Recruitment of functionally distinct membrane proteins to chromatin mediates nuclear envelope formation in vivo

Formation of the nuclear envelope (NE) around segregated chromosomes occurs by the reshaping of the endoplasmic reticulum (ER), a reservoir for disassembled nuclear membrane components during mitosis. In this study, we show that inner nuclear membrane proteins such as lamin B receptor (LBR), MAN1, Lap2β, and the trans-membrane nucleoporins Ndc1 and POM121 drive the spreading of ER membranes into the emerging NE via their capacity to bind chromatin in a collaborative manner. Despite their redundant functions, decreasing the levels of any of these trans-membrane proteins by RNAi-mediated knockdown delayed NE formation, whereas increasing the levels of any of them had the opposite effect. Furthermore, acceleration of NE formation interferes with chromosome separation during mitosis, indicating that the time frame over which chromatin becomes membrane enclosed is physiologically relevant and regulated. These data suggest that functionally distinct classes of chromatin-interacting membrane proteins, which are present at nonsaturating levels, collaborate to rapidly reestablish the nuclear compartment at the end of mitosis

Age Mosaicism across Multiple Scales in Adult Tissues

Most neurons are not replaced during an animal’s lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using ^(15)N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization

The Conserved Nup107-160 Complex Is Critical for Nuclear Pore Complex Assembly

AbstractNuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex

Single Bead Affinity Detection (SINBAD) for the Analysis of Protein-Protein Interactions

We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment

p120-catenin prevents multinucleation through control of MKLP1-dependent RhoA activity during cytokinesis.

Spatiotemporal activation of RhoA and actomyosin contraction underpins cellular adhesion and division. Loss of cell-cell adhesion and chromosomal instability are cardinal events that drive tumour progression. Here, we show that p120-catenin (p120) not only controls cell-cell adhesion, but also acts as a critical regulator of cytokinesis. We find that p120 regulates actomyosin contractility through concomitant binding to RhoA and the centralspindlin component MKLP1, independent of cadherin association. In anaphase, p120 is enriched at the cleavage furrow where it binds MKLP1 to spatially control RhoA GTPase cycling. Binding of p120 to MKLP1 during cytokinesis depends on the N-terminal coiled-coil domain of p120 isoform 1A. Importantly, clinical data show that loss of p120 expression is a common event in breast cancer that strongly correlates with multinucleation and adverse patient survival. In summary, our study identifies p120 loss as a driver event of chromosomal instability in cancer

ER membrane–bending proteins are necessary for de novo nuclear pore formation

Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) embedded in pores formed by inner and outer nuclear membrane fusion. The mechanism for de novo pore and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER) and the postmitotic nuclear envelope. In this study, we report that members of the RTN and Yop1/DP1 families are required for nuclear pore formation. Analysis of Saccharomyces cerevisiae prp20-G282S and nup133Δ NPC assembly mutants revealed perturbations in Rtn1–green fluorescent protein (GFP) and Yop1-GFP ER distribution and colocalization to NPC clusters. Combined deletion of RTN1 and YOP1 resulted in NPC clustering, nuclear import defects, and synthetic lethality with the additional absence of Pom34, Pom152, and Nup84 subcomplex members. We tested for a direct role in NPC biogenesis using Xenopus laevis in vitro assays and found that anti-Rtn4a antibodies specifically inhibited de novo nuclear pore formation. We hypothesize that these ER membrane–bending proteins mediate early NPC assembly steps