Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder

Article3 August 2020Open Access Transparent process Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder Lovisa Stenström orcid.org/0000-0002-2387-3491 Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Diana Mahdessian Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Christian Gnann Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Chan Zuckerberg Biohub, San Francisco, CA, USA Search for more papers by this author Anthony J Cesnik Chan Zuckerberg Biohub, San Francisco, CA, USA Department of Genetics, Stanford University, Stanford, CA, USA Search for more papers by this author Wei Ouyang Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Manuel D Leonetti Chan Zuckerberg Biohub, San Francisco, CA, USA Search for more papers by this author Mathias Uhlén orcid.org/0000-0002-4858-8056 Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Sara Cuylen-Haering Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Peter J Thul Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Emma Lundberg Corresponding Author [email protected] orcid.org/0000-0001-7034-0850 Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Chan Zuckerberg Biohub, San Francisco, CA, USA Department of Genetics, Stanford University, Stanford, CA, USA Search for more papers by this author Lovisa Stenström orcid.org/0000-0002-2387-3491 Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Diana Mahdessian Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Christian Gnann Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Chan Zuckerberg Biohub, San Francisco, CA, USA Search for more papers by this author Anthony J Cesnik Chan Zuckerberg Biohub, San Francisco, CA, USA Department of Genetics, Stanford University, Stanford, CA, USA Search for more papers by this author Wei Ouyang Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Manuel D Leonetti Chan Zuckerberg Biohub, San Francisco, CA, USA Search for more papers by this author Mathias Uhlén orcid.org/0000-0002-4858-8056 Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Sara Cuylen-Haering Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Peter J Thul Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Emma Lundberg Corresponding Author [email protected] orcid.org/0000-0001-7034-0850 Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Chan Zuckerberg Biohub, San Francisco, CA, USA Department of Genetics, Stanford University, Stanford, CA, USA Search for more papers by this author Author Information Lovisa Stenström1, Diana Mahdessian1, Christian Gnann1,2, Anthony J Cesnik2,3, Wei Ouyang1, Manuel D Leonetti2, Mathias Uhlén1, Sara Cuylen-Haering4, Peter J Thul1 and Emma Lundberg *,1,2,3 1Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden 2Chan Zuckerberg Biohub, San Francisco, CA, USA 3Department of Genetics, Stanford University, Stanford, CA, USA 4Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany *Corresponding author. Tel: +4687906000; E-mail: [email protected] Mol Syst Biol (2020)16:e9469https://doi.org/10.15252/msb.20209469 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas. Synopsis Spatiotemporal characterization of the human nucleolar proteome reveals spatial partitioning into fibrillar components and nucleoli rim. A subset of proteins with high intrinsic disorder show temporal relocation to the chromosomal periphery during mitosis. The human nucleolar proteome is large and functionally diverse with precise partitioning in time and space. The nucleolus rim is a subcompartment with a distinct proteomic composition. 65 nucleolar proteins (36 uncharacterized), many with high intrinsic disorder, relocate to the chromosomal periphery during mitosis. The recruitment of proteins to the chromosomal periphery is dependent on MKI67 and is partitioned into two phenotypes: early (prometaphase) and late (after metaphase) recruitment, suggesting phase-specific functions. Introduction One of the most prominent nuclear substructures is the nucleolus, the cellular site for ribosome synthesis and assembly. In addition, the nucleoli comprise proteins involved in cell cycle regulation and stress response (Visintin & Amon, 2000; Boisvert et al, 2007). Nucleoli form around nucleolar organizing regions on the ribosomal DNA sites (rDNA). As opposed to membrane-bound organelles, nucleoli and other nuclear bodies lack enclosing membranes. This allows for dynamic cellular responses as these structures can change in size and protein composition when needed. The size and number of nucleoli change throughout the cell cycle as they fuse together, a process recently suggested to be aided by interactions with the nucleoplasm (Caragine et al, 2019). The formation of these membrane-less yet spatially distinct structures is the result of reversible liquid–liquid phase transitions similar to oil-in-water emulsions (Brangwynne et al, 2009, 2011; Lin et al, 2015). In interphase, the nucleolus is structurally partitioned into three droplet-like layers with different miscibility (Feric et al, 2016). This separation facilitates a sequential production of ribosomes, from transcription of rDNA at the fibrillar center border (FC) followed by rRNA processing in the dense fibrillar component (DFC) and ribosome assembly in the granular component (GC). Phase separation is a dynamic process dependent on external factors such as pH, temperature, protein posttranslational modifications (PTMs) but most importantly protein composition and concentration. One common trait among proteins forming liquid-like droplets has shown to be the presence of low complexity sequence domains (LCDs) and protein disorder. Intrinsically disordered proteins (IDPs) are characterized by being fully or partially unfolded, making them flexible in terms of interaction, and have been suggested to be a strong driver of phase separation (Li et al, 2012; Berry et al, 2015; Elbaum-Garfinkle et al, 2015; Molliex et al, 2015; Nott et al, 2015). Intrinsically disordered proteins have shown to be central in various diseases such as cancer, cardiovascular diseases, and Alzheimer's disease. Mutations in disordered regions can drastically change the conformation of the protein, and since many IDPs function as hub proteins, altered protein function could initiate a loss-of-function cascade in the cell (Uversky et al, 2008). The inherently high density of the nucleoli enables its isolation and purification. Several studies using mass spectrometry (MS)-based proteomics have been focused on identifying proteins residing in the nucleoli (Andersen et al, 2002, 2005; Scherl et al, 2002; Leung et al, 2006). Together, they assign a nucleolar localization to over 700 proteins. Another intriguing finding is that the nucleolar proteome seems dynamic rather than static and contains many types of proteins not only related to ribosome biogenesis, indicating that the nucleolar proteome may be larger and more diverse than previously expected. As of today, there has been no effort to spatiotemporally map the human nucleolar proteome and its subcompartments throughout the cell cycle. When the nucleolus disassembles during mitosis, most nucleolar proteins leak to the cytoplasm. However, a majority of the mitotic chromosomal mass is not chromatin but other proteins residing in the perichromosomal layer including at least 50 known nucleolar proteins (Gautier et al, 1992a,b,c; Angelier et al, 2005; Van Hooser et al, 2005; Takata et al, 2007; Ohta et al, 2010; Booth et al, 2016). One example is the proliferation marker MKI67, a highly disordered nucleolar protein shown to be important for chromosome segregation by acting as an emulsifying shield around the chromosomes in mitosis (Booth et al, 2014; Cuylen et al, 2016). In this study, we used an antibody-based microscopy approach to generate a spatiotemporal map of the human nucleolar proteome in interphase and mitosis. We present a resource containing localization data for 1,318 nucleolar proteins including spatial sublocalization to nucleolar subcompartments such as the fibrillar center and the nucleoli rim, accessible as part of the Human Protein Atlas (HPA) database (www.proteinatlas.org; Uhlen et al, 2010; Thul et al, 2017). We also propose that the nucleoli rim is a dynamic nucleolar subcompartment with a distinct proteomic composition. Additionally, we show evidence for 65 nucleolar proteins being recruited to the chromosomal periphery during mitosis. Based on this subcellular map, we performed the first systematic analysis of intrinsic protein disorder for the human nucleolar proteome, experimentally confirming what has been conceptualized by others (Brangwynne et al, 2011; Nott et al, 2015; Feric et al, 2016), that a majority of the proteins have long intrinsically disordered domains. Results A detailed spatial map of the nucleolar proteome Despite the nucleolus being an intensively studied organelle, there is currently no resource offering a complete map of the human nucleolar proteome. To address this, we used the immunofluorescence (IF) and confocal microscopy workflow developed within the HPA Cell Atlas to systematically map all human nucleolar proteins (Thul et al, 2017). Out of the 12,393 proteins included in the HPA Cell Atlas (v19), we identified 1,318 nucleolar proteins, of which 287 localized to the fibrillar center or dense fibrillar component (from now on collectively denoted as fibrillar center) and 1,031 localized to the whole nucleolus (Dataset EV1). A schematic image of the tripartite nucleolar organization is shown in Fig 1A, while Fig 1B and C highlight typical confocal images of proteins localizing to whole nucleoli and fibrillar centers. UTP6, a protein thought to be involved in pre-18S rRNA based on its yeast homolog, is known to localize to nucleoli (Dragon et al, 2002; Fig 1B). UBTF activates RNA polymerase I-mediated transcription in the fibrillar center (Kwon & Green, 1994; Fig 1C). Functional enrichment analysis of the nucleolar proteome shows that the enriched Gene Ontology (GO) terms for biological process are well in line with the known functions of the nucleoli (e.g., ribosome biogenesis, rRNA processing, and transcription; Dataset EV2). We demonstrated the robustness of distinguishing nucleolar subcompartments by extracting features from all images in the HPA Cell Atlas using a machine-learning model (Ouyang et al, 2019), and then visualizing those features using a UMAP, a uniform manifold approximation and projection for dimensionality reduction (preprint: McInnes et al, 2018). The clustering of the IF microscopy images for the different nucleolar substructures confirms that they show distinct IF staining patterns that are consistent and robust across cell lines and antibodies, and the distance between the clusters shows that they easily can be distinguished both from each other and from other punctate nuclear substructures, such as nuclear speckles and nuclear bodies (Fig 1D). Figure 1. A detailed spatial map of the nucleolar human proteome Schematic overview of the nucleolus and its substructures: fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936). Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385). UMAP visualization of the IF images generated in the HPA Cell Atlas (also shown in Fig 3A). The images from singularly localizing nuclear proteins are highlighted in purple (nucleoli), blue (fibrillar center), brown (nuclear speckles), and orange (nuclear bodies). Multilocalizing nucleolar proteins are highlighted in yellow. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green). The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol, and ER in MCF-7 cells (HPA051702). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue, and the reference marker of microtubules in red. Scale bar 10 μm. Download figure Download PowerPoint Most nucleolar proteins are multilocalizing One advantage with image-based proteomics is the ability to study the in situ protein localization in single cells, including multilocalizing proteins (proteins localized to multiple compartments concurrently). In total, 54% of all proteins in the HPA Cell Atlas are detected in more than one cellular compartment, while as much as 87% of the nucleolar proteins (n = 1,145) are reported as multilocalizing (P < 2.2 × 10−16, using a one-tailed binomial test). Multilocalizing nucleolar proteins are visibly scattered throughout a majority of the organelle clusters in the UMAP (Fig 1D), with 33% simultaneously localizing to other nuclear locations only. However, this level of multilocalization is similar for other nuclear structures, such as the nuclear membrane and other nuclear bodies. The actin filament and plasma membrane proteome also have above 80% multilocalizing proteins (Thul et al, 2017). The most commonly shared localization for nucleolar proteins is the nucleoplasm, cytosol, or mitochondria. Multilocalizing nucleolar protein observations also provide the first evidence for nucleolar localization for several proteins. LEO1 is a nucleoplasmic protein which is part of the PAF1 complex. It is involved in transcription but has no previous experimental evidence for a nucleolar localization (Rozenblatt-Rosen et al, 2005; Zhao et al, 2005). However, using both antibodies and a GFP-tagged cell line we detected LEO1 in the nucleoplasm and the fibrillar center (Fig 1E, antibody staining in wild-type cells Appendix Fig S1). A protein association network of the shared proteomes between the nucleolus and cytosol shows a tightly connected cluster, indicating association with the same biological functions (Appendix Fig S2). Given that nucleoli synthesize and assemble ribosomes for export to the cytoplasm, multilocalizing nucleolar and cytosolic proteins could be involved in translation. The ribosomal protein cluster in the core of the network supports this. One such example is the ribosomal protein RPL13 (Fig 1F, siRNA antibody validation; Appendix Fig S3). The high number of multilocalizing nucleolar proteins suggests a functional versatility of these proteins, likely not only relating to ribosome biogenesis. The nucleolar proteome is larger than previously thought Based on our data, the nucleolus comprises around 7% of the human proteome, a higher number than previously proposed using MS-based methods (Andersen et al, 2002, 2005; Scherl et al, 2002; Leung et al, 2006). To assess the data reliability, we introduced a scoring system for each protein localization: enhanced, supported, approved, or uncertain (Thul et al, 2017). The score depends not only on available additional validation methods, such as siRNA knockdown, but also similarity in immunostaining patterns between independent antibodies or consistency with available experimental localization data in the UniProtKB/Swiss-Prot database (The UniProt Consortium, 2019). The requirements for each score are described in Materials and Methods, and the distribution of localization scores for the nucleolar proteins is shown in Appendix Fig S4. At least 700 proteins have been reported as nucleolar in the literature (Andersen et al, 2002, 2005; Scherl et al, 2002), while currently only 266 nucleolar proteins are reported as experimentally verified in GO (Ashburner et al, 2000; Gene Ontology Consortium, 2019). This highlights the need for an updated resource detailing the nucleolar proteome. We compared the HPA dataset to nucleolar proteins detected in other studies (Andersen et al, 2002, 2005; Scherl et al, 2002). To simplify the comparison, these datasets were merged since they show a high overlap (Appendix Fig S5). A total of 550 proteins are reported as nucleolar in these studies but not in the HPA Cell Atlas, possibly because of obsolete gene nomenclature resulting in incomplete mapping, potentially impure nucleolar fractions, or a lack of a specific antibody in the HPA library. In total, 237 of the HPA nucleolar proteins are verified in the studies mentioned above (Appendix Fig S5, Dataset EV1). When adding data from Orre et al (2019) and the experimentally verified nucleolar proteins in GO (Binns et al, 2009), 72 additional nucleolar proteins could be confirmed. Taken together, 1,009 nucleolar proteins are uniquely identified by the HPA dataset (Dataset EV1). Of these, 292 are not expressed in HeLa cells, which explains why they are absent from the mass spectrometry analysis on HeLa lysates (Andersen et al, 2002, 2005; Scherl et al, 2002). The HPA cell line panel consists of 64 cell lines expressing 98% (n = 19,216) of all protein-coding genes, enabling the detection of most proteins. The reasons why the HPA data contains so many uniquely identified nucleolar proteins are presumably the ability to detect multilocalizing proteins with lower abundance in the nucleolus and the ability to pick up single-cell variations obscured by bulk methods. Although it cannot be ruled out that the HPA dataset also contains false-positive nucleolus localizations, these proteins should at a maximum be a subset of the category labeled with the “Uncertain” reliability score (n = 122). Note that, no protein data is included in the HPA Cell Atlas, if only contradictory localization data exists. A total of 541 of the HPA nucleolar proteins have no previous human experimental data reported for any cellular component in GO, such as the nucleolar protein KRI1 (Fig 2A and Appendix Fig S6 for independent antibody staining and siRNA antibody validation) that may be required for ribosome biogenesis based on data from its yeast homolog (Sasaki et al, 2000; Huh et al, 2003). GPATCH4 has no localization data reported in GO, and we can validate it being nucleolar using two independent antibodies (Fig 2B and Appendix Fig S6 for independent antibody staining). The previously uncharacterized proteins FOXL2NB and FAM71E1 were localized to the fibrillar center (Fig 2C and D, Appendix Fig S6 for FOXL2NB-independent antibody staining), and LRRC23, ZNF853, and METTL5 (Fig 2E–G and Appendix Fig S6 for LRRC23-independent antibody staining) were localized to the whole nucleolus. Figure 2. Previously unknown nucleolar proteins KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue, and the reference marker of microtubules in red. Scale bar 10 μm. Download figure Download PowerPoint The nucleolus rim has a distinct proteomic composition and can be considered a subcompartment of the nucleolus Despite the lack of an enclosing membrane, 157 of the nucleolar proteins show a characteristic rim-like staining pattern in at least one cell line, denoted as nucleoli rim (Dataset EV1). When highlighting the single localizing nucleoli rim proteins in the UMAP, a distinct cluster adjacent to the nucleolar cluster appears, being significantly closer together than to the other nucleolar proteins (nrim = 27 and nnucleoli = 208, P = 0.0294 using nearest neighbor analysis followed by a two-tailed binomial test). This shows that the staining pattern relates to proteins in a distinct part of the nucleolus separated from the rest of the nucleolus and fibrillar centers (Fig 3A). For instance, MKI67 as well as GNL3 localize to the nucleoli rim using independent antibodies in all cell lines stained (Fig 3B and C and Appendix Fig S7). The rim localization pattern has previously been proposed to be an antibody artifact related to abundant proteins sterically hindering the antibody to penetrate the whole nucleoli; this could in theory result in a staining gradient similar to the rim localization (Sheval et al, 2005; Svistunova et al, 2012). To investigate this, we compared transcriptomics (Thul et al, 2017) and MS proteomic data from the U-2 OS cell line (Beck et al, 2011) for the rim proteins in relation to the non-rim nucleolar proteins. The analysis shows that the rim proteins on a group level are marginally more abundant compared with the non-rim nucleolar proteins (P = 2.975 × 10−5, nrim = 157, and nnon-rim = 1,161 for transcriptomics and P = 0.01992, nrim = 96, and nnon-rim = 613 for proteomic data, two-tailed unpaired Wilcoxon tests). However, the expression levels between the classes are still greatly overlapping (Appendix Fig S8) with both highly and lowly expressed proteins in both groups, showing that protein abundance is not the only factor driving the rim localization. To further confirm that the rim staining is not an antibody artifact, we created two cell lines expressing mNeonGreen-tagged (mNG) MKI67 and GNL3 at endogenous levels, tagged at the N-terminus and C-termini, respectively. Live cell imaging revealed a dimmer, but still visible, rim localization for MKI67 also in the tagged cell line that was further enhanced upon PFA fixation (Fig 3D and E). The rim localization was also confirmed for GNL3 in the tagged cells (Fig 3F). Since the rim localization is visible in the tagged cell lines, we conclude that proteins localizing to the nucleolar rim cannot simply be considered antibody artifacts but are instead a sign of a fourth nucleolar subcompartment with a distinct proteomic profile. The occurrence of this staining pattern seems to be complex, and these proteins have common molecular or functional features giving rise to this spatial organization. Figure 3. The nucleoli rim localization UMAP visualization of the IF images generated in the HPA Cell Atlas (also shown in Fig 1D), specifically highlighting the nucleolar protein clusters. The images from singularly localizing nucleolar proteins are highlighted in purple, fibrillar center proteins in blue, and nucleoli rim proteins in green. Multilocalizing nucleolar proteins are highlighted in yellow. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743). HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue, and microtubule reference marker in red. Scale bar 10 μm. Download figure Download PowerPoint Nucleolar proteins recruited to mitotic chromosomes As the cell enters mitosis, nucleoli are disassembled to enable the separation of the chromosomes. Most nucleolar proteins leak to the cytoplasm, while at least 50 nucleolar proteins have been shown to instead adhere to the periphery of the chromosomes (Gautier et al, 1992a,b,c; Angelier et al, 2005; Van Hooser et al, 2005; Takata et al, 2007; Ohta et al, 2010). To better understand the nucleolar dynamics during mitosis, we performed a single-cell spatial characterization of nucleolar proteins during cell division. MKI67 is one of the more prominent perichromosomal constituents. Thus, we generated a list of 150 targets with protein–protein association with MKI67, its interacting protein NIFK, or proteins showing a similar staining pattern in interphase as MKI67 (i.e., nucleoli rim; Dataset EV3). A mitotic shake-off protocol was used to enrich mitotic cells from an asynchronous cell population. A total of 85 nucleolar proteins could not be detected on the chromosomal periphery during cell division (Dataset EV3) as exemplified by the ribosomal protein RPS19BP1 (Appendix Fig S9). 65 proteins including MKI67 (Fig 4A) relocated to the chromosomal periphery of which 36 have, to our knowledge, no experimental data for being localized to chromosomes during cell division (Dataset EV3 and the HPA Cell Atlas, www.proteinatlas.org, for image data), exemplified by the proteins NOC2L, EMG1, BMS1, BRIX1, and RSL1D1 (Fig 4B–F, Appendix Fig S10 for independent antibody stainings of NOC2L and BMS1). Of the already known perichromosomal constituents, seven have been localized to chromosomes in chicken cells only (Ohta et al, 2010) and we provide experimental evidence for such a localization in human cells. For the 22 remaining known proteins, we confirmed their previously observed localization to condensed mitotic chromosomes (Gerdes et al, 1984; Weisenberger & Scheer, 1995; Magoulas et al, 1998; Westendorf et al, 1998; Olson et al, 2000; Lerch-Gaggl et al, 2002; Angelier et al, 2005; Takata et al, 2007; Amin et al, 2008; Gambe et al, 2009; Hirano et al, 2009; Hirai et al, 2013; Booth et al, 2014). Interestingly, a large fraction of the nucleoli rim proteins also relocate to the perichromosomal layer during mitosis. Assuming that the probability of a rim and non-rim protein localizing to mitotic chromosomes is equal and that the fraction of rim and non-rim proteins should be the same between the mitotic chromosome and the cytoplasmic leakage groups (73 rim proteins of 150 proteins stained, 49%), the actual distribution of rim proteins being relocated to mitotic chromosomes is significantly higher (49 of 65 mitotic chromosome proteins, 75%. P = 1 × 10−5, one-tailed binomial test). Figure 4. Nucleolar proteins recruited to mitotic chromosomes MKI67 (HPA000451). NOC2L (HPA044258). EMG1 (HPA039304). BMS1 (HPA043081). BRIX1 (HPA039614). RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules