation of HP1? leading to prometaphase arrest. Our results reveal that NDR kinase catalyses the hinge-specific phosphorylation of human HP1? during G2/M in vivo and this orchestrates accurate chromosome alignment and mitotic progression.
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Differential host cell gene expression and regulation of cell cycle progression by nonstructural protein 11 of porcine reproductive and respiratory syndrome virus.
Biomed Res Int
PUBLISHED: 01-07-2014
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Nonstructural protein 11 (nsp11) of porcine reproductive and respiratory syndrome virus (PRRSV) is a viral endoribonuclease with an unknown function. The regulation of cellular gene expression by nsp11 was examined by RNA microarrays using MARC-nsp11 cells constitutively expressing nsp11. In these cells, the interferon-?, interferon regulatory factor 3, and nuclear factor-? B activities were suppressed compared to those of parental cells, suggesting that nsp11 might serve as a viral interferon antagonist. Differential cellular transcriptome was examined using Affymetrix exon chips representing 28,536 transcripts, and after statistical analyses 66 cellular genes were shown to be upregulated and 104 genes were downregulated by nsp11. These genes were grouped into 5 major signaling pathways according to their functional relations: histone-related, cell cycle and DNA replication, mitogen activated protein kinase signaling, complement, and ubiquitin-proteasome pathways. Of these, the modulation of cell cycle by nsp11 was further investigated since many of the regulated genes fell in this particular pathway. Flow cytometry showed that nsp11 caused the delay of cell cycle progression at the S phase and the BrdU staining confirmed the cell cycle arrest in nsp11-expressing cells. The study provides insights into the understanding of specific cellular responses to nsp11 during PRRSV infection.
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Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB.
PLoS Genet.
PUBLISHED: 01-21-2013
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The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle-regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation.
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Spatial light interference tomography (SLIT).
Opt Express
PUBLISHED: 10-15-2011
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We present spatial light interference tomography (SLIT), a label-free method for 3D imaging of transparent structures such as live cells. SLIT uses the principle of interferometric imaging with broadband fields and combines the optical gating due to the micron-scale coherence length with that of the high numerical aperture objective lens. Measuring the phase shift map associated with the object as it is translated through focus provides full information about the 3D distribution associated with the refractive index. Using a reconstruction algorithm based on the Born approximation, we show that the sample structure may be recovered via a 3D, complex field deconvolution. We illustrate the method with reconstructed tomographic refractive index distributions of microspheres, photonic crystals, and unstained living cells.
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A BEN-domain-containing protein associates with heterochromatin and represses transcription.
J. Cell. Sci.
PUBLISHED: 09-15-2011
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In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression.
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Optical measurement of cycle-dependent cell growth.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-25-2011
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Determining the growth patterns of single cells offers answers to some of the most elusive questions in contemporary cell biology: how cell growth is regulated and how cell size distributions are maintained. For example, a linear growth in time implies that there is no regulation required to maintain homeostasis; an exponential pattern indicates the opposite. Recently, there has been great effort to measure single cells using microelectromechanical systems technology, and several important questions have been explored. However, a unified, easy-to-use methodology to measure the growth rate of individual adherent cells of various sizes has been lacking. Here we demonstrate that a newly developed optical interferometric technique, known as spatial light interference microscopy, can measure the cell dry mass of many individual adherent cells in various conditions, over spatial scales from micrometers to millimeters, temporal scales ranging from seconds to days, and cell types ranging from bacteria to mammalian cells. We found evidence of exponential growth in Escherichia coli, which agrees very well with other recent reports. Perhaps most importantly, combining spatial light interference microscopy with fluorescence imaging provides a unique method for studying cell cycle-dependent growth. Thus, by using a fluorescent reporter for the S phase, we measured single cell growth over each phase of the cell cycle in human osteosarcoma U2OS cells and found that the G2 phase exhibits the highest growth rate, which is mass-dependent and can be approximated by an exponential.
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"ORCanization" on heterochromatin: linking DNA replication initiation to chromatin organization.
Epigenetics
PUBLISHED: 06-01-2011
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Specialized complexes in eukaryotic cells recognize defined epigenetic histone marks to mediate chromatin organization. DNA replication, cell cycle progression and chromatin organization are intimately linked to one another. In addition to having roles in DNA replication initiation, the human Origin Recognition Complex (ORC) binds along with ORC-associated proteins ORCA/ LRWD1 to prominent transcriptional repressive lysine methylation marks and localizes to HP1-containing heterochromatic structures. In humans, Drosophila and Xenopus, ORC associates with HP1, and this interaction is crucial for heterochromatin organization. Further, several subunits of human ORC are required for centromere and telomere function and participate in chromosome segregation. The conserved function of ORC in replication initiation as well as in organization and maintenance of chromosome structure suggests that these cellular events are well coordinated.
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Polypurine-repeat-containing RNAs: a novel class of long non-coding RNA in mammalian cells.
J. Cell. Sci.
PUBLISHED: 10-12-2010
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In higher eukaryotic cells, long non-protein-coding RNAs (lncRNAs) have been implicated in a wide array of cellular functions. Cell- or tissue-specific expression of lncRNA genes encoded in the mammalian genome is thought to contribute to the complex gene networks needed to regulate cellular function. Here, we have identified a novel species of polypurine triplet repeat-rich lncRNAs, designated as GAA repeat-containing RNAs (GRC-RNAs), that localize to numerous punctate foci in the mammalian interphase nuclei. GRC-RNAs consist of a heterogeneous population of RNAs, ranging in size from ~1.5 kb to ~4 kb and localize to subnuclear domains, several of which associate with GAA.TTC-repeat-containing genomic regions. GRC-RNAs are components of the nuclear matrix and interact with various nuclear matrix-associated proteins. In mitotic cells, GRC-RNAs form distinct cytoplasmic foci and, in telophase and G1 cells, localize to the midbody, a structure involved in accurate cell division. Differentiation of tissue culture cells leads to a decrease in the number of GRC-RNA nuclear foci, albeit with an increase in size as compared with proliferating cells. Conversely, the number of GRC-RNA foci increases during cellular transformation. We propose that nuclear GRC-RNAs represent a novel family of mammalian lncRNAs that might play crucial roles in the cell nucleus.
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Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-05-2010
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The origin recognition complex (ORC) is a DNA replication initiator protein also known to be involved in diverse cellular functions including gene silencing, sister chromatid cohesion, telomere biology, heterochromatin localization, centromere and centrosome activity, and cytokinesis. We show that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) alpha- and HP1beta-containing heterochromatic foci. Fluorescent bleaching studies indicate that multiple subcomplexes of ORC exist at heterochromatin, with Orc1 stably associating with heterochromatin in G1 phase, whereas other ORC subunits have transient interactions throughout the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1alpha, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1alpha; however, both are essential for in vivo localization of Orc3 to heterochromatic foci. Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and Orc3 subunits by siRNA caused loss of HP1alpha association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1alpha distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1alpha from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins are mutually required for each other to bind to heterochromatin. Similar to HP1alpha-depleted cells, Orc2 and Orc3 siRNA-treated cells also show loss of compaction at satellite repeats, suggesting that ORC together with HP1 proteins may be involved in organizing higher-order chromatin structure and centromere function.
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The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation.
Mol. Cell
PUBLISHED: 02-08-2010
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Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.
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A WD-repeat protein stabilizes ORC binding to chromatin.
Mol. Cell
PUBLISHED: 01-23-2010
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Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.
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Orc1 controls centriole and centrosome copy number in human cells.
Science
PUBLISHED: 02-07-2009
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Centrosomes, each containing a pair of centrioles, organize microtubules in animal cells, particularly during mitosis. DNA and centrosomes are normally duplicated once before cell division to maintain optimal genome integrity. We report a new role for the Orc1 protein, a subunit of the origin recognition complex (ORC) that is a key component of the DNA replication licensing machinery, in controlling centriole and centrosome copy number in human cells, independent of its role in DNA replication. Cyclin A promotes Orc1 localization to centrosomes where Orc1 prevents Cyclin E-dependent reduplication of both centrioles and centrosomes in a single cell division cycle. The data suggest that Orc1 is a regulator of centriole and centrosome reduplication as well as the initiation of DNA replication.
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Emerging players in the initiation of eukaryotic DNA replication.
Cell Div
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Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression.
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Orc2 protects ORCA from ubiquitin-mediated degradation.
Cell Cycle
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Origin recognition complex (ORC) is highly dynamic, with several ORC subunits getting posttranslationally modified by phosphorylation or ubiquitination in a cell cycle-dependent manner. We have previously demonstrated that a WD repeat containing protein ORC-associated (ORCA/LRWD1) stabilizes the ORC on chromatin and facilitates pre-RC assembly. Further, ORCA levels are cell cycle-regulated, with highest levels during G(1), and progressively decreasing during S phase, but the mechanism remains to be elucidated. We now demonstrate that ORCA is polyubiquitinated in vivo, with elevated ubiquitination observed at the G(1)/S boundary. ORCA utilizes lysine-48 (K48) ubiquitin linkage, suggesting that ORCA ubiquitination mediates its regulated degradation. Ubiquitinated ORCA is re-localized in the form of nuclear aggregates and is predominantly associated with chromatin. We demonstrate that ORCA associates with the E3 ubiquitin ligase Cul4A-Ddb1. ORCA is ubiquitinated at the WD40 repeat domain, a region that is also recognized by Orc2. Furthermore, Orc2 associates only with the non-ubiquitinated form of ORCA, and Orc2 depletion results in the proteasome-mediated destabilization of ORCA. Based on the results, we suggest that Orc2 protects ORCA from ubiquitin-mediated degradation in vivo.
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Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation.
Mol. Cell. Biol.
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In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G(1)-to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G(1), and with geminin in post-G(1) cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G(1) cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.
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Replicating and transcribing on twisted roads of chromatin.
Brief Funct Genomics
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Chromatin, a complex of DNA and proteins in the eukaryotic cell nucleus governs various cellular processes including DNA replication, DNA repair and transcription. Chromatin architecture and dynamics dictates the timing of cellular events by regulating proteins accessibility to DNA as well as by acting as a scaffold for protein-protein interactions. Nucleosome, the basic unit of chromatin consists of a histone octamer comprised of (H3-H4)2 tetramer and two H2A-H2B dimers on which 146 bp of DNA is wrapped around ~1.6 times. Chromatin changes brought about by histone modifications, histone-modifying enzymes, chromatin remodeling factors, histone chaperones, histone variants and chromatin dynamics influence the regulation and timing of gene expression. Similarly, the timing of DNA replication is dependent on the chromatin context that in turn dictates origin selection. Further, during the process of DNA replication, not only does an organisms DNA have to be accurately replicated but also the chromatin structure and the epigenetic marks have to be faithfully transmitted to the daughter cells. Active transcription has been shown to repress replication while at the same time it has been shown that when origins are located at promoters, because of enhanced chromatin accessibility, they fire efficiently. In this review, we focus on how chromatin modulates two fundamental processes, DNA replication and transcription.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.