In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.
The centrosome acts as a microtubule-organizing center (MTOC) from the G to G phases of the cell cycle; it can mature into a spindle pole during mitosis and/or transition into a cilium by elongating microtubules (MTs) from the basal body on cell differentiation or cell cycle arrest. New studies hint that the centrosome functions in more than MT organization. For instance, it has recently been shown that a specific substructure of the centrosome-the mother centriole appendages-are required for the recycling of endosomes back to the plasma membrane. This alone could have important implications for a renaissance in our understanding of the development of primary cilia, endosome recycling, and the immune response. Here, we review newly identified roles for the centrosome in directing membrane traffic, the immunological synapse, and the stress response.
In most vertebrates, mitotic spindles and primary cilia arise from a common origin, the centrosome. In non-cycling cells, the centrosome is the template for primary cilia assembly and, thus, is crucial for their associated sensory and signaling functions. During mitosis, the duplicated centrosomes mature into spindle poles, which orchestrate mitotic spindle assembly, chromosome segregation, and orientation of the cell division axis. Intriguingly, both cilia and spindle poles are centrosome-based, functionally distinct structures that require the action of microtubulemediated, motor-driven transport for their assembly. Cilia proteins have been found at non-cilia sites, where they have distinct functions, illustrating a diverse and growing list of cellular processes and structures that utilize cilia proteins for crucial functions. In this review, we discuss cilia-independent functions of cilia proteins and re-evaluate their potential contributions to "cilia" disorders. See the Glossary for abbreviations used in this article. Cilia proteins and cilia disorders-is there more?Primary cilia (henceforth called "cilia") are present in almost all cell types, lymphoid cells being one exception. Thus, cilia proteins have the potential to adversely affect numerous organs and tissues when disrupted. Over the past 10-15 years, cilia loss and/or dysfunction have been linked to numerous human disorders, collectively termed ciliopathies. Phenotypes associated with cilia dysfunction are often syndromic and include cystic kidneys, polydactyly, situs inversus, obesity, and encephalocele [1], to name a few. For example, disruption of cilia-based Hedgehog (Hh) signaling has been implicated in polydactyly [2,3] and disruption of cilia-based signaling through Ca 2+ or planar cell polarity (PCP) has been linked to cystogenesis [4,5]. However, a growing body of evidence suggests that cilia proteins are, in fact, multifunctional. In addition to their localization to cilia, many localize to and are bona fide components of centrosomes, as well as other cellular organelles and structures. At these non-cilia sites, cilia proteins perform functions distinct from their ciliary roles. Thus, we contend that disruption of cilia proteins-historically named for their localization to cilia, as well as their association with cilia defects and cilia-related disorderscan additionally disrupt spindle poles and an expanding list of cellular structures that impact numerous cellular functions. As a result, it has become increasingly difficult to determine which of these cellular functions and which of the these non-cilia organelles truly contribute to ciliopathies when cilia proteins are disrupted [6,7]. In this review, we first explore the diversity of cilia-independent processes that involve cilia proteins and then discuss alternative hypotheses for the etiology of "ciliopathies".The centrosome, a common thread between cilia and mitosisMost vertebrate centrosomes are comprised of two centriole barrels surrounded by pericentriolar material (PCM). A...
In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining Nucleolus-Associated Domains (NADs) and Lamina-Associated Domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, due to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and with the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3 and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Interestingly, Type II NADs are enriched in distinct gene classes, notably factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, present in MEFs but not in undifferentiated embryonic stem (ES) cells. microscopy images (Supplemental Fig. 1B; see also the "NAD-seq" QC files at the NIH 4DN Data Portal (data.4dnucleome.org)). Quantitative PCR suggested that rDNA sequences were enriched relative to the input total genomic DNA > 20-fold in the non-crosslinked preparations, and > 7-fold in crosslinked preparations ( Supplemental Fig. 1C). Immunoblot analyses showed enrichment of nucleolar protein fibrillarin relative to non-nucleolar proteins such as actin, a nucleoporin (Nup62), or lamin A/C (Supplemental Fig.1D). Additionally, we characterized the small RNAs present in the non-crosslinked preparation, observing that the nucleoli were highly enriched for the nucleolar U3 RNA (Supplemental Fig.1E). Small RNAs could not be recovered from crosslinked samples (data not shown).Bioinformatic analysis of nucleolar-associated DNA. We performed three biological replicate experiments for both the crosslinked and non-crosslinked protocols. For each experiment, we purified DNA from the isolated nucleoli, alongside genomic DNA from a sample of whole, unfractionated cells from the same population for normalization of the nucleolar sequencing data. These DNAs were used to generate sequencing libraries via PCR-free protocols, yielding genome-scale maps. Visual inspection of these "NAD-seq" data showed that the total genomic DNA was largely evenly distributed across the genome (Fig. 1B, C), occasionally interspersed with short, poorly represented regions. The nucleolar DNA was distributed with distinct pea...
The centrosome is critical for cell division. Heat stress (HS) causes degradation of all centrosome substructures by centrosome-bound proteasomes. HS-activated degradation is centrosome specific and can be rescued by targeting Hsp70 to the centrosome. Centrosome inactivation is a physiological event, as centrosomes in leukocytes of febrile patients are disrupted.
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