Defining a core configuration for human centromeres during mitosis.

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.

Defining a core configuration for human centromeres during mitosis.

The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including cohesin, the centromere-specific H3 variant CENP-A, and centromeric DNA. Centromeric DNA is highly variable between chromosomes yet must accomplish a similar function. Moreover, how the 50 nm cohesin ring, proposed to encircle sister chromatids, accommodates inter-sister centromeric distances of hundreds of nanometers on the metaphase spindle is a conundrum. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We used ChIP-seq and super-resolution microscopy to examine the geometry of essential centromeric components on human chromosomes. ChIP-seq demonstrates that cohesin subunits are depleted in α-satellite arrays where CENP-A nucleosomes and kinetochores assemble. Cohesin is instead enriched at pericentromeric DNA. Structured illumination microscopy of sister centromeres is consistent, revealing a non-overlapping pattern of CENP-A and cohesin. We used single particle averaging of hundreds of mitotic sister chromatids to develop an average centromere model. CENP-A clusters on sister chromatids, connected by α-satellite, are separated by ~562 nm with a perpendicular intervening ~190 nM wide axis of cohesin. Two differently sized α-satellite arrays on chromosome 7 display similar inter-sister CENP-A cluster distance, demonstrating different sized arrays can achieve a common spacing. Our data suggest a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes at the outer edge of extensible α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation for future studies of additional components required for centromere function.

Nucleolin modulates compartmentalization and dynamics of histone 2B-ECFP in the nucleolus.

Eukaryotic cells have 2 to ​3 discrete nucleoli required for ribosome synthesis. Nucleoli are phase separated nuclear sub-organelles. Here we examined the role of nuclear Lamins and nucleolar factors in modulating the compartmentalization and dynamics of histone 2B (H2B-ECFP) in the nucleolus. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H2B-ECFP mobility in the nucleolus. Furthermore, Nucleolin knockdown significantly decreases H2B-ECFP compartmentalization in the nucleolus, while H2B-ECFP residence and mobility in the nucleolus was prolonged upon Nucleolin overexpression. Co-expression of N-terminal and RNA binding domain (RBD) deletion mutants of Nucleolin or inhibiting 45S rRNA synthesis reduces the sequestration of H2B-ECFP in the nucleolus. Taken together, these studies reveal a crucial role of Nucleolin-rRNA complex in modulating the compartmentalization, stability and dynamics of H2B within the nucleolus.

Lamin B2 Modulates Nucleolar Morphology, Dynamics, and Function.

The nucleolus is required for ribosome biogenesis. Human cells have 2 or 3 nucleoli associated with nucleolar organizer region (NOR)-bearing chromosomes. An increase in number and altered nucleolar morphology define cancer cells. However, the mechanisms that modulate nucleolar morphology and function are unclear. Here we show that in addition to localizing at the nuclear envelope, lamin B2 localizes proximal to nucleolin at the granular component (GC) of the nucleolus and associates with the nucleolar proteins nucleolin and nucleophosmin. Lamin B2 knockdown severely disrupted the nucleolar morphology, which was rescued to intact and discrete nucleoli upon lamin B2 overexpression. Furthermore, two mutually exclusive lamin B2 deletion mutants, ΔHead and ΔSLS, rescued nuclear and nucleolar morphology defects, respectively, induced upon lamin B2 depletion, suggesting independent roles for lamin B2 at the nucleolus and nuclear envelope. Lamin B2 depletion increased nucleolin aggregation in the nucleoplasm, implicating lamin B2 in stabilizing nucleolin within the nucleolus. Lamin B2 knockdown upregulated nucleolus-specific 45S rRNA and upstream intergenic sequence (IGS) transcripts. The IGS transcripts colocalized with aggregates of nucleolin speckles, which were sustained in the nucleoplasm upon lamin B2 depletion. Taken together, these studies uncover a novel role for lamin B2 in modulating the morphology, dynamics, and function of the nucleolus.

A Small Molecule for Controlled Generation of Peroxynitrite.

2-Methyl-3-[1-(N,N-dimethylamino)diazen-1-ium-1,2-diol-2-ato-methyl]-naphthalene-1,4-dione 1 (HyPR-1), a small molecule containing a superoxide generator strategically linked to a diazeniumdiolate-based nitric oxide donor, is reported. Evidence for HyPR-1's ability to generate peroxynitrite in the presence of an enzyme as well as enhance peroxynitrite within cells is provided. The utility of this tool in generating peroxynitrite for cellular studies is demonstrated.