Biomedical subjects

Yun Wah Lam

Publications and source records attributed to Yun Wah Lam.

11 recordsLinked to original sources

PubMed2007

Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions.

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division, signaling, differentiation, and metabolism. It is expressed in mammalian cells as three closely related isoforms: alpha, beta/delta, and gamma1. These isoforms differ in their relative affinities for proteins, termed targeting subunits, that mediate their intracellular localization and substrate specificity. Because of the dynamic nature of these interactions, it is important to find experimental approaches that permit direct analyses of PP1 localization and PP1-targeting subunit interactions in live cells. When transiently or stably expressed as fluorescent protein (FP) fusions, the three isoforms are active phosphatases with distinct localization patterns and can interact with both endogenous and exogenous targeting subunits. Their changing spatio-temporal distributions can be monitored both throughout the cell cycle and following cellular perturbations by time-lapse fluorescence microscopy, and turnover rates of intracellular pools of the protein calculated by fluorescence recovery after photobleaching (FRAP). Interactions with targeting subunits can be visualized in vivo by fluorescence resonance energy transfer (FRET), using techniques such as sensitized emission, acceptor photobleaching, or fluorescence lifetime imaging.

Fluorescence Recovery After Photobleaching↗

PubMed2006

Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability.

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1alpha and -gamma are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1gamma is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1gamma onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1alpha and -gamma in vivo. When overexpressed, Repo-Man can also recruit PP1alpha to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference-induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1gamma and is required for the recruitment of PP1 to chromatin.

Amino Acid Sequence↗

PubMed2006

NOPdb: Nucleolar Proteome Database.

The Nucleolar Proteome Database (NOPdb) archives data on >700 proteins that were identified by multiple mass spectrometry (MS) analyses from highly purified preparations of human nucleoli, the most prominent nuclear organelle. Each protein entry is annotated with information about its corresponding gene, its domain structures and relevant protein homologues across species, as well as documenting its MS identification history including all the peptides sequenced by tandem MS/MS. Moreover, data showing the quantitative changes in the relative levels of approximately 500 nucleolar proteins are compared at different timepoints upon transcriptional inhibition. Correlating changes in protein abundance at multiple timepoints, highlighted by visualization means in the NOPdb, provides clues regarding the potential interactions and relationships between nucleolar proteins and thereby suggests putative functions for factors within the 30% of the proteome which comprises novel/uncharacterized proteins. The NOPdb (http://www.lamondlab.com/NOPdb) is searchable by either gene names, nucleotide or protein sequences, Gene Ontology terms or motifs, or by limiting the range for isoelectric points and/or molecular weights and links to other databases (e.g. LocusLink, OMIM and PubMed).

Cell Nucleolus↗

PubMed2005

The nucleolus.

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Animals↗

PubMed2004

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions.

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.

Arabidopsis↗

PubMed2004

Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells.

One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.

Cell Nucleolus↗

PubMed2004

3D3/lyric: a novel transmembrane protein of the endoplasmic reticulum and nuclear envelope, which is also present in the nucleolus.

We previously performed a gene-trap screen in mouse cells with particular focus on clones in which the trapped protein-reporter fusions localise to compartments of the nucleus. Here we describe one such gene-trap line in which the fusion protein showed a unique, patchy distribution at the nuclear periphery. We have cloned the endogenous mouse and human cDNAs encoding the protein trapped in the F9/3D3 cell line. The predicted proteins (64 kDa) encoded by this novel gene are highly conserved and similar to an unpublished rat protein in sequence databases called p80 or lyric. The amino acid sequence of 3D3/lyric indicates that it may be a type-1b membrane protein with a single transmembrane domain (TMD). Antibodies against the endogenous protein recognise multiple isoforms, consistent with multiple 3D3/lyric mRNAs detected by Northern blot analysis. Subcellular fractionation and immunostaining show that 3D3/lyric is located not only principally in the endoplasmic reticulum (ER), but also in the nuclear envelope (NE), which is contiguous with this compartment. Furthermore, 3D3/lyric is also found in the nucleolus and is therefore a rare example of a protein that suggests a possible connection between this compartment and the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed2003

Time-lapse imaging reveals dynamic relocalization of PP1gamma throughout the mammalian cell cycle.

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, alpha, beta/delta, and gamma1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1gamma or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1gamma to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1gamma shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1gamma also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1gamma revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.

Cell Cycle↗

PubMed2002

Paraspeckles: a novel nuclear domain.

BACKGROUND: The cell nucleus contains distinct classes of subnuclear bodies, including nucleoli, splicing speckles, Cajal bodies, gems, and PML bodies. Many nuclear proteins are known to interact dynamically with one or other of these bodies, and disruption of the specific organization of nuclear proteins can result in defects in cell functions and may cause molecular disease. RESULTS: A proteomic study of purified human nucleoli has identified novel proteins, including Paraspeckle Protein 1 (PSP1) (see accompanying article, this issue of Current Biology). Here we show that PSP1 accumulates in a new nucleoplasmic compartment, termed paraspeckles, that also contains at least two other protein components: PSP2 and p54/nrb. A similar pattern of typically 10 to 20 paraspeckles was detected in all human cell types analyzed, including primary and transformed cells. Paraspeckles correspond to discrete bodies in the interchromatin nucleoplasmic space that are often located adjacent to splicing speckles. A stable cell line expressing YFP-PSP1 has been established and used to demonstrate that PSP1 interacts dynamically with nucleoli and paraspeckles in living cells. The three paraspeckle proteins relocalize quantitatively to unique cap structures at the nucleolar periphery when transcription is inhibited. CONCLUSIONS: We have identified a novel nuclear compartment, termed paraspeckles, found in both primary and transformed human cells. Paraspeckles contain at least three RNA binding proteins that all interact dynamically with the nucleolus in a transcription-dependent fashion.

Amino Acid Sequence↗

PubMed2002

Directed proteomic analysis of the human nucleolus.

BACKGROUND: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. RESULTS: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. CONCLUSIONS: This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

Amino Acid Sequence↗

PubMed2002

Large-scale isolation of Cajal bodies from HeLa cells.

The Cajal body (CB) is a conserved, dynamic nuclear structure that is implicated in various cellular processes, such as the maturation of splicing small nuclear ribonucleoproteins and the assembly of transcription complexes. Here, we report the first procedure for the large-scale purification of CBs from HeLa cell nuclei, resulting in an approximately 750-fold enrichment of the CB marker protein p80-coilin. Immunofluorescence, immunoblotting, and mass spectrometric analyses showed that the composition of the isolated CBs was similar to that of CBs in situ. The morphology and structure of the isolated CBs, as judged by transmission and scanning electron microscopy analysis, are also similar to those of CBs in situ. This protocol demonstrates the feasibility of isolating intact distinct classes of subnuclear bodies from cultured cells in sufficient yield and purity to allow detailed characterization of their molecular composition, structure, and properties.

Animals↗