Mutations in the nucleophosmin (NPM1) gene represent the most common genetic alteration in acute myeloid leukaemia (AML) and result in mis-localisation of the mutated protein from a predominantly nucleolar localisation to a predominantly cytoplasmic distribution. Here, we use high resolution imaging to demonstrate that NPM1 is crucial for maintaining normal nucleoli architecture and specifically the integrity of the enigmatic nucleoli rim, the least understood nucleolar compartment. We demonstrate that cell lines and primary cells with NPM1 mutations from individuals with AML have aberrant nucleoli architecture; intriguingly this abnormal nucleolar phenotype is reversible. Using a surrogate for rRNA synthesis, we show that the aberrant phenotype is associated with differences in nucleolar function; specifically, activity of RNA polymerase I is increased in NPM1 mutated cells. Perinucleolar chromatin organisation is also markedly different in NPM1 mutant cells. Finally, we report the novel finding that NPM1 mutated protein forms distinct aggregates and characterise these for the first time. This work reveals how nucleolar organisation contributes to the molecular mechanisms underpinning NPM1-driven AML, revealing novel therapeutic vulnerabilities.

Using two optical traps we explore the biophysical properties of individual chromosomes, studying the role of the chromosome periphery, and present a novel analysis method to explore mechanical properties over 7 decades of frequency.

In dividing cells, chromosomes are coated in a sheath of proteins and RNA called the mitotic chromosome periphery. This sheath is thought to confer biophysical properties to chromosomes, critical for successful cell division. However, the details of chromosome mechanics, and specifically, if and how the chromosome periphery contributes to them, remain poorly understood. In this study, we present a comprehensive characterisation of single-chromosome mechanics using optical tweezers and an improved broadband microrheology analysis. We extend this analysis to direct measurements of the chromosome periphery by manipulating levels of Ki-67, its chief organiser, and apply a rheological model to isolate its contribution to chromosome mechanics. We report that the chromosome periphery governs dynamic self-reorganisation of chromosomes and acts as a structural constraint, providing force-damping properties. This work provides significant insight into chromosome mechanics and will inform our understanding of the mitotic chromosome periphery's role in cell division.

Chromosome compaction is a key feature of mitosis and critical for accurate chromosome segregation. However, a precise quantitative analysis of chromosome geometry during mitotic progression is lacking. Here, we use volume electron microscopy to map, with nanometer precision, chromosomes from prometaphase through telophase in human RPE1 cells. During prometaphase, chromosomes acquire a smoother surface, their arms shorten, and the primary centromeric constriction is formed. The chromatin is progressively compacted, ultimately reaching a remarkable nucleosome concentration of over 750 µM in late prometaphase that remains relatively constant during metaphase and early anaphase. Surprisingly, chromosomes then increase their volume in late anaphase prior to deposition of the nuclear envelope. The plateau of total chromosome volume from late prometaphase through early anaphase described here is consistent with proposals that the final stages of chromatin condensation in mitosis involve a limit density, such as might be expected for a process involving phase separation.

Mutations in the NPM1 gene represent the most common (>30% of patients) genetic alteration in Acute Myeloid Leukaemia (AML) and results in the mis-localisation of the mutated NPM1 protein from a predominantly nucleolar localisation to a predominantly cytoplasmic distribution. Numerous studies of NPM1 mutated AML have focussed on the aberrant cytoplasmic localisation of the mutated protein but efforts to reverse this mis-localisation therapeutically have so far resulted in limited clinical benefit. More recently, attention has shifted towards the nucleus with studies showing that mutant NPM1 binds to specific chromatin regions, where it directly regulates oncogenic gene expression. Here, we use high resolution imaging to demonstrate that Nucleophosmin (NPM1) is critical for maintaining normal nucleoli architecture and specifically the integrity of the nucleoli rim. We report for the first time that NPM1 mutated cell lines and primary samples have aberrant nucleoli architecture and demonstrate that the abnormal nucleoli phenotype is reversible. We also report the novel finding that NPM1 mutated protein forms distinct aggregates in NPM1 mutated cells and characterise these for the first time. This work reveals how nucleolar organisation contributes to the molecular mechanisms underpinning NPM1 driven AML and reveals unexpected novel vulnerabilities to be exploited for therapeutic intervention. ### Competing Interest Statement The authors have declared no competing interest.

Chromosomes and their constituent compartments are core elements of the cell division machinery. In mammalian oocytes, defects in the structure or composition of chromosomes are a leading cause of aberrant or failed meiosis, and by extension, can cause miscarriage and infertility. The underlying mechanisms are poorly understood, but are critical for development of novel diagnostics and treatments. The chromosome periphery, the least understood chromosome compartment, has recently emerged as an essential component of mitotic chromosomes and somatic cell division. However, in female meiosis it remains completely unexplored. This study provides the first comprehensive survey of a meiotic chromosome periphery compartment in both human and mouse oocytes. Using a combination of time-lapse microscopy, super-resolution imaging and 3DCLEM we show that removing the chromosome periphery, via Ki67 depletion, has substantial negative impact on chromosome structure, spatial organization and positional awareness, with many oocytes stalling and arresting in meiosis I. Importantly, we also reveal key differences between the mitotic and oocyte meiotic chromosome periphery compartments, most remarkably in the retention of Ki67 in the oocyte through anaphase I, where unwanted chromosomes are stripped of Ki67 before being ejected from the oocyte. This work presents the discovery of an exciting new pathway operating during female meiosis and a provides a platform for future work exploring the meiotic chromosome periphery for therapeutic vulnerabilities. ### Competing Interest Statement The authors have declared no competing interest.

Mitotic chromosomes are specialised packets of condensed genetic material with dynamic mechanical properties. Each chromosome is coated by a sheath of proteins and RNA, the mitotic chromosome periphery (MCP). The MCP is widely considered as an essential chromosome compartment where its multiple functions bestow material properties important for successful cell division. However, the details of the micromechanical properties of mitotic chromosomes, and specifically if and how the MCP contributes to these features, remain poorly understood. In this study, we present the most comprehensive characterisation of single-chromosome mechanics to date spanning a broadband frequency range, using optical tweezers and a novel microrheology technique. We extend this analysis to the first direct measurements of MCP micromechanics by manipulating levels of Ki-67, the chief organiser of this compartment, and apply a rheological model to isolate its contribution to chromosome dynamics. We report that the MCP governs high-frequency self-reorganisation dynamics and acts as a structural constraint, providing force-damping properties that mitigate mitotic stress. This work significantly advances our understanding of chromosome micromechanics and how the MCP contributes to the fundamental properties of chromosomes. ### Competing Interest Statement The authors have declared no competing interest.

Voltage-gated sodium channels cluster in macromolecular complexes at nodes of Ranvier to promote rapid nerve impulse conduction in vertebrate nerves. Node assembly in peripheral nerves is thought to be initiated at heminodes at the extremities of myelinating Schwann cells, and fusion of heminodes results in the establishment of nodes. Here we show that assembly of 'early clusters' of nodal proteins in the murine axonal membrane precedes heminode formation. The neurofascin (Nfasc) proteins are essential for node assembly, and the formation of early clusters also requires neuronal Nfasc. Early clusters are mobile and their proteins are dynamically recruited by lateral diffusion. They can undergo fusion not only with each other but also with heminodes, thus contributing to the development of nodes in peripheral axons. The formation of early clusters constitutes the earliest stage in peripheral node assembly and expands the repertoire of strategies that have evolved to establish these essential structures.

Disruption of axon-glia interactions in the peripheral nervous system has emerged as a major cause of arthrogryposis multiplex congenita (AMC), a condition characterized by multiple congenital postural abnormalities involving the major joints. Several genes crucially important to the biology of Schwann cells have now been implicated with AMC. One such gene is LGI4 which encodes a secreted glycoprotein. LGI4 is expressed and secreted by Schwann cells and binds its receptor ADAM22 on the axonal membrane to drive myelination. Homozygous mutations in LGI4 or ADAM22 results in severe congenital hypomyelination and joint contractures in mice. Recently bi-allelic LGI4 loss of function mutations has been described in three unrelated families with severe AMC. Two individuals in a fourth, non-consanguineous family were found to be compound heterozygous for two LGI4 missense mutations. It is not known how these missense mutations affect the biology of LGI4. Here we investigated whether these missense mutations affected the secretion of the protein, its ADAM22 binding capacity, or its myelination-promoting function. We demonstrate that the mutations largely affect the progression of the mutant protein through the endomembrane system resulting in severely reduced expression. Importantly, binding to ADAM22 and myelination-promoting activity appear largely unaffected, suggesting that treatment with chemical chaperones to improve secretion of the mutant proteins might prove beneficial.

The rapid evolution of super-resolution light microscopy has narrowed the gap between light and electron microscopy, allowing the imaging of molecules and cellular structures at high resolution within their normal cellular and tissue context. Multimodal imaging approaches such as correlative light electron microscopy (CLEM) combine these techniques to create a tool with unique imaging capacity. However, these approaches are typically reserved for specialists, and their application to the analysis of neural tissue is challenging. Here we present SuperCLEM, a relatively simple approach that combines super-resolution fluorescence light microscopy (FLM), 3D electron microscopy (3D-EM) and rendering into 3D models. We demonstrate our workflow using neuron-glia cultures from which we first acquire high-resolution fluorescent light images of myelinated axons. After resin embedding and re-identification of the region of interest, serially aligned EM sections are acquired and imaged using a serial block face scanning electron microscope (SBF-SEM). The FLM and 3D-EM datasets are then combined to render 3D models of the myelinated axons. Thus, the SuperCLEM imaging pipeline is a useful new tool for researchers pursuing similar questions in neuronal and other complex tissue culture systems.

The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a “promiscuous” biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is “frozen.” This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.

The requirement for condensin in chromosome formation in somatic cells remains unclear as imperfectly condensed chromosomes do form in conventional condensindepleted cells. Here we have dissected the role of condensin at different stages of vertebrate mitosis by combining auxin-mediated rapid depletion of condensin subunit SMC2 with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells. We analysed the outcomes by live and fixed-cell microscopy methods, including 3D correlative light and serial block face scanning electron microscopy. Following rapid depletion of condensin, chromosomal defects were obvious. The chromatin was compacted normally, but formed a single mass of mitotic chromosomes clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments titrating the auxin concentration suggest a previously unsuspected dual role of condensin, as different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Kumiko Samejima and Daniel Booth are co-first authors on ‘Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis’, published in Journal of Cell Science. Kumiko is a post-doctoral fellow in the lab of William C. Earnshaw at the University of Edinburgh, UK, investigating the mechanism of mitotic chromosome assembly and segregation. Daniel is a senior post-doctoral fellow in the lab of Dies Meijer at the University of Edinburgh, UK, and combines basic cell biology with translational medicine to explore the contribution of cell division errors to disease states.

During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other members of the constitutive centromere associated network (CCAN) participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analyzing unfolded centrochromatin from CENP-depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multipeak fitting algorithms revealed the presence of an organized pattern of centrochromatin packing consistent with arrangement of CENPA- containing nucleosomes into up to five chromatin "subunits" each containing roughly 20-30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.

The chromosome periphery is a complex network of proteins and RNA molecules (many derived from nucleoli) that covers the outer surface of chromosomes and whose function remains mysterious. Although it was first described over 130 years ago, technological advances and the recent discovery that Ki-67 acts as an organiser of this region have allowed the chromosome periphery to be dissected in previously unattainable detail, leading to a revival of interest in this obscure chromosomal compartment. Here, we review the most recent advances into the composition, structure and function of the chromosome periphery, discuss possible roles of Ki-67 during mitosis and consider why this structure is likely to remain the focus of ongoing attention in the future.

Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.